Effect of selective thiol-group derivatization on enzyme kinetics of (R)-3-hydroxybutyrate dehydrogenase.
Effect of selective thiol-group derivatization on enzyme kinetics of (R)-3-hydroxybutyrate dehydrogenase.
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DOI：
10.1042/bj2960563
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发表时间：
1993-12
期刊：
影响因子：
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通讯作者：
L. Dalton;J. Mcintyre;S. Fleischer
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文献类型：
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作者：
L. Dalton;J. Mcintyre;S. Fleischer
(R)-3-羟基丁酸脱氢酶（BDH）是一种需要磷脂酰胆碱的四聚体酶，每个原聚体有两个巯基（SH-1和SH-2）。首先用二胺[1,1'-偶氮-（NN'-二甲基甲酰胺）DM]保护反应更快的巯基（SH-1）形成DM(SH-1)BDH， SH-2可以通过与马来酰亚胺试剂如4-马来酰亚胺-2,2,6,6-四甲基哌啶- n -氧基（MSL）选择性衍生，得到DM(SH-1)MSL(SH-2)BDH。二硫苏糖醇（DTT）还原生成SH-1，生成MAL(SH-2)BDH （MAL是MSL-BDH和DTT的抗磁还原产物）。DM(SH-1)BDH酶活性降低至约。相对于天然纯化酶的4%，底物KmBOH的表观Km增加了大约。100倍。DTT减少DM(SH-1)BDH再生SH-1并恢复正常的酶功能。用哌啶基马来酰亚胺修饰SH-2 [MAL(SH-2)BDH]使酶活性降低到大约。但对表观KmBOH无显著影响。双衍生酶DM(SH-1)MSL(SH-2)BDH具有较低的酶活性[约为DM(SH-2)BDH的一半]，但表观KmBOH高于DM(SH-1)BDH。SH-2与不同马来酰亚胺试剂的衍生化导致活性的降低与马来酰亚胺取代基的大小成正比，表明这种抑制是位阻的。SH-1的修饰会导致动力学参数的显著变化（表观Km增加，表观Vmax降低），SH-2的衍生化对酶功能的影响较小。因此，假设SH-1比SH-2更接近活性中心，尽管两者都不参与催化，因为：(1)衍生酶的活性没有被取消；(2)可以通过增加底物（和辅因子）浓度来增强活性。
(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a phosphatidylcholine-requiring tetrameric enzyme with two thiol groups (SH-1 and SH-2) per protomer. By first protecting the more rapidly reacting thiol group (SH-1) with diamide [1,1'-azobis-(NN'-dimethylformamide), DM] to form DM(SH-1)BDH, SH-2 can be selectively derivatized by reaction with maleimide reagents such as 4-maleimido-2,2,6,6-tetramethyl-piperidine-N-oxyl (MSL), which gives DM(SH-1)MSL(SH-2)BDH. Reduction with dithiothreitol (DTT) regenerates SH-1, yielding MAL(SH-2)BDH (where MAL is the diamagnetic reduction product of MSL-BDH and DTT). The enzymic activity of DM(SH-1)BDH is decreased to approx. 4% relative to the native purified enzyme, and the apparent Km for substrate, KmBOH, is increased approx. 100-fold. Reduction of DM(SH-1)BDH with DTT regenerates SH-1 and restores normal enzymic function. Modification of SH-2 with piperidinylmaleimide [MAL(SH-2)BDH] diminishes enzymic activity to approx. 35% of its original value, but has no significant effect on apparent KmBOH. The doubly derivatized enzyme, DM(SH-1)MSL(SH-2)BDH, has lower enzymic activity [about half that for DM(SH-2)BDH] and a yet higher apparent KmBOH than DM(SH-1)BDH. Derivatization of SH-2 with different maleimide reagents results in diminished activity approximately proportional to the size of the maleimide substituent, suggesting that this inhibition is steric. Whereas modification of SH-1 results in marked changes in kinetic parameters (increased apparent Km and reduced apparent Vmax), derivatization of SH-2 has a lesser effect on enzymic function. Thus SH-1 is postulated to be closer to the active centre than is SH-2, although neither is involved in catalysis, since: (1) the activity of the derivatized enzyme is not abolished; and (2) activity can be enhanced by increasing substrate (and cofactor) concentrations.