4‐Chloroacetylpyridine adenine dinucleotide

The analogue of NAD + , 4‐chloroacetylpyridine‐adenine dinucleotide (clac 4 PdAD + ), invactivated the glyceraldehyde‐3‐phosphate dehydrogenase from sturgeon at a high rate. An affinity labeling was shown to occur with clac 4 PdAD + . The mononucleotide 4‐chloroacetylpyridine 1‐β‐ d ‐ribose 5′‐phosphate (clac 4 PdMN + ) reacted with the enzyme in a second‐order reaction whose rate was much smaller than that calculated for clac 4 PdAD + taken as a second‐order rate reagent. The rate of the reaction of clac 4 PdAD + 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 clac 4 PdAD + 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 clac 4 PdAD + /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 dinucieotide (clac 3 PdAD + ) with the same enzyme [ Eur. J. Biochem (1982) 127 , 519–524; 129 , 437–446], enzyme inactivated with clac 4 PdAD + ) and clac 4 PdMN + exhibited an absorption maximum at long wavelength which was still present after denaturation. The chromophore is proposed to be the enol form of the α‐thioether ketone produced by alkylation of the thiolate of Cys‐149 by the chloroacetyl group.