Mannervik B, Marmstål E, Ekwall K, Górna-Hall B
Eur J Biochem. 1975 May 6;53(2):327-33. doi: 10.1111/j.1432-1033.1975.tb04072.x.
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.
来自猪红细胞和酵母的乙二醛酶I可被氨基试剂1-氟-2,4-二硝基苯、5-二甲氨基萘-1-磺酰氯和2,4,6-三硝基苯磺酸（N-3ph-S）灭活。灭活遵循假一级动力学，表观一级速率常数随pH值升高而增加，表明亲核基团的碱性形式被修饰。用N-3PH-S测试了增加灭活剂浓度的影响，发现表观速率常数增加到一个极限值。这样的结果与实际灭活前形成可逆的灭活剂x酶复合物的机制一致。与之前对酵母酶的研究结果相反，用红细胞乙二醛酶I和多种巯基试剂进行的实验未能表明催化活性对巯基的依赖性。这些实验似乎排除了红细胞酶的必需巯基被氨基试剂修饰的可能性。酵母乙二醛酶I不能重新激活，以及与红细胞酶的相似性表明，酵母乙二醛酶I也不会被后一种试剂修饰其必需巯基。涉及氨基和巯基试剂同时灭活的实验进一步支持了这一假设。结果与乙二醛酶I的氨基对催化活性至关重要的解释一致。发现作为乙二醛酶I可逆竞争性抑制剂的谷胱甘肽衍生物可保护该酶免受氨基试剂的灭活。然而，半最大保护所需的浓度远高于可逆抑制的抑制常数，这表明在获得完全保护之前，至少有两个保护剂分子必须与酶结合。
