Amino acid glyceraldehyde-3-phosphate dehydrogenases

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. 

Inactivation of alcohol dehydrogenase from yeast with 14C-labeled [3-(3-bromoacetylpyridinio)-propyl]-adenosine pyrophosphate followed by oxidation showed the presence of 1-carboxymethyl histidine66. After inactivation of the enzyme with labeled [3-(4-bromoacetylpyridinio)-propyl]-adenosine pyrophosphate followed by oxidation, S-carboxymethyl cysteine was identified in the protein. In the case of glyceraldehyde-3-phosphate dehydrogenase, treatment with either coenzyme analogue leads to the modification of the cysteine residue. Treatment with [14C]nicotinamide-5-bromo-4-methylimidazole dinucleotide did not reveal any modified amino-acid-residues. The labeled nicotinamide residue split off during the recovery of the inactivated enzyme. Attempts to synthesize an inactivator labeled with a 14C-acetyl residue did not give satisfactory yields. If the enzyme-coenzyme derivative was treated with tritiated sodium boron hydride, tritium could be introduced (Fig. 22). Studies with... 

The subunit-subunit interaction must be important in the assembly process. It is worth noting that in the case of glyceraldehyde-3-phosphate dehydrogenase the amino acids in the subunit interface are far more conserved than other amino acids elsewhere in that enzyme 189). If this... 

Dedman, J. R., Gracy, R. W. and Harris, B. G. (1974) A method for estimating sequence homology from amino acid comparisons. The evolution of Ascaris employing adolase and glyceraldehyde-3-phosphate dehydrogenase. Comp. Biochem. Physiol. 49B 715-731. 

A cartoon representation of the oxidation path used in Equation 9.5 is shown in Scheme 9.5. The process is catalyzed by the enzyme glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12). A large body of evidence has been accumulated about the role of the enzyme and, in particular, that the catalytic site on the enzyme that is used to help the oxidation occur bears a thiol (-SH, Chapter 8) group (from the amino acid cysteine. Chapter 12). Further, it has been shown that hydride (H ) transfer to nicotinamide dinucleotide (NAD ") (see Chapter 12), a required cofactor that accounts for the transfer of the hydride anion and, thus, the oxidation itself, is also involved. 

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