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The analogue of NAD + , 4-chloroacetylpyridine -adenine dinucleotide (clac4PdAD +), inactivated the glyceraldehyde-3-phosphate dehydrogenase from sturgeon at a high rate. An affinity labeling was shown to occur with clac4PdAD+. The mononucleotide 4-chloroacetylpyridine 1 -B-D-ribose 5'-phosphate (clac4PdMN+) reacted with the enzyme in a second-order reaction whose rate was much smaller than that calculated for clac4PdAD+ taken as a second-order rate reagent. The rate of the reaction of clac4PdAD+ with the enzyme was determined by stopped flow, using as a probe the long-wavelength absorption maximum (430 nm) formed concomitantly with inactivation of the enzyme. Computer-assisted graphic simulation showed that the clac4PdADf analogue could bind to the active site of the enzyme from Bacillus stearothermophilus in a similar manner to that of NAD', and that the reactive carbon and the reactive thiolate of Cys-149 were within bonding distance. The absorption at 430 nm was linearly proportional to the substoichiometric concentration of clac4PdAD+/mole subunit. Thiol titration suggested the modification of one thiol residue per subunit. The modified thiol was identified by degradation as Cys-149. In contrast to the absorption band generated during the reaction of the 3-chloroacetylpyridine -adenine dinucleotide (clac3PdAD+) with the same enzyme [Eur. J . Biochem. (1 982) 127, 519 -524; 129, 437-4461, enzyme inactivated with clac4PdAD+ and clac4PdMN+ exhibited an absorption maximum at long wavelength which was still present after denaturation. The chromophore is proposed to be the enol form of the a-thioether ketone produced by alkylation of the thiolate of Cys-149 by the chloroacetyl group.Glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate : NAD + oxidoreductase (phosphorylating)] an enzyme of the glycolytic pathway, has been extensively studied [l]. All NAD +-dependent glyceraldehyde-3-phosphate dehydrogenases are tetramers of four identical subunits. Amino acid and gene sequences for the enzyme from various sources have been published [2 -181 showing a high degree of similarity between molecules from different species. Amino acids which are postulated to be directly implicated in the NAD' binding or in the catalytic mechanism are highly conserved. Crystallographic studies of glyceraldehyde-3-phosphate dehydrogenase from several sources have been reported revealing similar three-dimensional structures [19 -241. The refinement of these structures, already undertaken for the Bacillus steurothermophilus holo-enzyme [25], as well as sitedirected mutagenesis [26] or affinity labelling studies, will contribute to complete the attainment about the reaction Abbreviutions. clac4PdAD +, 4-chloroacetylpyridine -adenine dinucleotide; clac4PdMN+, 4-chloroacetylpyridine 1 -P-D-ribose 5'-phosphate; clac3PdAD +, 3-chloroacetylpyridine -adenine dinucleotide.Enzymes. Glyceraldehyde-3-phosphate dchydrogenase, D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating) (EC 1.2.1.12). mechanism, the coenzyme bind...

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