Effects of pH and thiols on the kinetics of yeast glyoxalase I. An evaluation of the random pathway mechanism.
Effects of pH and thiols on the kinetics of yeast glyoxalase I. An evaluation of the random pathway mechanism.
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pH 值和硫醇对酵母乙二醛酶 I 动力学的影响。随机途径机制的评估。
DOI：
10.1021/bi00687a024
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发表时间：
1975
期刊：
影响因子：
2.9
通讯作者：
L. Han
中科院分区：
文献类型：
--
作者：
D. V. Vander Jagt;E. Daub;J. Krohn;L. Han
酵母乙二醛酶I催化的α-酮醛歧化反应是一种随机途径机制，其中一个分支以一种底物途径利用谷胱甘肽和α-酮醛的半合成，另一个分支以有序的两种底物途径首先利用谷胱甘肽和α-酮醛。以甲基乙醛和苯乙醛为代表的脂肪族和芳香族α-酮醛，通过比较无酶条件下半乳糖醛的初始生成速率和高酶浓度下的初始产物生成速率，在pH值为3-7的范围内，评价了这两种途径的相对重要性。如果没有最后加入酶，即使在水合的α-酮醛的脱水不完全是速率决定的条件下，产物的初始形成速率也与加合物的初始形成速率相同。如果酶是在半头形成之后加入的，则会有一个相当于半头形成量的产物形成的“爆发”，然后是一个较慢的反应，这与单底物途径一致。通过研究添加的硫醇试剂对“爆发”动力学的影响，获得了对这一途径的额外支持。酵母乙二醛酶I对脂肪族和芳香族α-酮醛的广泛专一性反映在对α-酮醛的性质不敏感的Vmax值上，如果α-酮醛的侧链是空间拥挤的，则突然下降。叔丁基乙二醛的半封端的Vmax比甲基乙二醛的Vmax小300倍；2，4，6-三甲基苯乙醛作为底物基本上没有活性，尽管密切相关的化合物2，4-二甲基苯乙醛是正常底物。对这些α-酮醛的Vmax和Km(或Ki)值的分析表明，空间拥挤的侧链既影响酶底物的形成，也影响催化反应。
The disproportionation of alpha-ketoaldehydes, catalyzed by yeast glyoxalase I, has been reported to involve a random pathway mechanism where one branch utilizes the hemimercaptal of glutathione and the alpha-ketoaldehyde in a one-substrate pathway, and the other branch utilizes first glutathione and then the alpha-ketoaldehyde in an ordered two-substrate pathway. The relative importance of the two pathways has been evaluated at 5 degrees in the pH range 3-7, using methylglyoxal and phenylglyoxal as representative aliphatic and aromatic alpha-ketoaldehydes, by comparing initial rates of hemimercaptal formation in the absence of enzyme with initial rates of product formation in the presence of high enzyme concentrations. If the enzyme is not added last, the initial rates of product formation are the same as the initial rates of adduct formation even under conditions where it could be shown that dehydration of the hydrated alpha-ketoaldehyde is not entirely rate determining. If the enzyme is added after hemimercaptal formation, there is a "burst" of product formation equivalent to the amount of hemimercaptal, followed by a slower reaction, consistent with the one-substrate pathway. Additional support for this pathway was obtained from a study of the effects of added thiol reagents on the "burst" kinetics. The broad specificity of yeast glyoxalase I for both aliphatic and aromatic alpha-ketoaldehydes, reflected in Vmax values which are insensitive to the nature of the alpha-ketoaldehyde drops abruptly if the side chain of the alpha-ketoaldehyde is sterically crowded. The hemimercaptal of tert-butylglyoxal has a Vmax 300-fold smaller than Vmax for methylglyoxal; 2,4,6-trimethylphenylglyoxal is essentially inactive as a substrate even though the closely related compound 2,4-dimethylphenylglyoxal is a normal substrate. Analysis of the Vmax and Km (or Ki) values of these alpha-ketoaldehydes suggests that sterically crowded side chains affect both enzyme-substrate formation and the catalytic reaction.