Glyoxalase I from porcine erythrocytes and from yeast is inactivated by the amino-group reagents 1-fluoro-2,4-dinitrobenzene, 5-dimethylaminonaphthalene-1-sulfonyl chloride, and 2,4,6-trinitrobenzenesulfonate (N-3ph-S). The inactivation follows pseudo-first-order kinetics, and the apparent first-order rate constant increases with pH, indicating that the basic form of a nucleophilic group is modified. The effect of increasing the inactivator concentration was tested with N-3PH-S, and it was found that the apparent rate constant increased to a limiting value. Such a result is consistent with a mechanism involving formation of a reversible inactivator x enzyme complex prior to the actual inactivation. Experiments with erythrocyte glyoxalase I and a variety of sulfhydryl-group reagents failed to show a dependence on sulfhydryl groups for catalytic activity, in contrast to previous results with the yeast enzyme. These experiments seem to exclude the possibility that essential sulfhydryl groups of the erythrocyte enzyme are modified by the amino-group reagents. Failure of reactivation of yeast glyoxalase I, and the similarities with the erythrocyte enzyme suggest that yeast glyoxalase I is not modified at essential sulfhydryl groups either by the latter reagents. This assumption has further support from experiments involving simultaneous inactivation with amino and sulfhydryl-group reagents. The results are consistent with the interpretation that amino groups of glyoxalase I are essential for catalytic activity. Glutathione derivatives, which are reversible competitive inhibitors of glyoxalase I, were found to protect the enzyme against inactivation by amino-group reagents. However, the concentration required for half-maximal protection was considerably higher than the inhibition constant of the reversible inhibition, which indicates that at least two molecules of the protector must be bound to the enzyme before full protection is obtained.

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Journal Article

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Mannervik B, Marmstål E, Ekwall K, Górna-Hall B

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