Glyceraldehyde-3-phosphate active site

The transaldolase functions primarily to make a useful glycolytic substrate from the sedoheptulose-7-phosphate produced by the first transketolase reaction. This reaction (Figure 23.35) is quite similar to the aldolase reaction of glycolysis, involving formation of a Schiff base intermediate between the sedohep-tulose-7-phosphate and an active-site lysine residue (Figure 23.36). Elimination of the erythrose-4-phosphate product leaves an enamine of dihydroxyacetone, which remains stable at the active site (without imine hydrolysis) until the other substrate comes into position. Attack of the enamine carbanion at the carbonyl carbon of glyceraldehyde-3-phosphate is followed by hydrolysis of the Schiff base (imine) to yield the product fructose-6-phosphate. 

Thus, an initial drop in ATP is followed by increases in Ca2+, which inhibits ATP synthase and increases ROS and reactive nitrogen species (RNS) formation via xanthine oxidase. These inhibit thiol-dependent Ca2+ transport. The reactive molecules can also inhibit the electron transport chain (by reacting with Fe at the active sites) and enzymes in glycolysis, notably glyceraldehyde 3-phosphate dehydrogenase, leading to further losses of ATP. The depleted ATP exacerbates the intracellular Ca2 increase as a result of reduced transport out and sequestration into the endoplasmic reticulum. 

Irreversible inhibitors often provide clues to the nature of the active site. Enzymes that are inhibited by iodo-acetamide, for example, frequently have a cysteine in the active site, and the cysteinyl sulfhydryl group often plays an essential role in the catalytic mechanism (fig. 7.18). An example is glyceraldehyde 3-phosphate dehydrogenase, in which the catalytic mechanism begins with a reaction of the cysteine with the aldehyde substrate (see fig. 12.21). As we discuss in chapter 8, trypsin and many related proteolytic enzymes are inhibited irreversibly by diisopropyl-fluorophosphate (fig. 7.18), which reacts with a critical serine residue in the active site. 

Fig. 7.1 Reactions catalyzed at the active sites of the a subunit (a reaction) and of the 0 subunit (0 reaction) and the coupled, physiological reaction (a0 reaction). In the a0 reaction, indole produced by cleavage of indole-3-glycerol phosphate at the a site diffuses through an intramolecular tunnel to the 0 site 25-30 A distant where it undergoes a pyridoxal phosphate-dependent /3-replacement reaction with L-serine to form L-tryptophan. Abbreviations used IGP, indole-3-glyceroI phosphate G-3-P, o-glyceraldehyde 3-phosphate, IND, indole [IND], indole intermediate PLP, pyridoxal phosphate.

Glyceraldehyde-3-phosphate dehydrogenase has an essential cysteine residue in its active site. The enzyme forms a transient acyl compound with its substrate, glyceraldehyde 3-phosphate, (a) What is the general chemical name of the compound (b) Draw its likely structure. 

Fig. 19. The active sites of lactate dehydrogenase (A) and glyceraldehyde-3-phosphate dehydrogenase (B). From the work of Rossmann and colleagues [52],...

M22. Mohr, S., Stamler, J. S., and Brune, B., Mechanism of covalent modification of glyceraldehyde-3-phosphate dehydrogenase at its active site thiol by nitric oxide, peroxynitrite and related nitrosating agents. FEBS Lett. 348, 223-227 (1994). 

Like transketolase, transaldolase (TA, E.C. 2.2.1.2) is an enzyme in the oxidative pentose phosphate pathway. TA is a class one lyase that operates through a Schiff-base intermediate and catalyzes the transfer of the C(l)-C(3) aldol unit from D-sedoheptulose 7-phosphate to glyceraldehyde-3-phosphate (G3P) to produce D-Fru 6-P and D-erythrose 4-phosphate (Scheme 5.59). TA from human as well as microbial sources have been cloned.110 111 The crystal structure of the E. coliu and human112 transaldolases have been reported and its similarity to the aldolases is apparent, since it consists of an eight-stranded (o /(3)s or TIM barrel domain as is common to the aldolases. As well, the active site lysine residue that forms a Schiff base with the substrate was identified.14112 Thus, both structurally and mechanistically it is related to the type I class of aldolases. 

After fructose-1,6-bisphosphate is cleaved by aldolase to afford dihydroxyacetone phosphate (122) and glyceraldehyde-3-phosphate (124), the former is further converted to the latter by trlosephosphate isomerase (TIM). The structure of this enzyme has the prototypical TIM fold as an (a/ )s barrel (Figure 12), with the active site in one side of the barrel. This protein folding pattern is also found in the structures of hexose isomerases. 

Several different amino acid side chains can act as nucleophiles in enzyme catalysis. The most powerful nucleophile is the thiol side chain of cysteine, which can be deproto-nated to form the even more nucleophilic thiolate anion. One example in which cysteine is used as a nucleophile is the enzyme glyceraldehyde 3-phosphate dehydrogenase, which uses the redox coenzyme NAD+. As shown in Fig. 10, the aldehyde substrate is attacked by an active site cysteine, Cys-149, to form a hemi-thioketal intermediate, which transfers hydride to NAD+ to form an oxidized thioester intermediate (7). Attack of phosphate anion generates an energy-rich intermediate 3-phosphoglycerate. 

Figure 16.7. Structure of Glyceraldehyde 3-Phosphate Dehydrogenase. The active site includes a cysteine residue and a histidine residue adjacent to a hound NAD+. 

An enzyme that has been the subject of intensive experimental and theoretical studies is triosephosphate isomerase (TIM), which catalyses the interconversion of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (GAP), an essential step in the glycolytic pathway (Fersht 1985). The mechanism of the enzyme has been examined by QM/MM calculations which we do not describe here because it falls outside the topic ofthis review (Bash et al. 1991). However, an additional aspect of the overall mechanism is the conformational change of an 11-residue loop region (residues 166-176) which moves more than 7 A and closes over the active site when substrate binds (Joseph et al. 1990 Lolis et al. 1990). Mutagenesis experiments have... 

Enzymes that contain free sulfhydryl groups at the active site (e.g., glyceraldehyde-3-phosphate dehydrogenase see Chapter 13) react with an alkylating reagent, iodoacetic acid, resulting in inactivation of the enzyme. 

Dietze, E. C., Schafer, A., Omichinski, J. G., Nelson, S. D. Inactivation of glyceraldehyde-3-phosphate dehydrogenase by a reactive metabolite of acetaminophen and mass spectral characterization of an arylated active site peptide. Chertr Res. Toxicol. 1997,10,1097-1103. 

Figure 16.6 Structure of glyceraldehyde 3-phosphate dehydrogenase. Notice that the active site includes a cysteine residue and a histidine residue adjacent to a bound NAD molecule. The sulfur atom of cysteine will link with the substrate to form a transitory thioester intermediate. [Drawn from IGAD.pdb,]...

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