Enzymatic mechanism for the hydrolysis of 5,10-methenyltetrahydropteroylglutamate to 5-formyltetrahydropteroylglutamate by serine hydroxymethyltransferase.
Enzymatic mechanism for the hydrolysis of 5,10-methenyltetrahydropteroylglutamate to 5-formyltetrahydropteroylglutamate by serine hydroxymethyltransferase.
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丝氨酸羟甲基转移酶将 5,10-次甲基四氢蝶酰谷氨酸水解为 5-甲酰四氢蝶酰谷氨酸的酶机制。
DOI：
10.1021/bi00122a037
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Schirch,V
中科院分区：
文献类型：
--
作者：
Stover,P;Schirch,V
Department of Biochemistry and Molecular Biophysics，Virginia Commonwealth University，里士满，Virginia 23298 Received August 9，1991; Revised Mandarin pt Received October 18，1991摘要：丝氨酸羟甲基转移酶在甘氨酸存在下催化（6 i？）- 5，10-亚甲基四氢蝶酰聚谷氨酸盐至（6S）-5-甲酰基四氢蝶酰聚谷氨酸盐。该酶还催化从与（6 R）-N-（4-）氨基）乙基）氨基）苯基）氨基）乙基）氨基）苯甲酸酯平衡的化合物形成（6S）-5-甲酰基四氢蝶酰基聚谷氨酸酯。5，10-亚甲基四氢蝶酰聚谷氨酸被认为是（6 J？，1里？）- 5，10-羟亚甲基四氢蝶酰聚谷氨酸盐，在5，10-亚甲基四氢蝶酰谷氨酸盐非酶促水解成5-甲酰基四氢蝶酰谷氨酸盐中的一种推定中间体[秸秆，P.，& Schirch，V.（1992）生物化学（在此问题上的前一篇论文）]。探讨了由这些底物生成（6S）-5-甲酰四氢蝶酰聚谷氨酸的酶促反应机理及甘氨酸在反应中的作用。有证据表明，（6 i？，l li？）- 5，10-羟亚甲基四氢蝶酰四谷氨酸是（6 J？）- 5，10-亚甲基四氢蝶酰四谷氨酸盐。该酶显示出40 pM的高Km（6 J？）- 5，10-亚甲基四氢蝶酰基四谷氨酸的Km，而（6 R，11 R）-5，10-羟亚甲基四氢蝶酰基四谷氨酸的Km低于0.5pM。两个反应的kat值是相同的，等于在502 nm处吸收的酶三元复合物的形成速率，该复合物是由甘氨酸和（bS）-S-甲酰四氢蝶酰聚谷氨酸形成的。（6 R）-5，10-亚甲基四氢蝶酰四谷氨酸酯的C11甲酰基质子交换进行水解反应，表明该酶催化反应与非酶催化反应一样是通过C11碳负离子转化机制进行的。使用[2- 3 H]甘氨酸和差示扫描量热法数据的同位素交换实验表明，在酶促反应中甘氨酸的催化和构象作用。根据SHMT催化丝氨酸的逆向醛醇裂解和（6 i？）-丝氨酸水解的相似机理对结果进行了讨论。丝氨酸羟甲基转移酶（SHMT）1催化5，10-CH 2-H4 PteGlu和甘氨酸可逆转化形成丝氨酸和四氢蝶酰谷氨酸（H4 PteGlu）（反应1）。尽管已经提出了该反应的几种机理，但有利的机理涉及5，10-CH 2-H4 PteGlu水解为H4 PteGlu和酶结合的甲醛（马修斯和Drummond，1991）。然后，结合的甲醛添加到甘氨酸阴离子上，甘氨酸阴离子作为具有吡啶的共振稳定的亚胺存在于活性位点处。
Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond, Virginia 23298 Received August 9, 1991; Revised Manuscript Received October 18, 1991 abstract: Serine hydroxymethyltransferase in the presence of glycine catalyzes the hydrolysis of (6i?)-5, 10-methenyltetrahydropteroylpolyglutamate to (6S)-5-formyltetrahydropteroylpolyglutamate. The enzyme also catalyzes the formation of (6S')-5-formyltetrahydropteroylpolyglutamate from a compound in equilibrium with (6/?)-5, 10-methenyltetrahydropteroylpolyglutamate believed to be (6J?, 1 li?)-5, 10-hydroxymethylenetetrahydropteroylpolyglutamate, a putative intermediate in the nonenzymatic hydrolysis of 5, 10-methenyltetrahydropteroylglutamate to 5-formyltetrahydropteroylglutamate [Stover, P., & Schirch, V.(1992) Biochemistry (preceding paper in this issue)]. The enzymatic mechanism for the formation of (6S)-5-formyltetrahydropteroylpolyglutamate from these substrates andthe role of glycine in the reaction was addressed. Evidence suggests that (6i?, l li?)-5, 10-hydroxymethylenetetrahydropteroyltetraglutamate is a catalytically competent intermediatein the enzyme-catalyzed hydrolysis of (6J?)-5, 10-methenyltetrahydropteroyltetraglutamate. The enzyme displays a high Km of 40 pM for (6J?)-5, 10-methenyltetrahydropteroyltetraglutamate, while the Km for (6R, llR)-5, 10-hydroxymethylenetetrahydropteroyltetra-glutamate is below 0.5 pM. The kat values for both reactions are identical and equal to the rate of formation of an enzyme ternary complex absorbingat 502 nm which is formed from glycine and (bS^-S-formyltetrahydropteroylpolyglutamate. The hydrolysis reaction proceeds with exchange of the C11 formyl proton of (6R)-5, 10-methenyltetrahydropteroyltetraglutamate, suggesting that theenzyme-catalyzed reaction occurs by the same C11 carbanion inversion mechanism as the nonenzymatic reaction. Isotope exchange experiments using [2-3H] glycine and differential scanning calorimetry data suggest both a catalytic and a conformational role for glycine in the enzymatic reaction. The results are discussed in terms of the similarity inmechanisms of the SHMT-catalyzed retroaldol cleavage of serine and hydrolysis of (6i?)-5, 10-methenyltetrahydropteroylpolyglutamates.Serine hydroxymethyltransferase (SHMT) 1 catalyzes the reversible conversion of 5, 10-CH2-H4PteGlu and glycine to form serine and tetrahydropteroylglutamate (H4PteGlu)(re-action 1). Although several mechanisms have been proposed for this reaction, a favored mechanism involves the hydrolysis of 5, 10-CH2-H4PteGluto H4PteGlu and enzyme-bound formaldehyde (Matthews & Drummond, 1991). The bound formaldehyde then adds to a glycine anion, which is present at the active site as a resonance-stabilized imine with pyri-