Dalton L A, McIntyre J O, Fleischer S
Department of Molecular Biology, Vanderbilt University, Nashville, TN 37235.
Biochem J. 1993 Dec 15;296 ( Pt 3)(Pt 3):563-9. doi: 10.1042/bj2960563.
(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.
（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，每个亚基含有两个硫醇基团（SH-1和SH-2）。通过首先用二酰胺[1,1'-偶氮双-（N，N'-二甲基甲酰胺），DM]保护反应更快的硫醇基团（SH-1）以形成DM（SH-1）BDH，SH-2可以通过与马来酰亚胺试剂如4-马来酰亚胺基-2,2,6,6-四甲基哌啶-N-氧基（MSL）反应而被选择性衍生化，得到DM（SH-1）MSL（SH-2）BDH。用二硫苏糖醇（DTT）还原可使SH-1再生，产生MAL（SH-2）BDH（其中MAL是MSL-BDH和DTT的抗磁性还原产物）。DM（SH-1）BDH的酶活性相对于天然纯化酶降低至约4％，并且底物的表观Km，即KmBOH，增加约100倍。用DTT还原DM（SH-1）BDH可使SH-1再生并恢复正常的酶功能。用哌啶基马来酰亚胺[MAL（SH-2）BDH]修饰SH-2可使酶活性降低至其原始值的约35％，但对表观KmBOH没有显著影响。双重衍生化的酶DM（SH-1）MSL（SH-2）BDH具有较低的酶活性[约为DM（SH-2）BDH的一半]，并且比DM（SH-1）BDH具有更高的表观KmBOH。用不同的马来酰亚胺试剂衍生化SH-2导致活性降低，其降低程度大致与马来酰亚胺取代基的大小成比例，这表明这种抑制是空间位阻的。虽然SH-1的修饰导致动力学参数发生明显变化（表观Km增加和表观Vmax降低），但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）衍生化酶的活性没有被消除；（2）通过增加底物（和辅因子）浓度可以提高活性。
