Evidence for the Presence of Anion‐Recognition Sites in Pig‐Liver Aldehyde Reductase

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Abstract
Aldehyde reductase from pig liver is completely inactivated by phenyl glyoxal, a substrate of this enzyme. Inactivation, exhibiting pseudo‐first‐order kinetics and a reaction order of nearly one, is observed in the presence or absence of the coenzyme analogue 1,4,5,6‐tetrahydronicotinamide‐adenine dinucleotide phosphate (h5.62NADPH). These results suggest that one single residue, not located in the coenzyme‐binding site, is modified. Loss of activity is directly proportional to incorporation of [14C]phenyl glyoxal. Amino acid analysis demonstrates the modification of one arginine residue, on average. Nevertheless, the ternary complex enzyme · h5.62NADPH ·p‐carboxybenzaldehyde does not afford complete protection against phenyl glyoxal inactivation. In contrast, (±)2,3‐dimethylsuccinic acid, a strong inhibitor of the reductase, completely prevents inactivation of reductase by phenyl glyoxal. A pseudo‐affinity labeling is observed with p‐carboxyphenyl glyoxal, used as a specific arginine‐modifying agent. Such a mechanism differs from an affinity labeling in that the formation of the binary complex enzyme ·p‐carboxyphenyl glyoxal does not precede the alkylating step. At high concentration, p‐carboxybenzaldehyde protects aldehyde reductase from inactivation by p‐carboxyphenyl glyoxal.All these results suggest the presence of two anion‐binding sites on this reductase, one being responsible for the better binding of substrates bearing a carboxyl group to the enzyme‐coenzyme complex, and the second one being modified by specific arginine reagents and implicated in the binding of uncompetitive inhibitors like (±)2,3‐dimethylsuccinic acid.Results obtained by W. S. Davidson and T. G. Flynn [J. Biol. Chem. 254, 3724–3729 (1979)] on the modification of pig kidney reductase by 2,3‐butanedione conflicted somewhat with our results. This has led us to compare these two reductases. No differences between the enzymes from either source could be detected, thus it is likely that they are both the same protein.