USE OF ISOTOPE EFFECTS AND PH STUDIES TO DETERMINE THE CHEMICAL MECHANISM OF BACILLUS-SUBTILIS L-ALANINE DEHYDROGENASE
USE OF ISOTOPE EFFECTS AND PH STUDIES TO DETERMINE THE CHEMICAL MECHANISM OF BACILLUS-SUBTILIS L-ALANINE DEHYDROGENASE
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DOI：
10.1021/bi00523a003
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发表时间：
1981-01-01
期刊：
影响因子：
2.9
通讯作者：
CLELAND, WW
中科院分区：
文献类型：
--
作者：
GRIMSHAW, CE;COOK, PF;CLELAND, WW
氘同位素效应（B.枯草杆菌L-丙氨酸脱氢酶）与L-丙氨酸-d4和L-丝氨酸-d3的反应，以及用相同底物的pH曲线，表明L-丙氨酸是粘性的（即，反应产生产物的速度是其解离速度的1-7倍），而L-丝氨酸则不然。pH曲线显示NH3和单阴离子氨基酸是底物;酶上的阳离子酸基团（可能是赖氨酸）在E-NAD中的pK为9.0-9.6，但在E-NADH中的pK远高于10，必须质子化以获得活性和抑制剂的良好结合，并且可能对保持酶的正确构象很重要。酶上的阳离子酸基团（可能是组氨酸）在E-NAD和E-NADH中的pK都在7左右，对于氨基酸的氧化必须是非质子化的，但对于丙酮酸的结合和反应必须是质子化的。后者是化学反应的酸碱催化剂。在E-NAD中，其位置使得其可与抑制剂的D-羟基或羰基形成氢键（且因此当质子化时增强其结合），但其质子化状态不影响L-乳酸盐或丙酸盐的结合。在E-NADH中，它的位置使得它可以与D-和L-羟基以及羰基形成氢键。一种化学机制被假定，其中的L-丙氨酸由NAD脱氢产生亚氨基丙酮酸，随后由水的攻击从相同的一侧，从该氢化物被删除。催化性组氨酸将质子从攻击水转移到所得甲醇胺的氨基，然后随着氨的消除从甲醇胺的羟基除去质子，得到丙酮酸盐。
Analysis of deuterium isotope effects (of B. subtilis L-alanine dehydrogenase) with L-alanine-d4 and L-serine-d3, and of pH profiles with the same substrates, shows that L-alanine is sticky (i.e., reacts to give products 1-7 times as fast as it dissociates) while L-serine is not. The pH profiles show that NH3 and monoanionic amino acids are the substrates; a cationic acid group on the enzyme (probably lysine) with a pK of 9.0-9.6 in E-NAD, but a pK well above 10 in E-NADH must be protonated for activity and good binding of inhibitors and is probably important for maintaining the proper conformation of the enzyme. A cationic acid group on the enzyme (probably histidine) with a pK around 7 in both E-NAD and E-NADH must be unprotonated for oxidation of amino acids but protonated for binding and reaction of pyruvate. This latter group is the acid-base catalyst for the chemical reaction. In E-NAD, it is so positioned that it can hydrogen bond to (and thus when protonated enhance the binding of) a D-hydroxy or a carbonyl group of an inhibitor, but its state of protonation does not affect the binding of L-lactate or propionate. In E-NADH, it is so placed that it can hydrogen bond to both D- and L-hydroxy groups, and carbonyl groups. A chemical mechanism is postulated in which the dehydrogenation of L-alanine by NAD to produce iminopyruvate is followed by attack of water from the same side from which the hydride was removed. The catalytic histidine transfers a proton from the attacking water to the amino group of the resulting carbinolamine and then removes a proton from the hydroxyl group of the carbinolamine as ammonia is eliminated to give pyruvate.