Catalytic mechanisms of glutamine synthetase enzymes. Studies with analogs of possible intermediates and transition states.
Catalytic mechanisms of glutamine synthetase enzymes. Studies with analogs of possible intermediates and transition states.
复制标题
谷氨酰胺合成酶的催化机制。
DOI：
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发表时间：
1976
影响因子：
4.8
通讯作者：
B. Horn
中科院分区：
文献类型：
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作者：
F. Wedler;B. Horn
谷氨酰胺合成酶分离豌豆种子和大肠杆菌中观察到不同的行为在实验中设计的探测反应机制。尽管发现两种酶都以随机顺序机制结合和释放底物（Wedler，F.C.（1974）J. Biol. Chem，247，5080-5087），与指示γ-谷氨酰磷酸中间体的部分反应系统的同位素交换仅由豌豆种子酶催化。急诊大肠杆菌系统不能以可感知的速率催化任何交换，除非所有底物都存在。该阴性结果意味着结合底物的绝对构象要求或推定复合物（E-Glu-P-MgADP）非常紧密。为了测试后者，已经合成了γ-谷氨酰-磷酸的非反应性结构类似物，即3-（膦酰基乙酰氨基）-L-丙氨酸（PA 2LA）。用E.发现大肠杆菌酶PA 2LA的结合不比L-谷氨酸更紧密，并且与L-谷氨酸严格竞争（Ki = 3 mM）。因此，未能催化指示γ-Glu-P的部分交换反应可能不归因于紧密复合物的形成。PA 2LA与豌豆种子酶的结合显然涉及两步过程：一个快速、可逆的步骤，其中PA 2LA比L-谷氨酸结合紧密10倍，然后是一个缓慢（但可逆）的过程，涉及非常紧密的PA 2LA结合，显然是核苷酸促进的酶异构化。这两种酶对L-蛋氨酸-SR-亚砜亚胺Met（O）（NH）的特异性也不同。
Glutamine synthetase enzymes isolated from pea seeds and from Escherichia coli are observed to behave differently in experiments designed to probe reaction mechanism. Although both enzymes were found to bind and release substrates in random order mechanisms (Wedler, F.C. (1974) J. Biol. Chem, 247, 5080-5087), isotopic exchanges with partial reaction systems indicative of a gamma-glutamylphosphate intermediate are catalyzed only by the pea seed enzyme. The E. coli system fails to catalyze any exchanges at appreciable rates unless all substrates are present. This negative result implies either an absolute conformational requirement for bound substrates or that the putative complex (E-Glu-P-MgADP) is exceedingly tight. To test the latter, a nonreactive structural analog of gamma-glutamyl-phosphate, namely 3-(phosphonoacetylamido)-L-alanine (PA2LA), has been synthesized. With the E. coli enzyme PA2LA was found to bind no more tightly than L-glutamate and is strictly competitive versus L-glutamate (Ki = 3 mM). Thus, failure to catalyze partial exchange reactions indicative of gamma-Glu-P is probably not attributable to tight complex formation. The binding of PA2LA with the pea seed enzyme apparently involves a two-step process: a rapid, reversible step in which PA2LA binds 10-fold more tightly than L-glutamate, followed by a slow (but reversible) process involving very tight PA2LA binding, apparently with enzyme isomerization promoted by nucleotide. The specificity of the two enzymes toward L-methionine-SR-sulfoximine, Met(O)(NH), was also different...