Glycolysis and Fermentation

http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/glycolysis.htm

Contents of this page:
Glycolysis pathway reactions
Summary of pathway
Fermentation
Regulation of glycolysis

Glycolysis Pathway
The Glycolysis pathway is described below and summarized in Fig. 17.3 p. 584 of Biochemistry, by Voet & Voet, 3rd Edition.

The reactions of Glycolysis take place in the cytosol of cells.

Glucose enters the Glycolysis pathway by conversion to glucose-6-phosphate. Initially, there is energy input corresponding to cleavage of two ~P bonds of ATP.

1. Hexokinase catalyzes: glucose + ATPà glucose-6-phosphate + ADP

The Hexokinase reaction involves nucleophilic attack of the C6 hydroxyl oxygen of glucose on the phosphorous of the terminal phosphate of ATP. ATP binds to the enzyme as a complex with Mg⁺⁺.

Glycolysis and Fermentation - student2.ru

The positively charged Mg⁺⁺ interacts with negatively charged phosphate oxygen atoms of ATP, providing charge compensation and promoting a favorable conformation of ATP at the active site. (See also diagram p. 585.) The reaction catalyzed by Hexokinase is highly spontaneous. A phosphoanhydride bond of ATP (~P) is cleaved. The phosphate ester formed in glucose-6-phosphate has a lower DG of hydrolysis.

Induced fit: Glucose binding to Hexokinase stabilizes a conformation in which

The C6 hydroxyl of the bound glucose is close to the terminal phosphate of ATP, promoting catalysis.
Water is excluded from the active site. This prevents the enzyme from catalyzing ATP hydrolysis, rather than transfer of phosphate to glucose.

It is a common motif for an enzyme active site to be located at an interface between protein domains that are connected by a flexible hinge region. The structural flexibility allows access to the active site, while permitting precise positioning of active site residues, and in some cases exclusion of water, as substrate binding promotes a particular conformation.

2. Phosphoglucose Isomerase catalyzes:

glucose-6-phosphate(aldose) ßàfructose-6-phosphate(ketose)

The Phosphoglucose Isomerase mechanism involves acid/base catalysis, with ring opening, isomerization via an enediolate intermediate, and then ring closure (diagram p. 587). A similar reaction catalyzed by Triose Phosphate Isomerase is presented in more detail below.

3. Phosphofructokinase catalyzes:

fructose-6-phosphate + ATPàfructose-1,6-bisphosphate + ADP

This highly spontaneous reaction has a mechanism similar to that of Hexokinase. The Phosphofructokinase reaction is the rate-limiting step of Glycolysis. The enzyme is highly regulated, as will be discussed later.

4. Aldolase catalyzes:

fructose-1,6-bisphosphate ßà dihydroxyacetone phosphate + glyceraldehyde-3-phosphate.

The Aldolase reaction is an aldol cleavage, the reverse of an aldol condensation. Note that carbon atoms are renumbered in reaction products.

A lysine residue at the active site of the Aldolase enzyme functions in catalysis. The keto group of fructose-1,6-bisP reacts with the e-amino group of the active sitelysine, to form a protonated Schiff base intermediate. Cleavage of the bond between C3 and C4 follows. (See p. 590).

5. Triose Phosphate Isomerase (TIM) catalyzes (diagrams p. 591-594):

dihydroxyacetone phosphate (ketose) ßà glyceraldehyde-3-phosphate (aldose)

Glycolysis continues from glyceraldehyde-3-phosphate.The equilibrium constant (K_eq) for the TIM reaction favors dihydroxyacetone phosphate, but removal of glyceraldehyde-3-phosphate by a subsequent spontaneous reaction allows throughput.

The ketose/aldose conversion of TIM involves acid/base catalysis, and is thought to proceed via an enediol intermediate, as with Phosphoglucose Isomerase, Active site Glu and His residues are thought to extract and donate protons during catalysis.

2-Phosphoglycolate is an example of a transition state analog that binds tightly at the active site of Triose Phosphate Isomerase. This inhibitor of catalysis by TIM is similar in structure to the proposed enediolate intermediate. TIM is considered a "perfect enzyme", because the reaction rate is limited only by the rate at which substrate collides with the enzyme.

The structure of Triose Phosphate Isomerase is an ab barrel, or TIM barrel. In an ab barrel there are 8 parallel b-strands surrounded by 8 a-helices. Short loops connect alternating b-strands and a-helices. TIM barrels serve as scaffolds for active site residues in a diverse array of enzymes. Residues that form the active site are always located at the same end of the barrel, associated with the C-terminal ends of b-strands and the loops connecting these to a-helices. There is debate over whether the many different TIM barrel enzymes are evolutionarily related, since in spite of the structural similarities there is tremendous diversity in catalytic functions of these enzymes and little sequence homology.

Explore at right the structure of the Triosephosphate Isomerase (TIM) homodimer, with the transition state inhibitor 2-phosphoglycolatebound to one of the TIM monomers. Note the structure of the TIM barrel, and the loop that forms a lid that closes over the active site after binding of the substrate.

Triose Phosphate Isomerase

6. Glyceraldehyde-3-phosphate Dehydrogenase catalyzes:

glyceraldehyde-3-phosphate + NAD⁺ + P_ißà1,3,bisphosphoglycerate + NADH + H⁺

Exergonic oxidation of the aldehyde in glyceraldehyde-3-phosphate, to a carboxylic acid, drives formation of an acyl phosphate, a "high energy" bond (~P), in 1,3-bisphosphoglycerate. This is the only step in Glycolysis in which NAD⁺ is reduced to NADH.

A cysteine thiolat the active site of Glyceraldehyde-3-phosphate Dehydrogenase has a role in catalysis (p. 596). Glycolysis and Fermentation - student2.ru

The aldehyde of glyceraldehyde-3-phosphate reacts with the active site cysteine thiol to form a thiohemiacetal intermediate. Oxidation to a carboxylic acid (in a "high energy" thioester) occurs, as NAD⁺ is reduced to NADH. The "high energy" acyl thioester is attacked by P_i to yield the acyl phosphate (~P) product.