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Pentose Shunt, Electron Transfer, Galactose, more Lipids in brief


Pentose Shunt, Electron Transfer, Galactose, more Lipids in brief

Reviewer and Curator: Larry H. Bernstein, MD, FCAP

Pentose Shunt, Electron Transfer, Galactose, and other Lipids in brief

This is a continuation of the series of articles that spans the horizon of the genetic
code and the progression in complexity from genomics to proteomics, which must
be completed before proceeding to metabolomics and multi-omics.  At this point
we have covered genomics, transcriptomics, signaling, and carbohydrate metabolism
with considerable detail.In carbohydrates. There are two topics that need some attention –
(1) pentose phosphate shunt;
(2) H+ transfer
(3) galactose.
(4) more lipids
Then we are to move on to proteins and proteomics.

Summary of this series:

The outline of what I am presenting in series is as follows:

  1. Signaling and Signaling Pathways
    https://pharmaceuticalintelligence.com/2014/08/12/signaling-and-signaling-pathways/
  2. Signaling transduction tutorial.
    https://pharmaceuticalintelligence.com/2014/08/12/signaling-transduction-tutorial/
  3. Carbohydrate metabolism
    https://pharmaceuticalintelligence.com/2014/08/13/carbohydrate-metabolism/

Selected References to Signaling and Metabolic Pathways published in this Open Access Online Scientific Journal, include the following: 

https://pharmaceuticalintelligence.com/2014/08/14/selected-references-to-signaling-
and-metabolic-pathways-in-leaders-in-pharmaceutical-intelligence/

  1. Lipid metabolism

4.1  Studies of respiration lead to Acetyl CoA
https://pharmaceuticalintelligence.com/2014/08/18/studies-of-respiration-lead-to-acetyl-coa/

4.2 The multi-step transfer of phosphate bond and hydrogen exchange energy
https://pharmaceuticalintelligence.com/2014/08/19/the-multi-step-transfer-of-phosphate-
bond-and-hydrogen-exchange-energy/

5.Pentose shunt, electron transfers, galactose, and other lipids in brief

6. Protein synthesis and degradation

7.  Subcellular structure

8. Impairments in pathological states: endocrine disorders; stress
hypermetabolism; cancer.

Section I. Pentose Shunt

Bernard L. Horecker’s Contributions to Elucidating the Pentose Phosphate Pathway

Nicole Kresge,     Robert D. Simoni and     Robert L. Hill

The Enzymatic Conversion of 6-Phosphogluconate to Ribulose-5-Phosphate
and Ribose-5-Phosphate (Horecker, B. L., Smyrniotis, P. Z., and Seegmiller,
J. E.      J. Biol. Chem. 1951; 193: 383–396

Bernard Horecker

Bernard Leonard Horecker (1914) began his training in enzymology in 1936 as a
graduate student at the University of Chicago in the laboratory of T. R. Hogness.
His initial project involved studying succinic dehydrogenase from beef heart using
the Warburg manometric apparatus. However, when Erwin Hass arrived from Otto
Warburg’s laboratory he asked Horecker to join him in the search for an enzyme
that would catalyze the reduction of cytochrome c by reduced NADP. This marked
the beginning of Horecker’s lifelong involvement with the pentose phosphate pathway.

During World War II, Horecker left Chicago and got a job at the National Institutes of
Health (NIH) in Frederick S. Brackett’s laboratory in the Division of Industrial Hygiene.
As part of the wartime effort, Horecker was assigned the task of developing a method
to determine the carbon monoxide hemoglobin content of the blood of Navy pilots
returning from combat missions. When the war ended, Horecker returned to research
in enzymology and began studying the reduction of cytochrome c by the succinic
dehydrogenase system.

Shortly after he began these investigation changes, Horecker was approached by
future Nobel laureate Arthur Kornberg, who was convinced that enzymes were the
key to understanding intracellular biochemical processes
. Kornberg suggested
they collaborate, and the two began to study the effect of cyanide on the succinic
dehydrogenase system. Cyanide had previously been found to inhibit enzymes
containing a heme group, with the exception of cytochrome c. However, Horecker
and Kornberg found that

  • cyanide did in fact react with cytochrome c and concluded that
  • previous groups had failed to perceive this interaction because
    • the shift in the absorption maximum was too small to be detected by
      visual examination.

Two years later, Kornberg invited Horecker and Leon Heppel to join him in setting up
a new Section on Enzymes in the Laboratory of Physiology at the NIH. Their Section on Enzymes eventually became part of the new Experimental Biology and Medicine
Institute and was later renamed the National Institute of Arthritis and Metabolic
Diseases.

Horecker and Kornberg continued to collaborate, this time on

  • the isolation of DPN and TPN.

By 1948 they had amassed a huge supply of the coenzymes and were able to
present Otto Warburg, the discoverer of TPN, with a gift of 25 mg of the enzyme
when he came to visit. Horecker also collaborated with Heppel on 

  • the isolation of cytochrome c reductase from yeast and 
  • eventually accomplished the first isolation of the flavoprotein from
    mammalian liver.

Along with his lab technician Pauline Smyrniotis, Horecker began to study

  • the enzymes involved in the oxidation of 6-phosphogluconate and the
    metabolic intermediates formed in the pentose phosphate pathway.

Joined by Horecker’s first postdoctoral student, J. E. Seegmiller, they worked
out a new method for the preparation of glucose 6-phosphate and 6-phosphogluconate, 
both of which were not yet commercially available.
As reported in the Journal of Biological Chemistry (JBC) Classic reprinted here, they

  • purified 6-phosphogluconate dehydrogenase from brewer’s yeast (1), and 
  • by coupling the reduction of TPN to its reoxidation by pyruvate in
    the presence of lactic dehydrogenase
    ,
  • they were able to show that the first product of 6-phosphogluconate oxidation,
  • in addition to carbon dioxide, was ribulose 5-phosphte.
  • This pentose ester was then converted to ribose 5-phosphate by a
    pentose-phosphate isomerase.

They were able to separate ribulose 5-phosphate from ribose 5- phosphate and demonstrate their interconversion using a recently developed nucleotide separation
technique called ion-exchange chromatography. Horecker and Seegmiller later
showed that 6-phosphogluconate metabolism by enzymes from mammalian
tissues also produced the same products
.8

Bernard Horecker

Bernard Horecker

http://www.jbc.org/content/280/29/e26/F1.small.gif

Over the next several years, Horecker played a key role in elucidating the

  • remaining steps of the pentose phosphate pathway.

His total contributions included the discovery of three new sugar phosphate esters,
ribulose 5-phosphate, sedoheptulose 7-phosphate, and erythrose 4-phosphate, and
three new enzymes, transketolase, transaldolase, and pentose-phosphate 3-epimerase.
The outline of the complete pentose phosphate cycle was published in 1955
(2). Horecker’s personal account of his work on the pentose phosphate pathway can
be found in his JBC Reflection (3).1

Horecker’s contributions to science were recognized with many awards and honors
including the Washington Academy of Sciences Award for Scientific Achievement in
Biological Sciences (1954) and his election to the National Academy of Sciences in
1961. Horecker also served as president of the American Society of Biological
Chemists (now the American Society for Biochemistry and Molecular Biology) in 1968.

Footnotes

  • 1 All biographical information on Bernard L. Horecker was taken from Ref. 3.
  • The American Society for Biochemistry and Molecular Biology, Inc.

References

  1. ↵Horecker, B. L., and Smyrniotis, P. Z. (1951) Phosphogluconic acid dehydrogenase
    from yeast. J. Biol. Chem. 193, 371–381FREE Full Text
  2. Gunsalus, I. C., Horecker, B. L., and Wood, W. A. (1955) Pathways of carbohydrate
    metabolism in microorganisms. Bacteriol. Rev. 19, 79–128  FREE Full Text
  3. Horecker, B. L. (2002) The pentose phosphate pathway. J. Biol. Chem. 277, 47965–
    47971 FREE Full Text

The Pentose Phosphate Pathway (also called Phosphogluconate Pathway, or Hexose
Monophosphate Shunt) is depicted with structures of intermediates in Fig. 23-25
p. 863 of Biochemistry, by Voet & Voet, 3rd Edition. The linear portion of the pathway
carries out oxidation and decarboxylation of glucose-6-phosphate, producing the
5-C sugar ribulose-5-phosphate.

Glucose-6-phosphate Dehydrogenase catalyzes oxidation of the aldehyde
(hemiacetal), at C1 of glucose-6-phosphate, to a carboxylic acid in ester linkage
(lactone). NADPserves as electron acceptor.

6-Phosphogluconolactonase catalyzes hydrolysis of the ester linkage (lactone)
resulting in ring opening. The product is 6-phosphogluconate. Although ring opening
occurs in the absence of a catalyst, 6-Phosphogluconolactonase speeds up the
reaction, decreasing the lifetime of the highly reactive, and thus potentially
toxic, 6-phosphogluconolactone.

Phosphogluconate Dehydrogenase catalyzes oxidative decarboxylation of
6-phosphogluconate, to yield the 5-C ketose ribulose-5-phosphate. The
hydroxyl at C(C2 of the product) is oxidized to a ketone. This promotes loss
of the carboxyl at C1 as CO2.  NADP+ again serves as oxidant (electron acceptor).

pglucose hd

pglucose hd

https://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/images/pglucd.gif

Reduction of NADP+ (as with NAD+) involves transfer of 2e- plus 1H+ to the
nicotinamide moiety.

nadp

NADPH, a product of the Pentose Phosphate Pathway, functions as a reductant in
various synthetic (anabolic) pathways, including fatty acid synthesis.

NAD+ serves as electron acceptor in catabolic pathways in which metabolites are
oxidized. The resultant NADH is reoxidized by the respiratory chain, producing ATP.

nadnadp

https://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/images/nadnadp.gif

Regulation: 
Glucose-6-phosphate Dehydrogenase is the committed step of the Pentose
Phosphate Pathway. This enzyme is regulated by availability of the substrate NADP+.
As NADPH is utilized in reductive synthetic pathways, the increasing concentration of
NADP+ stimulates the Pentose Phosphate Pathway, to replenish NADPH.

The remainder of the Pentose Phosphate Pathway accomplishes conversion of the
5-C ribulose-5-phosphate to the 5-C product ribose-5-phosphate, or to the 3-C
glyceraldehyde -3-phosphate and the 6-C fructose-6-phosphate (reactions 4 to 8
p. 863).

Transketolase utilizes as prosthetic group thiamine pyrophosphate (TPP), a
derivative of vitamin B1.

tpp

tpp

https://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/images/tpp.gif

Thiamine pyrophosphate binds at the active sites of enzymes in a “V” conformation.The amino group of the aminopyrimidine moiety is close to the dissociable proton,
and serves as the proton acceptor. This proton transfer is promoted by a glutamate
residue adjacent to the pyrimidine ring.

The positively charged N in the thiazole ring acts as an electron sink, promoting
C-C bond cleavage. The 3-C aldose glyceraldehyde-3-phosphate is released.
2-C fragment remains on TPP.

FASEB J. 1996 Mar;10(4):461-70.   http://www.ncbi.nlm.nih.gov/pubmed/8647345

Reviewer

The importance of this pathway can easily be underestimated.  The main source for
energy in respiration was considered to be tied to the

  • high energy phosphate bond in phosphorylation and utilizes NADPH, converting it to NADP+.

glycolysis n skeletal muscle in short term, dependent on muscle glycogen conversion
to glucose, and there is a buildup of lactic acid – used as fuel by the heart.  This
pathway accounts for roughly 5% of metabolic needs, varying between tissues,
depending on there priority for synthetic functions, such as endocrine or nucleic
acid production.

The mature erythrocyte and the ocular lens both are enucleate.  85% of their
metabolic energy needs are by anaerobic glycolysis.  Consider the erythrocyte
somewhat different than the lens because it has iron-based hemoglobin, which
exchanges O2 and CO2 in the pulmonary alveoli, and in that role, is a rapid
regulator of H+ and pH in the circulation (carbonic anhydrase reaction), and also to
a lesser extent in the kidney cortex, where H+ is removed  from the circulation to
the urine, making the blood less acidic, except when there is a reciprocal loss of K+.
This is how we need a nomogram to determine respiratory vs renal acidosis or
alkalosis.  In the case of chronic renal disease, there is substantial loss of
functioning nephrons, loss of countercurrent multiplier, and a reduced capacity to
remove H+.  So there is both a metabolic acidosis and a hyperkalemia, with increased
serum creatinine, but the creatinine is only from muscle mass – not accurately
reflecting total body mass, which includes visceral organs.  The only accurate
measure of lean body mass would be in the linear relationship between circulating
hepatic produced transthyretin (TTR).

The pentose phosphate shunt is essential for

  • the generation of nucleic acids, in regeneration of red cells and lens – requiring NADPH.

Insofar as the red blood cell is engaged in O2 exchange, the lactic dehydrogenase
isoenzyme composition is the same as the heart. What about the lens of and cornea the eye, and platelets?  The explanation does appear to be more complex than
has been proposed and is not discussed here.

Section II. Mitochondrial NADH – NADP+ Transhydrogenase Reaction

There is also another consideration for the balance of di- and tri- phospopyridine
nucleotides in their oxidized and reduced forms.  I have brought this into the
discussion because of the centrality of hydride tranfer to mitochondrial oxidative
phosphorylation and the energetics – for catabolism and synthesis.

The role of transhydrogenase in the energy-linked reduction of TPN 

Fritz HommesRonald W. Estabrook∗∗

The Wenner-Gren Institute, University of Stockholm
Stockholm, Sweden
Biochemical and Biophysical Research Communications 11, (1), 2 Apr 1963, Pp 1–6
http://dx.doi.org:/10.1016/0006-291X(63)90017-2

In 1959, Klingenberg and Slenczka (1) made the important observation that incubation of isolated

  • liver mitochondria with DPN-specific substrates or succinate in the absence of phosphate
    acceptor resulted in a rapid and almost complete reduction of the intramitochondrial TPN.

These and related findings led Klingenberg and co-workers (1-3) to postulate

  • the occurrence of an ATP-controlled transhydrogenase reaction catalyzing the reduction of
    mitochondrial TPN by DPNH. A similar conclusion was reached by Estabrook and Nissley (4).

The present paper describes the demonstration and some properties of an

  • energy-dependent reduction of TPN by DPNH, catalyzed by submitochondrial particles.

Preliminary reports of some of these results have already appeared (5, 6 ) , and a
complete account is being published elsewhere (7).We have studied the energy- dependent reduction of TPN by PNH with submitochondrial particles from both
rat liver and beef heart. Rat liver particles were prepared essentially according to
the method of Kielley and Bronk (8), and beef heart particles by the method of
Low and Vallin (9).

PYRIDINE NUCLEOTIDE TRANSHYDROGENASE  II. DIRECT EVIDENCE FOR
AND MECHANISM OF THE
 TRANSHYDROGENASE REACTION*

BY  NATHAN 0. KAPLAN, SIDNEY P. COLOWICK, AND ELIZABETH F. NEUFELD
(From the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore,
Maryland)  J. Biol. Chem. 1952, 195:107-119.
http://www.jbc.org/content/195/1/107.citation

NO Kaplan

NO Kaplan

Sidney Colowick

Sidney Colowick

Elizabeth Neufeld

Elizabeth Neufeld

Kaplan studied carbohydrate metabolism in the liver under David M. Greenberg at the
University of California, Berkeley medical school. He earned his Ph.D. in 1943. From
1942 to 1944, Kaplan participated in the Manhattan Project. From 1945 to 1949,
Kaplan worked with Fritz Lipmann at Massachusetts General Hospital to study
coenzyme A. He worked at the McCollum-Pratt Institute of Johns Hopkins University
from 1950 to 957. In 1957, he was recruited to head a new graduate program in
biochemistry at Brandeis University. In 1968, Kaplan moved to the University of
California, San Diego
, where he studied the role of lactate dehydrogenase in cancer. He also founded a colony of nude mice, a strain of laboratory mice useful in the study
of cancer and other diseases. [1] He was a member of the National Academy of
Sciences.One of Kaplan’s students at the University of California was genomic
researcher Craig Venter.[2]3]  He was, with Sidney Colowick, a founding editor of the scientific book series Methods
in Enzymology
.[1]

http://books.nap.edu/books/0309049768/xhtml/images/img00009.jpg

Colowick became Carl Cori’s first graduate student and earned his Ph.D. at
Washington University St. Louis in 1942, continuing to work with the Coris (Nobel
Prize jointly) for 10 years. At the age of 21, he published his first paper on the
classical studies of glucose 1-phosphate (2), and a year later he was the sole author on a paper on the synthesis of mannose 1-phosphate and galactose 1-phosphate (3). Both papers were published in the JBC. During his time in the Cori lab,

Colowick was involved in many projects. Along with Herman Kalckar he discovered
myokinase (distinguished from adenylate kinase from liver), which is now known as
adenyl kinase. This discovery proved to be important in understanding transphos-phorylation reactions in yeast and animal cells. Colowick’s interest then turned to
the conversion of glucose to polysaccharides, and he and Earl Sutherland (who
will be featured in an upcoming JBC Classic) published an important paper on the
formation of glycogen from glucose using purified enzymes (4). In 1951, Colowick
and Nathan Kaplan were approached by Kurt Jacoby of Academic Press to do a
series comparable to Methodem der Ferment Forschung. Colowick and Kaplan
planned and edited the first 6 volumes of Methods in Enzymology, launching in 1955
what became a series of well known and useful handbooks. He continued as
Editor of the series until his death in 1985.

The Structure of NADH: the Work of Sidney P. Colowick

Nicole KresgeRobert D. Simoni and Robert L. Hill

On the Structure of Reduced Diphosphopyridine Nucleotide

(Pullman, M. E., San Pietro, A., and Colowick, S. P. (1954)

J. Biol. Chem. 206, 129–141)

Elizabeth Neufeld
·  Born: September 27, 1928 (age 85), Paris, France
·  EducationQueens College, City University of New YorkUniversity of California,
Berkeley

http://fdb5.ctrl.ucla.edu/biological-chemistry/institution/photo?personnel%5fid=45290&max_width=155&max_height=225

In Paper I (l), indirect evidence was presented for the following transhydrogenase
reaction, catalyzed by an enzyme present in extracts of Pseudomonas
fluorescens:

TPNHz + DPN -+ TPN + DPNHz

The evidence was obtained by coupling TPN-specific dehydrogenases with the
transhydrogenase and observing the reduction of large amounts of diphosphopyridine nucleotide (DPN) in the presence of catalytic amounts of triphosphopyridine
nucleotide (TPN).

In this paper, data will be reported showing the direct

  • interaction between TPNHz and DPN, in thepresence of transhydrogenase alone,
  • to yield products having the propertiesof TPN and DPNHZ.

Information will be given indicating that the reaction involves

  • a transfer of electrons (or hydrogen) rather than a phosphate 

Experiments dealing with the kinetics and reversibility of the reaction, and with the
nature of the products, suggest that the reaction is a complex one, not fully described
by the above formulation.

Materials and Methods [edited]

The TPN and DPN used in these studies were preparations of approximately 75
percent purity and were prepared from sheep liver by the chromatographic procedure
of Kornberg and Horecker (unpublished). Reduced DPN was prepared enzymatically with alcohol dehydrogenase as described elsewhere (2). Reduced TPN was prepared by treating TPN with hydrosulfite. This treated mixture contained 2 pM of TPNHz per ml.
The preparations of desamino DPN and reduced desamino DPN have been
described previously (2, 3). Phosphogluconate was a barium salt which was kindly
supplied by Dr. B. F. Horecker. Cytochrome c was obtained from the Sigma Chemical Company.

Transhydrogenase preparations with an activity of 250 to 7000 units per mg. were
used in these studies. The DPNase was a purified enzyme, which was obtained
from zinc-deficient Neurospora and had an activity of 5500 units per mg. (4). The
alcohol dehydrogenase was a crystalline preparation isolated from yeast according to the procedure of Racker (5).

Phosphogluconate dehydrogenase from yeast and a 10 per cent pure preparation of the TPN-specific cytochrome c reductase from liver (6) were gifts of Dr. B. F.
Horecker.

DPN was assayed with alcohol and crystalline yeast alcohol dehydrogenase. TPN was determined By the specific phosphogluconic acid dehydrogenase from yeast and also by the specific isocitric dehydrogenase from pig heart. Reduced DPN was
determined by the use of acetaldehyde and the yeast alcohol dehydrogenase.
All of the above assays were based on the measurement of optical density changes
at 340 rnp. TPNHz was determined with the TPN-specific cytochrome c reductase system. The assay of the reaction followed increase in optical density at 550 rnp  as a measure of the reduction of the cytochrome c after cytochrome c
reductase was added to initiate the reaction. The changes at 550 rnp are plotted for different concentrations of TPNHz in Fig. 3, a. The method is an extremely sensitive and accurate assay for reduced TPN.

Results
[No Figures or Table shown]

Formation of DPNHz from TPNHz and DPN-Fig. 1, a illustrates the direct reaction between TPNHz and DPN to form DPNHZ. The reaction was carried out by incubating TPNHz with DPN in the presence of the
transhydrogenase, yeast alcohol dehydrogenase, and acetaldehyde. Since the yeast dehydrogenase is specific for DPN,

  • a decrease in absorption at340 rnp can only be due to the formation of reduced DPN. It can
    be seen from the curves in Fig. 1, a that a decrease in optical density occurs only in the
    presence of the complete system.

The Pseudomonas enzyme is essential for the formation of DPNH2. It is noteworthy
that, under the conditions of reaction in Fig. 1, a,

  • approximately 40 per cent of theTPNH, reacted with the DPN.

Fig. 1, a also indicates that magnesium is not required for transhydrogenase activity.  The reaction between TPNHz and DPN takes place in the absence of alcohol
dehydrogenase and acetaldehyde
. This can be demonstrated by incubating the
two pyridine nucleotides with the transhydrogenase for 4 8 12 16 20 24 28 32 36
minutes

FIG. 1. Evidence for enzymatic reaction of TPNHt with DPN.

  • Rate offormation of DPNH2.

(b) DPN disappearance and TPN formation.

(c) Identification of desamino DPNHz as product of reaction of TPNHz with desamino DPN.  (assaying for reduced DPN by the yeast alcohol dehydrogenase technique.

Table I (Experiment 1) summarizes the results of such experiments in which TPNHz was added with varying amounts of DPN.

  • In the absence of DPN, no DPNHz was formed. This eliminates the possibility that TPNH 2 is
    converted to DPNHz
  • by removal ofthe monoester phosphate grouping.

The data also show that the extent of the reaction is

  • dependent on the concentration of DPN.

Even with a large excess of DPN, only approximately 40 per cent of the TPNHzreacts to form reduced DPN. It is of importance to emphasize that in the above
experiments, which were carried out in phosphate buffer, the extent of  the reaction

  • is the same in the presence or absence of acetaldehyde andalcohol dehydrogenase.

With an excess of DPN and different  levels of TPNHZ,

  • the amount of reduced DPN which is formed is
  • dependent on the concentration of TPNHz(Table I, Experiment 2).
  • In all cases, the amount of DPNHz formed is approximately
    40 per cent of the added reduced TPN.

Formation of TPN-The reaction between TPNHz and DPN should yield TPN as well as DPNHz.
The formation of TPN is demonstrated in Table 1. in Fig. 1, b. In this experiment,
TPNHz was allowed to react with DPN in the presence of the transhydrogenase
(PS.), and then alcohol and alcohol dehydrogenase were added . This
would result in reduction of the residual DPN, and the sample incubated with the
transhydrogenase contained less DPN. After the completion of the alcohol
dehydrogenase reaction, phosphogluconate and phosphogluconic dehydrogenase (PGAD) were added to reduce the TPN. The addition of this TPN-specific
dehydrogenase results in an

  • increase inoptical density in the enzymatically treated sample.
  • This change represents the amount of TPN formed.

It is of interest to point out that, after addition of both dehydrogenases,

  • the total optical density change is the same in both

Therefore it is evident that

  • for every mole of DPN disappearing  a mole of TPN appears.

Balance of All Components of Reaction

Table II (Experiment 1) shows that,

  • if measurements for all components of the reaction are made, one can demonstrate
    that there is
  • a mole for mole disappearance of TPNH, and DPN, and
  • a stoichiometric appearance of TPN and DPNH2.
  1. The oxidized forms of the nucleotides were assayed as described
  2. the reduced form of TPN was determined by the TPNHz-specific cytochrome c reductase,
  3. the DPNHz by means of yeast alcohol dehydrogenase plus

This stoichiometric balance is true, however,

  • only when the analyses for the oxidized forms are determined directly on the reaction

When analyses are made after acidification of the incubated reaction mixture,

  • the values found forDPN and TPN are much lower than those obtained by direct analysis.

This discrepancy in the balance when analyses for the oxidized nucleotides are
carried out in acid is indicated in Table II (Experiment 2). The results, when
compared with the findings in Experiment 1, are quite striking.

Reaction of TPNHz with Desamino DPN

Desamino DPN

  • reacts with the transhydrogenase system at the same rate as does DPN (2).

This was of value in establishing the fact that

  • the transhydrogenase catalyzesa transfer of hydrogen rather than a phosphate transfer reaction.

The reaction between desamino DPN and TPNHz can be written in two ways.

TPN f desamino DPNHz

TPNH, + desamino DPN

DPNH2 + desamino TPN

If the reaction involved an electron transfer,

  • desamino DPNHz would be
  • Phosphate transfer would result in the production of reduced

Desamino DPNHz can be distinguished from DPNHz by its

  • slowerrate of reaction with yeast alcohol dehydrogenase (2, 3).

Fig. 1, c illustrates that, when desamino DPN reacts with TPNH2, 

  • the product of the reaction is desamino DPNHZ.

This is indicated by the slow rate of oxidation of the product by yeast alcohol
dehydrogenase and acetaldehyde.

From the above evidence phosphate transfer 

  • has been ruled out as a possible mechanism for the transhydrogenase reaction.

Inhibition by TPN

As mentioned in Paper I and as will be discussed later in this paper,

  • the transhydrogenase reaction does not appear to be readily reversible.

This is surprising, particularly since only approximately 

  • 40 per cent of the TPNHz undergoes reaction with DPN
    under the conditions described above. It was therefore thought that
  • the TPN formed might inhibit further transfer of electrons from TPNH2.

Table III summarizes data showing the

  • strong inhibitory effect of TPN on thereaction between TPNHz and DPN.

It is evident from the data that

  • TPN concentration is a factor in determining the extent of the reaction.

Effect of Removal of TPN on Extent of Reaction

A purified DPNase from Neurospora has been found

  • to cleave the nicotinamide riboside linkagesof the oxidized forms of both TPN and DPN
  • without acting on thereduced forms of both nucleotides (4).

It has been found, however, that

  • the DPNase hydrolyzes desamino DPN at a very slow rate (3).

In the reaction between TPNHz and desamino DPN, TPN and desamino DPNH:,

  • TPNis the only component of this reaction attacked by the Neurospora enzyme
    at an appreciable rate

It was  thought that addition of the DPNase to the TPNHZ-desamino DPN trans-
hydrogenase reaction mixture

  • would split the TPN formed andpermit the reaction to go to completion.

This, indeed, proved to be the case, as indicated in Table IV, where addition of
the DPNase with desamino DPN results in almost

  • a stoichiometric formation of desamino DPNHz
  • and a complete disappearance of TPNH2.

Extent of Reaction in Buffers Other Than Phosphate

All the reactions described above were carried out in phosphate buffer of pH 7.5.
If the transhydrogenase reaction between TPNHz and DPN is run at the same pH
in tris(hydroxymethyl)aminomethane buffer (TRIS buffer)

  • with acetaldehydeand alcohol dehydrogenase present,
  • the reaction proceeds muchfurther toward completion 
  • than is the case under the same conditions ina phosphate medium (Fig. 2, a).

The importance of phosphate concentration in governing the extent of the reaction
is illustrated in Fig. 2, b.

In the presence of TRIS the transfer reaction

  • seems to go further toward completion in the presence of acetaldehyde
    and 
    alcohol dehydrogenase
  • than when these two components are absent.

This is not true of the reaction in phosphate,

  • in which the extent is independent of the alcoholdehydrogenase system.

Removal of one of the products of the reaction (DPNHp) in TRIS thus

  • appears to permit the reaction to approach completion,whereas
  • in phosphate this removal is without effect on the finalcourse of the reaction.

The extent of the reaction in TRIS in the absence of alcohol dehydrogenase
and acetaldehyde
 is

  • somewhat greater than when the reaction is run in phosphate.

TPN also inhibits the reaction of TPNHz with DPN in TRIS medium, but the inhibition

  • is not as marked as when the reaction is carried out in phosphate buffer.

Reversibility of Transhydrogenase Reaction;

Reaction between DPNHz and TPN

In Paper I, it was mentioned that no reversal of the reaction could be achieved in a system containing alcohol, alcohol dehydrogenase, TPN, and catalytic amounts of
DPN.

When DPNH, and TPN are incubated with the purified transhydrogenase, there is
also

  • no evidence for reversibility.

This is indicated in Table V which shows that

  • there is no disappearance of DPNHz in such a system.

It was thought that removal of the TPNHz, which might be formed in the reaction,
could promote the reversal of the reaction. Hence,

  • by using the TPNHe-specific cytochrome c reductase, one could
  1. not only accomplishthe removal of any reduced TPN,
  2. but also follow the course of the reaction.

A system containing DPNH2, TPN, the transhydrogenase, the cytochrome c
reductase, and cytochrome c, however, gives

  • no reduction of the cytochrome

This is true for either TRIS or phosphate buffers.2

Some positive evidence for the reversibility has been obtained by using a system
containing

  • DPNH2, TPNH2, cytochrome c, and the cytochrome creductase in TRIS buffer.

In this case, there is, of course, reduction of cytochrome c by TPNHZ, but,

  • when the transhydrogenase is present.,there is
  • additional reduction over and above that due to the added TPNH2.

This additional reduction suggests that some reversibility of the reaction occurred
under these conditions. Fig. 3, b shows

  • the necessity of DPNHzfor this additional reduction.

Interaction of DPNHz with Desamino DPN-

If desamino DPN and DPNHz are incubated with the purified Pseudomonas enzyme,
there appears

  • to be a transfer of electrons to form desamino DPNHz.

This is illustrated in Fig. 4, a, which shows the

  • decreased rate of oxidation by thealcohol dehydrogenase system
  • after incubation with the transhydrogenase.
  • Incubation of desamino DPNHz with DPN results in the formation of DPNH2,
  • which is detected by the faster rate of oxidation by the alcohol dehydrogenase system
  • after reaction of the pyridine nucleotides with thetranshydrogenase (Fig. 4, b).

It is evident from the above experiments that

the transhydrogenase catalyzes an exchange of hydrogens between

  • the adenylic and inosinic pyridine nucleotides.

However, it is difficult to obtain any quantitative information on the rate or extent of
the reaction by the method used, because

  • desamino DPNHz also reacts with the alcohol dehydrogenase system,
  • although at a much slower rate than does DPNH2.

DISCUSSION

The results of the balance experiments seem to offer convincing evidence that
the transhydrogenase catalyzes the following reaction.

TPNHz + DPN -+ DPNHz + TPN

Since desamino DPNHz is formed from TPNHz and desamino DPN,

  • thereaction appears to involve an electron (or hydrogen) transfer
  • rather thana transfer of the monoester phosphate grouping of TPN.

A number of the findings reported in this paper are not readily understandable in
terms of the above simple formulation of the reaction. It is difficult to understand
the greater extent of the reaction in TRIS than in phosphate when acetaldehyde
and alcohol dehydrogenase are present.

One possibility is that an intermediate may be involved which is more easily converted
to reduced DPN in the TRIS medium. The existence of such an intermediate is also
suggested by the discrepancies noted in balance experiments, in which

  • analyses of the oxidized nucleotides after acidification showed
  • much lower values than those found by direct analysis.

These findings suggest that the reaction may involve

  • a 1 electron ratherthan a 2 electron transfer with
  • the formation of acid-labile free radicals as intermediates.

The transfer of hydrogens from DPNHz to desamino DPN

  • to yield desamino DPNHz and DPN and the reversal of this transfer
  • indicate the unique role of the transhydrogenase
  • in promoting electron exchange between the pyridine nucleotides.

In this connection, it is of interest that alcohol dehydrogenase and lactic
dehydrogenase cannot duplicate this exchange  between the DPN and
the desamino systems.3  If one assumes that desamino DPN behaves
like DPN,

  • one might predict that the transhydrogenase would catalyze an
    exchange of electrons (or hydrogen) 3.

Since alcohol dehydrogenase alone

  • does not catalyze an exchange of electrons between the adenylic
    and inosinic pyridine nucleotides, this rules out the possibility
  • that the dehydrogenase is converted to a reduced intermediate
  • during electron between DPNHz and added DPN.

It is hoped to investigate this possibility with isotopically labeled DPN.
Experiments to test the interaction between TPN and desamino TPN are
also now in progress.

It seems likely that the transhydrogenase will prove capable of

  • catalyzingan exchange between TPN and TPNH2, as well as between DPN and

The observed inhibition by TPN of the reaction between TPNHz and DPN may
therefore

  • be due to a competition between DPN and TPNfor the TPNH2.

SUMMARY

  1. Direct evidence for the following transhydrogenase reaction. catalyzedby an
    enzyme from Pseudomonas fluorescens, is presented.

TPNHz + DPN -+ TPN + DPNHz

Balance experiments have shown that for every mole of TPNHz disappearing
1 mole of TPN appears and that for each mole of DPNHz generated 1 mole of
DPN disappears. The oxidized nucleotides found at the end of the reaction,
however, show anomalous lability toward acid.

  1. The transhydrogenase also promotes the following reaction.

TPNHz + desamino DPN -+ TPN + desamino DPNH,

This rules out the possibility that the transhydrogenase reaction involves a
phosphate transfer and indicates that the

  • enzyme catalyzes a shift of electrons (or hydrogen atoms).

The reaction of TPNHz with DPN in 0.1 M phosphate buffer is strongly
inhibited by TPN; thus

  • it proceeds only to the extent of about40 per cent or less, even
  • when DPNHz is removed continuously by meansof acetaldehyde
    and alcohol dehydrogenase.
  • In other buffers, in whichTPN is less inhibitory, the reaction proceeds
    much further toward completion under these conditions.
  • The reaction in phosphate buffer proceedsto completion when TPN
    is removed as it is formed.
  1. DPNHz does not react with TPN to form TPNHz and DPN in the presence
    of transhydrogenase. Some evidence, however, has been obtained for
    the reversibility by using the following system:
  • DPNHZ, TPNHZ, cytochromec, the TPNHz-specific cytochrome c reductase,
    and the transhydrogenase.
  1. Evidence is cited for the following reversible reaction, which is catalyzed
    by the transhydrogenase.

DPNHz + desamino DPN fi DPN + desamino DPNHz

  1. The results are discussed with respect to the possibility that the
    transhydrogenase reaction may
  • involve a 1 electron transfer with theformation of free radicals as intermediates.

 

BIBLIOGRAPHY

  1. Coiowick, S. P., Kaplan, N. O., Neufeld, E. F., and Ciotti, M. M., J. Biol. Chem.,196, 95 (1952).
  2. Pullman, 111. E., Colowick, S. P., and Kaplan, N. O., J. Biol. Chem., 194, 593(1952).
  3. Kaplan, N. O., Colowick, S. P., and Ciotti, M. M., J. Biol. Chem., 194, 579 (1952).
  4. Kaplan, N. O., Colowick, S. P., and Nason, A., J. Biol. Chem., 191, 473 (1951).
  5. Racker, E., J. Biol. Chem., 184, 313 (1950).
  6. Horecker, B. F., J. Biol. Chem., 183, 593 (1950).

Section !II. 

Luis_Federico_Leloir_-_young

The Leloir pathway: a mechanistic imperative for three enzymes to change
the stereochemical configuration of a single carbon in galactose.

Frey PA.
FASEB J. 1996 Mar;10(4):461-70.    http://www.fasebj.org/content/10/4/461.full.pdf
PMID:8647345

The biological interconversion of galactose and glucose takes place only by way of
the Leloir pathway and requires the three enzymes galactokinase, galactose-1-P
uridylyltransferase, and UDP-galactose 4-epimerase.
The only biological importance of these enzymes appears to be to

  • provide for the interconversion of galactosyl and glucosyl groups.

Galactose mutarotase also participates by producing the galactokinase substrate
alpha-D-galactose from its beta-anomer. The galacto/gluco configurational change takes place at the level of the nucleotide sugar by an oxidation/reduction
mechanism in the active site of the epimerase NAD+ complex. The nucleotide portion
of UDP-galactose and UDP-glucose participates in the epimerization process in two ways:

1) by serving as a binding anchor that allows epimerization to take place at glycosyl-C-4 through weak binding of the sugar, and

2) by inducing a conformational change in the epimerase that destabilizes NAD+ and
increases its reactivity toward substrates.

Reversible hydride transfer is thereby facilitated between NAD+ and carbon-4
of the weakly bound sugars.

The structure of the enzyme reveals many details of the binding of NAD+ and
inhibitors at the active site
.

The essential roles of the kinase and transferase are to attach the UDP group
to galactose, allowing for its participation in catalysis by the epimerase. The
transferase is a Zn/Fe metalloprotein
, in which the metal ions stabilize the
structure rather than participating in catalysis. The structure is interesting
in that

  • it consists of single beta-sheet with 13 antiparallel strands and 1 parallel strand
    connected by 6 helices.

The mechanism of UMP attachment at the active site of the transferase is a double
displacement
, with the participation of a covalent UMP-His 166-enzyme intermediate
in the Escherichia coli enzyme. The evolution of this mechanism appears to have
been guided by the principle of economy in the evolution of binding sites.

PMID: 8647345 Free full text

Section IV.

More on Lipids – Role of lipids – classification

  • Energy
  • Energy Storage
  • Hormones
  • Vitamins
  • Digestion
  • Insulation
  • Membrane structure: Hydrophobic properties

Lipid types

lipid types

lipid types

nat occuring FAs in mammals

nat occuring FAs in mammals

Read Full Post »


Carbohydrate Metabolism

Author and Curator: Larry H. Bernstein, MD, FCAP

This is the portion of the discussion in a series of articles that began with signaling and signaling pathways. There are features on the functioning of enzymes and proteins, on sequential changes in a chain reaction, and on conformational changes that we shall return to.  These are critical to developing a more complete understanding of life processes.  I have indicated that many of the protein-protein interactions or protein-membrane interactions and associated regulatory features have been referred to previously, but the focus of the discussion or points made were different.  Even though I considered placing this after the discussion of proteins and how they play out their essential role, I needed to lay out the scope of metabolic reactions and pathways, and their complementary changes. These may not appear to be adaptive, if the circumstances and the duration is not clear. The metabolic pathways map in total is in interaction with environmental conditions – light, heat, external nutrients and minerals, and toxins – all of which give direction and strength to these reactions. I shall again take from Wikipedia, as needed, and also follow mechanisms and examples from the literature, which give insight into the developments in cell metabolism. A developing goal is to discover how views introduced by molecular biology and genomics don’t clarify functional cellular dynamics that are not related to the classical view.  The work is vast.

  1. Signaling and signaling pathways
  2. Signaling transduction tutorial.
  3. Carbohydrate metabolism
  4. Lipid metabolism
  5. Protein synthesis and degradation
  6. Subcellular structure
  7. Impairments in pathological states: endocrine disorders; stress hypermetabolism; cancer.

Carbohydrate metabolism

Carbohydrate metabolism denotes the various biochemical processes responsible for the formation, breakdown and interconversion of carbohydrates in living organisms.

The most important carbohydrate is glucose, a simple sugar (monosaccharide) that is metabolized by nearly all known organisms. Glucose and other carbohydrates are part of a wide variety of metabolic pathways across species: plants synthesize carbohydrates from carbon dioxide and water by photosynthesis storing the absorbed energy internally, often in the form of starch or lipids. Plant components are consumed by animals and fungi, and used as fuel for cellular respiration. Oxidation of one gram of carbohydrate yields approximately 4 kcal of energy and from lipids about 9 kcal. Energy obtained from metabolism (e.g. oxidation of glucose) is usually stored temporarily within cells in the form of ATP.[1] Organisms capable of aerobic respiration metabolize glucose and oxygen to release energy with carbon dioxide and water as byproducts.

Complex carbohydrates contain three or more sugar units linked in a chain, with most containing hundreds to thousands of sugar units. They are digested by enzymes to release the simple sugars.
I shall not go into the digestion, breakdown and absorption of these sugar molecules. Carbohydrates are used for short-term fuel, and the most important is glucose.  Even though they are simpler to metabolize than fats or those amino acids (components of proteins) that can be used for fuel, they do not produce as effect an energy yield measured by ATP.  In animals, The concentration of glucose in the blood is linked to the pancreatic endocrine hormone, insulin.

Carbohydrates are typically stored as long polymers of glucose molecules with glycosidic bonds for structural support (e.g. chitin, cellulose) or for energy storage (e.g. glycogen, starch). However, the strong affinity of most carbohydrates for water makes storage of large quantities of carbohydrates inefficient due to the large molecular weight of the solvated water-carbohydrate complex. In most organisms, excess carbohydrates are regularly catabolised to form acetyl-CoA, which is a feed stock for the fatty acid synthesis pathway; fatty acids, triglycerides, and other lipids are commonly used for long-term energy storage. The hydrophobic character of lipids makes them a much more compact form of energy storage than hydrophilic carbohydrates. However, animals, including humans, lack the necessary enzymatic machinery and so do not synthesize glucose from lipids, though glycerol can be converted to glucose.[6]

Metabolic pathways in eukaryotes

  • Carbon fixation, or photosynthesis, in which CO2 is reduced to carbohydrate.  [omitted]
  • Glycolysis – the metabolism of glucose molecules to obtain ATP and pyruvate[7] by way of first splitting a six-carbon into two three csrbon chains, which are converted to lactic acid from pyruvate in the lactic dehydrogenase reaction. The reverse conversion is by a separate unidirectional reaction back to pyruvate after moving through pyruvate dehydrogenase complex.[8]
  • Krebs, tricarboxylic acic, or citric acid cycle
    • Typically, a breakdown of one molecule of glucose by aerobic respiration (i.e. involving both glycolysis and Kreb’s cycle) is about 33-35 ATP.[1] This is categorized as:
  • Glycogenolysis – the breakdown of glycogen into glucose, which provides a glucose supply for glucose-dependent tissues.
    • Glycogenolysis in liver provides circulating glucose short term.
    • Glycogenolysis in muscle is obligatory for muscle contraction.
    •     Anaerobic breakdown by glycolysis – yielding 8-10 ATP
    •     Aerobic respiration by kreb’s cycle – yielding 25 ATP
  • The pentose phosphate pathway (shunt) converts hexoses into pentoses and regenerates NADPH.[9] NADPH is an essential antioxidant in cells which prevents oxidative damage and acts as precursor for production of many biomolecules.
  • Glycogenesis – the conversion of excess glucose into glycogen as a cellular storage mechanism; achieving low osmotic pressure.
  • Gluconeogenesisde novo synthesis of glucose molecules from simple organic compounds. An example in humans is the conversion of a few amino acids in cellular protein to glucose.
    Metabolic use of glucose is highly important as an energy source for muscle cells and in the brain, and red blood cells.

Glucoregulation

The hormone insulin is the primary glucose regulatory signal in animals. It mainly promotes glucose uptake by the cells,  and causes liver to store excess glucose as glycogen. Its absence turns off glucose uptake, reverses electrolyte adjustments, begins glycogen breakdown and glucose release into the circulation by some cells, begins lipid release from lipid storage cells, etc. The level of circulatory glucose (known informally as “blood sugar”) is the most important signal to the insulin-producing cells. Because the level of circulatory glucose is largely determined by the intake of dietary carbohydrates, diet controls major aspects of metabolism via insulin. In humans, insulin is made by beta cells in the pancreas, fat is stored in adipose tissue cells, and glycogen is both stored and released as needed by liver cells. Regardless of insulin levels, no glucose is released to the blood from internal glycogen stores from muscle cells.

The hormone glucagon, on the other hand, opposes that of insulin, forcing the conversion of glycogen in liver cells to glucose, and then release into the blood. Muscle cells, however, lack the ability to export glucose into the blood. The release of glucagon is precipitated by low levels of blood glucose. Other hormones, notably growth hormone, cortisol, and certain catecholamines (such as epinepherine) have glucoregulatory actions similar to glucagon.  These hormones are referred to as stress hormones because they are released under the influence of catabolic proinflammatory (stress) cytokines – interleukin-1 (IL1) and tumor necrosis factor α (TNFα).

metabolic pathways

metabolic pathways

Glycemic control in DM

Glycemic control in DM

  1. Catabolic proinflammatory cytokines. Argilés JM1López-Soriano FJ. Curr Opin Clin Nutr Metab Care.1998 May;1(3):245-51.
  2. Tumor necrosis factor as a mediator of shock, cachexia and inflammation. Cerami A. Blood Purif. 1993; 11(2):108-17.
  3. Mediators of cytokine-induced insulin resistance in obesity and other inflammatory settings. Marette A. Curr Opin Clin Nutr Metab Care. 2002 Jul; 5(4):377-83.
  4. Inflammation: the link between insulin resistance, obesity and diabetes. Dandona P, Aljada A, Bandyopadhyay A. Trends Immunol. 2004 Jan; 25(1):4-7
  5. Proinflammatory cytokines and skeletal muscle. Späte U1, Schulze PC. Curr Opin Clin Nutr Metab Care. 2004 May;7(3):265-9.
  6. Insulin-like growth factor-1 and muscle wasting in chronic heart failure. Schulze PC, Späte U. Int J Biochem Cell Biol. 2005 Oct; 37(10):2023-35.
  7. IGF-I stimulates muscle growth by suppressing protein breakdown and expression of atrophy-related ubiquitin ligases, atrogin-1 and MuRF1. Sacheck JM, Ohtsuka A, McLary SC, Goldberg AL. Am J Physiol Endocrinol Metab. 2004 Oct; 287(4):E591-601. Epub 2004 Apr 20.

Glycolysis – Animation and Notes

By Sweety Mehta – Sept 20, 2011  in: Animations, Biochemistry Animations, Biochemistry Notes

http://pharmaxchange.info/press/2011/09/glycolysis-animation-and-notes/

Cellular respiration involves breaking the bonds of glucose to produce energy in the form of ATP (adenosine triphosphate). The total energy produced during glucose metabolism is described at Energetics of Cellular Respiration. Glycolysis is the most critical phase in glucose metabolism during cellular respiration. The term “glycolysis” literally means breakdown of glucose and sugars. Biochemically, it involves the breakdown of glucose to pyruvate (or pyruvic acid) via a series of enzymes. Glycolysis does not require molecular oxygen and is hence considered anaerobic. Therefore, it is a common pathway for all living organisms.

Glycolysis is followed by

Kreb’s cycle in the stages of cellular respiration.

Glycolysis is said to occur in two phases:

  1. The Preparatory Phase: From glucose till formation of Glyceraldehyde 3-Phosphate (GADP)
  2. The Pay-off Phase: From Glyceraldehyde-3-Phosphate (GADP) to the final product pyruvate

.

.

glycolysis

The animation below gives an outline of the entire pathway of glucose metabolism by glycolysis

Note – The animation is best played in full screen. To go forward in the animation, press the Play button. To skip the whole section press the forward button. To go back press the rewind button.

The Preparatory Phase

In this stage of the cycle, ATP or energy is actually consumed and is hence also known as the investment phase of glycolysis.

Step 1, involves the conversion of glucose to glucose-6-phosphate (G6P) with the help of the enzyme hexokinase and the consumption of 1 molecule of ATP. This reaction helps keep the concentration of glucose low in the cell, allowing for more absorption of glucose into it. Additionally, G6P is not transported out of the cell as there are no G6P transporters on the cell.

Step 2 involves the rearrangement of glucose-6-phosphate to fructose-6-phosphate (F6P) with the help of the enzyme phosphohexose isomerase in a reversible manner. Fructose can directly enter the glycolysis pathway at this point. This isomerization to a keto-sugar such as fructose is essential for carbanion stabilization required for the next step.

Step 3 involves the phosphorylation of fructose-6-phosphate to fructose-1,6-biphosphate (F1,6BP) by the use of 1 molecule of ATP and the enzyme phosphofructokinase-1 (PPK1). This phosphorylation step destabilizes the molecule and helps drive the next reaction which ensures breakdown of the molecule to a 3-carbon unit.

Step 4 involves the breakdown of fructose-1,6-biphosphate (6 carbons) to two molecules of 3-carbon units i.e. glyceralde 3-phosphate (GADP) and Dihydroxyacetone phosphate (DHAP). The GADP can be interconverted to DHAP by enzyme triose phosphate isomerase.

The Pay-Off Phase

In this stage of the cycle, ATP or energy is produced either in the form of ATP alone or in the form of NADH + H+ which can be later converted to ATP via the electron transport chain (ETS). In this since energy is restored it is known as the pay-off phase of glycolysis. All steps in this phase occur with 2 molecules of the substrates each as indicated in the brackets by the name of the molecules.

Step 1, involves the dehydrogenation of glyceraldehyde-3-phosphate (GADP) to 1,3-biphophoglycerate (1,3BPG) by the use of 2 molecules of inorganic phosphate (Pi) with the production of 2 molecules of NADH + H+ in the presence of the enzyme glyceraldehyde 3-phosphate dehydrogenase.

Step 2, in this step dephosphorylation of 1,3-biphosphoglycerate (1,3BPG) to 3-phospoglycerate (3PG) produces 2 molecules of ATP by the enzyme phosphoglycerate kinase.

Step 3, involves the isomerisation of 3-phosphoglycerate (3PG) to 2-phosphoglycerate (2PG) by the enzyme phosphoglycerate mutase in a reversible manner.

Step 4 involves the enolization of 2-phosphoglycerate (2PG) to phosphoenolpyruvate (PEP) with the loss of one molecule of water in the presence of enzyme enolase.

Step 5 is the final step of the glycolysis pathway and it involves the dephosphorylation of the phosphoenolpyruvate (PEP) to pyruvate by enzyme pyruvate kinase to produce 2 more molecules of ATP.

Net Yield of Glycolysis

  1. The preparatory phase consumes 2 ATP
  2. The pay-off phase produces 4 ATP.
  3. The gross yield of glycolysis is therefore
    4 ATP – 2 ATP = 2 ATP
  4. The pay-off phase also produces 2 molecules of NADH + H+ which can be further converted to a total of 5 molecules of ATP* by the electron transport chain (ETC) during oxidative phosphorylation.
  5. Thus the net yield during glycolysis is 7 molecules of ATP.

* This is calculated assuming one NADH molecule gives 2.5 molecules of ATP during oxidative phosphorylation.

References

  1. David L. Nelson and Michael M. Cox, Lehninger Principles of Biochemistry, 4th Ed.
  2. Jeremy M. Berg, John L. Tymockzo and Luber Stryer, Biochemistry, 7th Ed.

Tags: cellular respiration, electron transport chain, etc, glucose, glycolysis, metabolism, pay-off phase

Kreb’s Cycle or Citric Acid Cycle or Tricarboxylic Acid Cycle (with Animation)

By Sweety Mehta  – Sept 21, 2013  in: Animations, Biochemistry Animations, Biochemistry Notes
http://pharmaxchange.info/press/2013/09/krebs-cycle-citric-acid-cycle-tricarboxylic-acid-cycle-animation/

Introduction

Cellular respiration involves 3 stages for the breakdown of glucose – glycolysis, Kreb’s cycle and the electron transport system. The total energy produced during glucose metabolism is described at Energetics of Cellular Respiration. We have seen the glycolysis pathway with animation previously. The Kreb’s cycle is named after Adolf Krebs who studied the utilization of oxygen in a pigeon. It is also commonly known as the citric acid cycle or the tricarboxylic acid cycle. Kreb’s cycle is a very important step in the metabolic pathway as it produces about 60-70% of ATP for release of energy in the body. It directly or indirectly connects with all the other individual pathways in the body too. It takes place in the mitochondria as all the enzymes and co-enzymes required are present there.

The Kreb’s Cycle occurs in two stages:

1. Conversion of Pyruvate to Acetyl CoA

Glycolysis of 1 molecule of glucose produces 2 molecules of pyruvate. Each pyruvate in the presence of pyruvate dehydrogenase (PDH) complex in the mitochondria gets converted to acetyl CoA which in turn enters the Kreb’s cycle. This reaction is called as oxidative  decarboxylation as the carboxyl group is removed from the pyruvate molecule in the form of CO2 thus yielding 2-carbon acetyl group which along with the coenzyme A forms acetyl CoA.

The pyruvate dehydrogenase complex (PDH) comprises of three enzymes – pyruvate dehydrogenase, dihydrolipoyl transacetylase and dihydrolipoyl dehydrogenase each one playing an important role in the reaction as shown below. The PDH requires the sequential action of five co-factors or co-enzymes for the combined action of dehydrogenation and decarboxylation to take place. These five are TPP (thiamine phosphate), FAD (flavin adenine dinucleotide), NAD (nicotinamide adenine dinucleotide), coenzyme A (denoted as CoA-SH at times to depict role of -SH group) and lipoamide.

Conversion of pyruvate to acetyl CoA by the pyruvate dehydrogenase complex

pyruvate_dehydrogenase_complex_new2

Conversion of pyruvate to acetyl CoA by the pyruvate dehydrogenase complex

Pyruvate reacts with the TPP (Thiamine Phosphate) bound part of pyruvate dehydrogenase and undergoes decarboxylation to give hydroxyethyl-TPP.

This hydroxyethyl-TPP in turn gets oxidised to acetyl lipoamide by the same enzyme pyruvate dehydrogenase by the transfer of two electrons. These electrons then reduce the disulfide bond of the enzyme dihydrolipoyl transacetylase with the transfer of the acetyl group as highlighted in purple.

Dihydrolipoyl transacetylase catalyses the transesterification forming acetyl CoA by transfer of acetyl group to coenzyme A.

When acetyl CoA is being formed, at the same time reduced lipoamide is getting converted to oxidised lipoamide due to enzyme dihydrolipoyl dehydrogenase by the transfer of 2 hydrogen atoms to FAD.

Dihydrolipoyl dehydrogenase transfers the reduced equivalents (2 hydrogen atoms) to FAD thus forming FADH2. FADH2 in turn transfers a hydride ion to NAD+ to form NADH+H+.

2. Acetyl CoA Enters the Kreb’s Cycle

The acetyl CoA produced from the pyruvate dehydrogenase complex enters the Kreb’s cycle.

The animation below describes the Kreb’s cycle in detail followed by the discussion. A static image of the cycle can be found next to the discussion for reference. Press the play button to progress in the animation.

Discussion

Krebs1 cycle

The Kreb’s Cycle or Citric Acid Cycle or Tricarboxylic Acid Cycle in a static image version of the animation.

Acetyl CoA condenses with oxaloacetate (4C) to form a citrate (6C) by transferring its acetyl group in the presence of enzyme citrate synthase. The CoA liberated in this reaction is ready to participate in the oxidative decarboxylation of another molecule of pyruvate by PDH complex.

  • Citrate is then isomerised to Isocitrate by the enzyme aconitase through the formation of the intermediate cis-aconitate. This is a reversible reaction as aconitase has an iron-sulfur center which can promote reversible addition of H2O to the double bond of enzyme-bound cis-aconitate in 2 different ways, one forming citrate and the other isocitrate.
  • Isocitrate undergoes oxidative decarboxylation by the enzyme isocitrate dehydrogenase to form oxalosuccinate (intermediate- not shown) which in turn forms α-ketoglutarate (also known as oxoglutarate) which is a five carbon compound. CO2 and NADH are released in this step.
  • α-ketoglutarate (5C) undergoes oxidative decarboxylation once again to form succinyl CoA (4C) catalysed by the enzyme α-ketoglutarate dehydrogenase complex. α-ketoglutarate dehydrogenase complex is similar to PDH complex and is made up of 3 enzymes and is dependent on five co-enzymes TPP, FAD, NAD, bound lipoate and conenzyme A. In this step once again NADH and CO2 are liberated. So in all 2 molecules of NADH and 2 molecules of CO2 is produced till now.
  • Succinyl CoA is then converted to succinate by succinate thiokinase or succinyl coA synthetase in a reversible manner. This reaction involves an intermediate step in which the enzyme gets phosphorylated and then the phosphoryl group which has a high group transfer potential is transferred to GDP to form GTP. This GTP is converted to ATP by the enzyme nucleoside diphosphate kinase by donating its phosphoryl group to ADP. This reaction which involves the formation of GTP is a substrate level phosphorylation as it happens by using the energy formed by the oxidative decarboxylation of α-ketoglutarate.
  • Succinate then gets oxidised reversibly to fumarate by succinate dehydrogenase. The enzyme contains iron-sulfur clusters and covalently bound FAD which when undergoes electron exchange in the mitochondria causes the production of FADH2.
  • Fumarate is then by the enzyme fumarase converted to malate by hydration(addition of H2O) in a reversible manner.
  • Malate is then reversibly converted to oxaloacetate by malate dehydrogenase which is NAD linked and thus produces NADH.
  • The oxaloacetate produced is now ready to be utilized in the next cycle by the citrate synthase reaction and thus the equilibrium of the cycle shifts to the right.
Schrodingers_cat

Schrodingers_cat

Energetics of the Kreb’s Cycle

Keeping in mind that 1 molecule of glucose would produce 2 molecules of pyruvate via glycolysis. Hence the net energy produced by the Kreb’s cycle for each molecule of pyruvate is doubled for each molecule of glucose. Thus net energy yield in Kreb’s cycle can be summarized as follows for each molecule of glucose:

Reaction                                                              Number of ATP or                                        Number of ATP
reduced coenzyme formed                        ultimately formed

2 Pyruvate → 2 acetyl CoA                                  2 NADH                                                             5

2 Isocitrate → 2 α- ketoglutarate                     2 NADH                                                              5

2 α- ketoglutarate → 2 succinyl CoA             2 NADH                                                               5

2 Succinyl CoA → 2 succinate                           2 ATP                                                                  2

2 Succinate → 2 fumarate                               2 FADH2                                                               3

2 Malate → 2 oxaloacetate                            2 NADH                                                                  5

TOTAL                                                                                                                                                 25 ATP

* Note- This is calculated as 2.5 ATP per NADH and 1.5 ATP per FADH2. This is because there are multiple electron transport shuttle pathways through which these can be broken to ATP.

Regulation of Kreb’s Cycle

The amount of ADP and ATP largely control the citric acid cycle along with the activity of three key enzymes within the cycle:

Availability of ADP: ADP is a key substrate which finally gets converted to ATP that is essential for the energetics of the cell. A drop in ADP levels would result in inhibition of the electron transport system leading to accumulation of NADH and FADH2. These in turn inhibit the enzymes below.

Citrate Synthase: inhibited by ATP, acetyl CoA, NADH, and succinyl CoA.
Isocitrate Dehydrogenase: activated by ADP, and inhibited by NADH and ATP.
α-ketoglutarate dehydrogenase: inhibited by NADH and succinyl CoA.

Recommended Texts

David L. Nelson and Michael M. Cox, Lehninger Principles of Biochemistry 6th Edition
Jeremy M. Berg, John L. Tymockzo and Luber Stryer, Biochemistry 7th Edition

Tags: acetyl coA, animation, cellular respiration, citric acid cycle, energy, kreb’s cycle, pyruvate, pyruvate dehydrogenase, TCA cycle, tricarboxylic acid cycle

Energetics of Cellular Respiration (Glucose Metabolism)

By Sweety Mehta   – Oct 9, 2013 in: Biochemistry Notes, Notes
http://pharmaxchange.info/press/2013/10/energetics-of-cellular-respiration-glucose-metabolism/

energetics-of-cellular-respiration

electron transport chain in the mitochondrion energetics-of-cellular-respiration

Important Note: The NADH formed in the cytosol can yield variable amounts of ATP depending on the shuttle system utilized to transport them into the mitochondrial matrix. This NADH, formed in the cytosol, is impermeable to the mitochondrial inner-membrane where oxidative phosphorylation takes place. Thus to carry this NADH to the mitochondrial matrix there are special shuttle systems in the body. The most active shuttle is the malate-aspartate shuttle via which 2.5 molecules of ATP are generated for 1 NADH molecule. This shuttle is mainly used by the heart, liver and kidneys. The brain and skeletal muscles use the other shuttle known as glycerol 3-phosphate shuttle which synthesizes 1.5 molecules of ATP for 1 NADH.

Note: The above calculations are done considering that one NADH molecules produces 2.5 ATP and one FADH2 molecule produces 1.5 ATP in the ETS cycle (See full reasoning above). This is because the Kreb’s cycle occurs within the mitochondria and therefore does not require any shuttle pathway for the transport of the NADH into the mitochondrial matrix. Hence there is optimal conversion of NADH to ATP.

Development of the acetylation problem: a personal account

FRITZ LI P M A N N  Nobel Prize  1953

After my  apprenticeship with Otto Meyerhof, a first interest on my own became the phenomenon we call the Pasteur effect, this peculiar depression of the wasteful fermentation in the respiring cell. By looking for a chemical explanation of this economy measure on the cellular level, I was prompted into a study of the mechanism of pyruvic acid oxidation, since it is at the pyruvic stage where respiration branches off from fermentation. For this study I chose as a  promising system a relatively simple looking pyruvic acid oxidation enzyme in a certain strain of Lactobacillus delbrueckii1.

The most important event during this whole period, I now feel, was the accidental observation that in the L. delbrueckii system, pyruvic acid oxidation was completely dependent on the presence of inorganic phosphate. This observation was made in the course of attempts to replace oxygen by methylene blue. To measure the methylene blue reduction manometrically,
I had to switch to a bicarbonate buffer instead of the otherwise routinely used In bicarbonate, to my surprise, as shown in Fig. 1, pyruvate oxidation was very slow, but the addition of a little phosphate caused a remarkable increase in rate. The next figure, Fig. 2, shows the phosphate effect more drastically, using a preparation from which all phosphate was removed by washing with acetate buffer. Then it appeared that the reaction was really fully dependent on phosphate.

In spite of such a phosphate dependence, the phosphate balance measured by the ordinary Fiske-Subbarow procedure did not at first indicate any phosphorylative step. Nevertheless, the suspicion remained that phosphate in  some manner was entering into the reaction and that a phosphorylated intermediary was formed. As a first approximation, a coupling of this pyruvate oxidation with adenylic acid phosphorylation was attempted. And,indeed, addition of adenylic acid to the pyruvic oxidation system brought out a net  disappearance of inorganic phosphate, accounted for as adenosine triphosphate (Table 11).

I  now concluded that the missing link in the reaction chain was acetyl phosphate. In partial confirmation it was shown that a crude preparation of acetyl phosphate, synthesized by the old method of Kämmerer and Carius 2 would transfer phosphate to adenylic acid (Table 2). However, it still took quite some time from then on to identify acetyl phosphate definitely as the initial product of the pyruvic oxidation in this system3,4

At the time when these observations were made, about a dozen years ago,there was, to say the least, a tendency to believe that phosphorylation was rather specifically coupled with the glycolytic reaction. Here, however, we had found a coupling of phosphorylation with a respiratory system. This observation immediately suggested a rather sweeping biochemical significance, of transformations of electron transfer potential, respiratory or fermentative, to phosphate bond energy and therefrom to a wide range of biosynthetic reactions7.

There was a further unusual feature in this pyruvate oxidation system in that the product emerging from the process not only carried an energy-rich phosphoryl radical such as already known, but the acetyl phosphate was even more impressive through its energy-rich acetyl. It rather naturally became a contender for the role of “active” acetate, for the widespread existence of which the isotope experience had already furnished extensive evidence. I became, therefore, quite attracted by the possibility that acetyl phosphate could serve two rather different purposes, either to transfer its phosphoryl group into the phosphate pool, or to supply its active acetyl for biosynthesisof carbon structures. Thus acetyl phosphate should be able to serve as acetyldonor as well as phosphoryl donor, transferring, as shown in Fig. 3, on either side of the oxygen center, such as indicated by Bentley’s early experiments on cleavage7a of acetyl phosphate in H218O.

Phosphate dependence of pyruvate oxidation

Phosphate dependence of pyruvate oxidation

These two novel aspects of the energy problem, namely

(1) the emergence of an energy-rich phosphate bond from a purely
respiratory reaction; and

(2) the presumed derivation of a metabolic building-block through this same
reaction, prompted me to propose not only

  • the generalization of the phosphate bond as a versatie energy distributing system, but also to
    aim from there towards
  • a general concept of transfer of activated groupings by carrier as the fundamental reaction in
    biosynthesis8,9.

Although in the related manner the appearance of acetyl phosphate as a
metabolic intermediary first

  •  focused attention to possible mechanisms for the metabolic elaboration of  group activation,

it soon turned out that the relationship between acetyl phosphate and
acetyl transfer was much more complicated than anticipated.

Acetyl phosphate as acetyl and phosphoryl donor.

Although acetylation was found with rabbit liver homogenate, the
reaction was rather weak. In search of a more active system,   Pigeon
liver homogenate was tried and found to harbour an exceedingly potent
acetylation system (Ref. 11, cf. also Ref. 12). This finding of a particularly
active acetylation reaction in cell-free pigeon liver preparations was most
fortunate and played a quite important part in the development of the
acetylation problem.

[portion of lecture]

The pentose phosphate pathway is the major source for the NADPH
required for anabolic processes.
Pentose Phosphate Pathway

http://chemwiki.ucdavis.edu/Biological_Chemistry/Metabolism/Pentose_
Phosphate_Pathway

  • There are three distinct phases each of which has a distinct outcome.
  • Depending on the needs of the organism the metabolites of that outcome
    can be fed into many other pathways.
  • Gluconeogenesis is directly connected to the pentose phosphate pathway.
  • As the need for glucose-6-phosphate (the beginning metabolite in the pentose
    phosphate pathway) increases so does the activity of gluconeogenesis.

 pentose-phosphate-pathway

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Introduction

The main molecule in the body that makes anabolic processes possible is NADPH.  Because of the structure of this molecule it readily donates hydrogen ions to metabolites thus reducing them and making them available for energy harvest at a later time. The PPP is the main source of synthesis for NADPH.  The pentose phosphate pathway (PPP) is also responsible for the production of Ribose-5-phosphate which is an important part of nucleic acids. Finally the PPP can also be used to produce glyceraldehyde-3-phosphate which can then be fed into the TCA and ETC cycles allowing for the harvest of energy. Depending on the needs of the cell certain enzymes can be regulated and thus increasing or decreasing the production of desired metabolites. The enzymes reasonable for catalyzing the steps of the PPP are found most abundantly in the liver (the major site of gluconeogenesis) more specifically in the cytosol. The cytosol is where fatty acid synthesis takes place which is a NADPH dependent process.

 

Oxidation Phase

  • The beginning molecule for the PPP is glucose-6-P which is the second intermediate metabolite in glycolysis. Glucose-6-P is oxidized in the presence of glucose-6-P dehydrogenase and NADP+.  This step is irreversible and is highly regulated.  NADPH and fatty acyl-CoA are strong negative inhibitors to this enzyme.  The purpose of this is to decrease production of NADPH when concentrations are high or the synthesis of fatty acids is no longer necessary.
  • The metabolic product of this step is gluconolactone which is hydrolytrically unstable.  Gluconolactonase causes gluconolactone to undergo a ring opening hydrolysis.  The product of this reaction is the more stable sugar acid, 6-phospho-D-gluconate.
  • 6-phospho-D-gluconate is oxidized by NADP+ in the presence of 6-phosphogluconate dehydrogenase which yields ribulose-5-phosphate.
  • The oxidation phase of the PPP is solely responsible for the production of the NADPH to be used in anabolic processes.

Isomerization Phase

  •  Ribulose-5-phosphate can then be isomerized by phosphopentose isomerase to produce ribose-5-phosphate.  Ribose-5-phosphate is one of the main building blocks of nucleic acids and the PPP is the primary source of production of ribose-5-phosphate.
  • If production of ribose-5-phosphate exceeds the needs of required ribose-5-phosphate in the organism, then phosphopentose epimerase catalyzes a chiralty rearrangement about the center carbon creating xylulose-5-phosphate.
  • The products of these two reactions can then be rearranged to produce many different length carbon chains.  These different length carbon chains have a variety of metabolic fates.

Rearrangement Phase 

  •  There are two main classes of enzymes responsible for the rearrangement and synthesis of the different length carbon chain molecules.  These are transketolase and transaldolase.
  • Transketolase is responsible for the cleaving of a two carbon unit from xylulose-5-P and adding that two carbon unit to ribose-5-P thus resulting in glyceraldehyde-3-P and sedoheptulose-7-P.
  • Transketolase is also responsible for the cleaving of a two carbon unit from xylulose-5-P and adding that two carbon unit to erythrose-4-P resulting in glyceraldehyde-3-P and fructose-6-P.
  • Transaldolase is responsible for cleaving the three carbon unit from sedoheptulose-7-P and adding that three carbon unit to glyceraldehyde-3-P thus resulting in erythrose-4-P and fructose-6-P.
  • The end results of the rearrangement phase is a variety of different length sugars which can be fed into many other metabolic processes.  For example, fructose-6-P is a key intermediate of glycolysis as well as glyceraldehyde-3-P.

References

  1. Garrett, H., Reginald and Charles Grisham. Biochemistry. Boston: Twayne Publishers, 2008.
  2. Raven, Peter. Biology. Boston: Twayne Publishers, 2005.

Glycogen Metabolism

Glycogen is a readily mobilized storage form of glucose. It is a very large, branched polymer of glucose residues (Figure 21.1) that can be broken down to yield glucose molecules when energy is needed. Most of the glucose residues in glycogen are linked by α-1,4-glycosidic bonds. Branches at about every tenth residue are created by α-1,6-glycosidic bonds. Recall that α-glycosidic linkages form open helical polymers, whereas β linkages produce nearly straight strands that form structural fibrils, as in cellulose (Section 11.2.3).

http://www.ncbi.nlm.nih.gov/books/NBK21190/bin/ch21f1.jpg

Figure 21.1

Glycogen Structure ch21f1

Glycogen Structure. In this structure of two outer branches of a glycogen molecule, the residues at the nonreducing ends are shown in red and residue that starts a branch is shown in green. The rest of the glycogen molecule is represented by R.

Glycogen is not as reduced as fatty acids are and consequently not as energy rich. Why do animals store any energy as glycogen? Why not convert all excess fuel into fatty acids? Glycogen is an important fuel reserve for several reasons. The controlled breakdown of glycogen and release of glucose increase the amount of glucose that is available between meals. Hence, glycogen serves as a buffer to maintain blood-glucose levels. Glycogen’s role in maintaining blood-glucose levels is especially important because glucose is virtually the only fuel used by the brain, except during prolonged starvation. Moreover, the glucose from glycogen is readily mobilized and is therefore a good source of energy for sudden, strenuous activity. Unlike fatty acids, the released glucose can provide energy in the absence of oxygen and can thus supply energy for anaerobic activity.

Gluconeogenesis
ChemWiki: The Dynamic Chemistry E-textbook > Biological Chemistry > Metabolism > Gluconeogenesis

Gluconeogenesis is much like glycolysis only the process occurs in reverse. However, there are exceptions. In glycolysis there are three highly exergonic steps (steps 1,3,10). These are also regulatory steps which include the enzymes hexokinase, phosphofructokinase, and pyruvate kinase. Biological reactions can occur in both the forward and reverse direction. If the reaction occurs in the reverse direction the energy normally released in that reaction is now required. If gluconeogenesis were to simply occur in reverse the reaction would require too much energy to be profitable to that particular organism. In order to overcome this problem, nature has evolved three other enzymes to replace the glycolysis enzymes hexokinase, phosphofructokinase, and pyruvate kinase when going through the process of gluconeogenesis:

  1. The first step in gluconeogenesis is the conversion of pyruvate to phosphoenolpyruvic acid (PEP). In order to convert pyruvate to PEP there are several steps and several enzymes required. Pyruvate carboxylase, PEP carboxykinase and malate dehydrogenase are the three enzymes responsible for this conversion. Pyruvate carboxylase is found on the mitochondria and converts pyruvate into oxaloacetate. Because oxaloacetate cannot pass through the mitochondria membranes it must be first converted into malate by malate dehydrogenase. Malate can then cross the mitochondria membrane into the cytoplasm where it is then converted back into oxaloacetate with another malate dehydrogenase. Lastly, oxaloacetate is converted into PEP via PEP carboxykinase. The next several steps are exactly the same as glycolysis only the process is in reverse.
  2. The second step that differs from glycolysis is the conversion of fructose-1,6-bP to fructose-6-P with the use of the enzyme fructose-1,6-phosphatase. The conversion of fructose-6-P to glucose-6-P uses the same enzyme as glycolysis, phosphoglucoisomerase.
  3. The last step that differs from glycolysis is the conversion of glucose-6-P to glucose with the enzyme glucose-6-phosphatase. This enzyme is located in the endoplasmic reticulum.

Glycolysis

File:Glycolysis overview.svg

Regulation

Because it is important for organisms to conserve energy, they have derived ways to regulate those metabolic pathways that require and release the most energy. In glycolysis and gluconeogenesis seven of the ten steps occur at or near equilibrium. In gluconeogenesis the conversion of pyruvate to PEP, the conversion of fructose-1,6-bP, and the conversion of glucose-6-P to glucose all occur very spontaneously which is why these processes are highly regulated. It is important for the organism to conserve as much energy as possible. When there is an excess of energy available, gluconeogenesis is inhibited. When energy is required, gluconeogenesis is activated.

  1. The conversion of pyruvate to PEP is regulated by acetyl-CoA. More specifically pyruvate carboxylase is activated by acetyl-CoA. Because acetyl-CoA is an important metabolite in the TCA cycle which produces a lot of energy, when concentrations of acetyl-CoA are high organisms use pyruvate carboxylase to channel pyruvate away from the TCA cycle. If the organism does not need more energy, then it is best to divert those metabolites towards storage or other necessary processes.
  2. The conversion of fructose-1,6-bP to fructose-6-P with the use of fructose-1,6-phosphatase is negatively regulated and inhibited by the molecules AMP and fructose-2,6-bP. These are reciprocal regulators to glycolysis’ phosphofructokinase. Phosphofructosekinase is positively regulated by AMP and fructose-2,6-bP. Once again, when the energy levels produced are higher than needed, i.e. a large ATP to AMP ratio, the organism increases gluconeogenesis and decreases glycolysis. The opposite also applies when energy levels are lower than needed, i.e. a low ATP to AMP ratio, the organism increases glycolysis and decreases gluconeogenesis.
  3. The conversion of glucose-6-P to glucose with use of glucose-6-phosphatase is controlled by substrate level regulation. The metabolite responsible for this type of regulation is glucose-6-P. As levels of glucose-6-P increase, glucose-6-phosphatase increases activity and more glucose is produced. Thus glycolysis is unable to proceed.

 

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