Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 438

Introductory Biochemistry

Spring 2010

Lecture Notes: 2 April

© R. Paselk 2006
 
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GLYCOLYSIS 3

Irreversible Reactions: Note that HK and PFK both catalyze biologically irreversible reactions. That is the enzymes are designed such that the concentrations of the products are far below the KM values under physiological conditions, so the reverse reactions are not catalyzed!

 

The next reaction involves an almost symmetrical cleavage of F-1,6-bisP to begin phase II:

4) Aldolase: F-1,6-bis P to Glyceraldehyde-3-Phosphate & Dihydroxyacetone Phosphate.

Structural diagram for aldolase reaction

This reaction is an aldol cleavage, the reverse of the aldol condensation discussed in organic chemistry:

Structural diagram for aldol cleavage mechanism

Recall that this reaction, where C-C bond making or breaking takes place, is only possible because of the acidity of C-2 (the alpha C), which allows the formation of the nucleophilic carbanion. This acidity can be explained by the resonance structures which may be drawn for the alkyl-carbonyl "group":

Structural diagram for alpha carbon resonance

Thus in thinking about a catalytic mechanism for this reaction we should look for ways to further stabilize the carbanion, making it an even better leaving group, and therefore making the transition state easier to achieve.

The molecular mechanism for this enzyme, a type I aldolase, is shown below:

Structural diagram for the molecular mechanism of type 1 aldolases

Note that the enzyme works on the open form of the sugar, and uses a protonated schiff base intermediate at the heart of the mechanism.

Aside: Multi-substrate Enzymes

Look at three common and easily understood types (p 333-5). We will use Cleland Nomenclature.

  • Ordered Sequential Bi Bi mechanism (two on; two off); Note: A must bind first, Q is released last.
  • Ping Pong Bi Bi (one on, one off; one on, one off); Note: have some sort of modified enzyme intermediate (often covalent intermediate)
  • Random Sequential Bi Bi (two on; two off); Note: A or B may bind first, P or Q may be released last.

Kinetic Mechanism

ordered sequential Bi Bi Kinetic mechanism diagram

Ordered Sequential Bi Bi

Random Sequential Bi Bi Kinetic mechanism diagram

Random Sequential Bi Bi

Ping Pong Bi Bi Kinetic mechanism diagram

Ping pong Bi Bi

The enzyme shows an Ordered Sequential Uni Bi kinetic mechanism:

Ordered Sequential Uni Bi Kinetic mechanism diagram for aldolase

5) Triose Phosphate Isomerase: DHAP to GA-3-P

Structural diagram for triose phosphate isomerase reaction

DHAP is more stable, so most of the aldolase product ends up in the DHAP pool in the cell. Need a high activity enzyme to assure the availability of this pool for proceeding through Glycolysis.

TPI turns out to have a very high turnover number (number of molecules processed per active site per time): approx. 1,000,000 mol/min/site, apparently diffusion controlled. That is this enzyme appears to operate as fast as physically possible: as soon as substrate arrives it is converted. Sometimes referred to as a "perfect catalyst."

As with G-6-P Isomerase it uses a LBE type mechanism with enediol intermediate. Again see 2 pKa's and bell shaped pH titration curve. (What must be different about this mechanism compared to G-6-P Isomerase?)

6) Glyceraldehyde 3-Phosphate Dehydrogenase: GA-3-P to 1,3-bis PGA

Structural diagram for glyceraldehyde 3-phosphate dehydrogenase reaction

GA-3-P DH shows an Ordered Sequential Ter Bi kinetic mechanism:

Oxidizing an aldehyde to an 'acid,' creating a mixed acid anhydride in the process: How? Go through an enzyme bound hemithioacetal which is then oxidized to an enzyme bound high energy thiolester. The thiolester can then be phosphorylized to give 1,3-bis PGA:

simplified molecular mechanism for Ga-3-P DH

Note in this mechanism that the thiol group of cysteine is used both as catalyst and to preserve and transfer the free energy of the oxidation reaction. Thus the carbon of the thiohemiacetal is less (+) than an acetal carbon and so it is easier to remove a hydride ion using NAD+, and the resulting thiol ester is a high energy compound which is readily attacked by phosphate. 

Now let's look at the detailed mechanism (A detailed mechanism for this enzyme is also shown as Figure 11.7 in your text): [overhead]

Structural diagram for the molecular mechanism of Ga-3-P DH

(Note: Arsenate can substitute for phosphate forming highly unstable 1-As-3-PGA, which readily hydrolyses, thus producing no ATP - one mechanism of As toxicity.

7) Phosphoglycerate Kinase: 1,3-bis PGA to 3-PGA

Structural equation for Phosphoglycerate Kinase

After a series of unfavorable or marginal reactions now we get a highly favorable reaction again - pulling the pathway forward.

 

These two reactions couple an oxidation (favorable) to a phosphorylation (unfavorable) to give a substrate level oxidative phosphorylation with the capture of the oxidative energy as ATP.

Note that the energy investment in Stage 1 has now been "paid back" and Glycolysis is now energy neutral. This brings us to the third stage of Glycolysis and our ATP energy "profit."

 

8) Phosphoglycerate Mutase: 3-PGA to 2-PGA, begins the third stage of Glycolysis and our ATP energy "profit."

Structural equation for PGA mutase

This enzyme requires 2,3-bis PGA (2,3BPG; DPG) as a cofactor to phosphorylate the enzyme and to maintain the E-P intermediate:

3-PGA + E-P equilibrium arrows 2,3-bisPGA + E equilibrium arrows 2-PGA + E-P

A detailed mechanism for this enzyme is shown below:

Molecular mechanism for PGA Mutase

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

Structural equation for Enolase

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

Molecular mechanism for Enolase

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

Structural equation for Pyruvate kinase reaction

Here we have an attack by ADP:

Molecular mechanism for PK (simplified)

The resulting enol then spontaneously tautomerizes to pyruvate.

FYI - PK Isozymes

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

  • 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.
  • M-PK (Muscle, brain): least regulation - product inhibition by Pyruvate and MgATP.
  • 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. Later we will look at this process for aerobic cells.

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

 

Structural equation for Lactate DH reaction

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

Simplified molecular mechanism for Lactate DH

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.

FYI - LD Isozymes

Lactate DH is a tetramer of two types of monomers, H & M. The kinetic properties of these pure monomer LD isozymes 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.
 

Pathway Diagrams

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Last modified 2 April 2010