Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 431	Biochemistry	Fall 2001
Lecture Notes:: 2 November	© R. Paselk 2001
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GLYCOLYSIS 4

Glycolysis Pathway

8) Phosphoglycerate Mutase: 3-PGA to 2-PGA, cont.

Note that we need another enzyme to produce the BPG cofactor: Bisphosphoglycerate mutase. This enzyme catalyses the interconversion of 1,3-bis PGA to 2,3-bis PGA, taking a high energy compound to a low energy compound: this enzyme is thus an obvious candidate for control, since if it had much activity it could drain Glycolysis of ATP production! Normally of very lo activity. (But enhanced in RBC's, since they use BPG to control the binding of oxygen by hemoglobin. RBC's also have another enzyme, 2,3-bis PGA Pase to bring BPG back into Glycolysis as 2-PGA, but without making ATP.)

9) Enolase: 2-PGA to PEP

This is an alcohol elimination reaction as you've seen in OChem, with catalysis by Magnesium and using general base catalysis by the enzyme:

Note that a low energy compound (2-PGA, approx 10 kJ) is converted to a high energy compound (PEP, greater than 60 kJ) with very little change in energy overall. Essentially have made the phosphate bond much less stable, while increasing the stability of other bonds in the molecule.

10) Pyruvate Kinase: PEP to Pyruvate

Here we have an attack by ADP:

The resulting enol then spontaneously tautomerizes to pyruvate.

PK is a regulatory enzyme in some tissues. There are three isozymes:

1. L-PK (Liver, Kidney, RBC's): greatest regulation. Sigmoidal vs. [PEP] & [K⁺]; F-1,6-bis P is a (+) effector to both. Hormonal control operates via phosphorylation to inactivate the enzyme.
2. M-PK (Muscle, brain): least regulation - product inhibition by Pyruvate and MgATP.
3. K-PK (Adipose, kidney, liver): intermediate regulation. Sigmoidal vs. [PEP] & [K⁺]; F-1,6-bis P is a (+) effector to both.

PK completes the reactions of Glycolysis. However, for Glycolysis to proceed NAD⁺ needs to be regenerated. For aerobic tissues this is done via the Kreb's TCA Cycle. Next time we will look at this process for anerobic cells.

Lactate DH is used to regenerate NAD⁺ in anaerobic tissue in mammals, and takes Pyruvate to Lactate:

 

 

Again the NAD⁺ abstracts a Hydride ion in the reverse reaction:

 

 

while a general base aids the formation of the carbonyl carbon, and a positive charge draws electron charge up to the carboxyl group and aids the removal of the hydride ion.

 

Lactate DH also has isozymes. It is a tetramer of two types of monomers, H & M. Can thus have 5 possible isomers: H₄, H₃M, H₂M₂, HM₃, & M₄, with one active site per monomer. The kinetic properties of these monomers are given in the Table:

 

Michaelis Constant (KM)   H M Pyruvate 1.4 x 10-4 5.2 x 10-4 Lactate 9 x 10-3 2.5 x 10-2 Pyruvate Inhibition? yes no

 

The properties of the H(eart) monomer, which predominates in aerobic tissues can be rationalized as better adapted to the aerobic environment. (Heart uses lactate from the serum as a fuel, but doesn't want to lose pyruvate produced in glycolysis to lactate production.)

GLYCOGEN METABOLISM

Start with G-6-P, again note that this molecule is at a metabolic crossroads. First convert to G-1-P using Phosphoglucomutase:

 

 

This reaction is very much like PGA Mutase, requiring the bis phosphorylated intermediate to form and to regenerate the phosphorylated enzyme intermediate. Again a separate "support" enzyme, Phosphoglucokinase, is required to form the intermediate, this time using ATP as the energy source:

 

 

Note that this reaction is easily reversible, though it favors G-6-P.

 

UDP-glucose pyrophosphorylase, which catalyzes the next reaction, has a near zero DG° ':

 

 

It is driven to completion by the hydrolysis of the PP_i to 2 P_i by Pyrophosphatase with a DG° ' of about -32 kJ (approx. one ATP's worth of energy).

Finally glycogen is synthesized with Glycogen Synthase:

This reaction is favored by a DG° ' of about 12 kcal, thus the overall synthesis of glycogen from G-1-P is favored by a standard free energy of about 40 kJ. Note that the glucose is added to the non-reducing end of a glycogen strand, and that there is a net investment of 2 ATP equivalents per glucose (ATP to ADP and UTP to UDP, regenerated with ATP to ADP). Note also that glycogen synthase requires a 'primer.' That is it needs to have a glycogen chain to add on to. What happens then in new cells to make now glycogen granules? Can use a special primer protein (glycogenin). Thus glycogen granules have a protein core.

 

These reactions will give linear glycogen strands, additional reactions are required to produce branching. Branching enzyme [amylo-a-(1,4) to a-(1,6)-transglycosylase] transfers a block of residues from the end of one chain to another chain making a 1,6-linkage (cannot be closer than 4 residues to a previous branch). (For efficient release of glucose residues it has been determined that the optimum branching pattern is a new branch every 13 residues, with two branchs per strand.)

 

Glycogen is broken down using Phosphorylase to phosphorylize off glucose residues:

Note that no ATP is required to recover Glucose phosphate from glycogen. This is a major advantage in anaerobic tissues, get one more ATP/glucose (3 instead of 2!). [Phosphorylase was originally thought to be the synthetic as well as breakdown enzyme since the reaction is readily reversible in vitro. However it was found that folks lacking this enzyme - McArdle's disease - can still make glycogen, though they can't break it down.]

Pathway Diagrams

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Last modified 2 November 2001