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首页> 外文期刊>Biochemistry >Serine Hydroxymethyltransferase:Role of Glu75 and Evidence that Serine Is Cleaved by a Retroaldol Mechanism
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Serine Hydroxymethyltransferase:Role of Glu75 and Evidence that Serine Is Cleaved by a Retroaldol Mechanism

机译：丝氨酸羟甲基转移酶：Glu75的作用和丝氨酸被逆转录醛缩酶裂解的证据

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摘要

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible interconversion of serine and glycine with tetrahydrofolate serving as the one-carbon carrier. SHMT also catalyzes the folate-independent retroaldol cleavage of allothreonine and 3-phenylserine and the irreversible conversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate. Studies of wild-type and site mutants of SHMT have failed to clearly establish the mechanism of this enzyme. The cleavage of 3-hydroxy amino acids to glycine and an aldehyde occurs by a retroaldol mechanism. However, the folate-dependent cleavage of serine can be described by either the same retroaldol mechanism with formaldehyde as an enzyme-bound intermediate or by a nucleophilic displacement mechanism in which N5 of tetrahydrofolate displaces the C3 hydroxyl of serine, forming a covalent intermediate. Glu75 of SHMT is clearly involved in the reaction mechanism; it is within hydrogen bonding distance of the hydroxyl group of serine and the formyl group of 5-formyltetrahydrofolate in complexes of these species with SHMT. This residue was changed to Leu and Gin, and the structures, kinetics, and spectral properties of the site mutants were determined. Neither mutation significantly changed the structure of SHMT, the spectral properties of its complexes, or the kinetics of the retroaldol cleavage of allothreonine and 3-phenylserine. However, both mutations blocked the folate-dependent serine-to-glycine reaction and the conversion of methenyltetrahydrofolate to 5-formyltetrahydrofolate. These results clearly indicate that interaction of Glu75 with folate is required for folate-dependent reactions catalyzed by SHMT. Moreover, we can now propose a promising modification to the retroaldol mechanism for serine cleavage. As the first step, N5 of tetrahydrofolate makes a nucleophilic attack on C3 of serine, breaking the C2-C3 bond to form N5-hydroxymethylene-tetrahydrofolate and an enzyme-bound glycine anion. The transient formation of formaldehyde as an intermediate is possible, but not required. This mechanism explains the greatly enhanced rate of serine cleavage in the presence of folate, and avoids some serious difficulties presented by the nucleophilic displacement mechanism involving breakage of the C3-OH bond.
机译：丝氨酸羟甲基转移酶（SHMT）催化丝氨酸和甘氨酸与四氢叶酸作为单碳载体的可逆相互转化。 SHMT还催化了不依赖叶酸的去甲肾上腺素和3-苯基丝氨酸的逆向羟醛裂解，以及5,10-亚甲基四氢叶酸不可逆地转化为5-甲酰基四氢叶酸。 SHMT的野生型和位点突变体的研究未能清楚地建立这种酶的机制。 3-羟基氨基酸裂解成甘氨酸和醛是通过逆醛醇缩合机理进行的。然而，丝氨酸的叶酸依赖裂解可以通过与甲醛作为酶结合中间体的相同的逆醛醇缩合机理或通过亲核置换机理来描述，其中四氢叶酸的N5置换丝氨酸的C3羟基，形成共价中间体。 SHMT的Glu75明显参与了反应机理。在这些物质与SHMT的络合物中，它在丝氨酸的羟基和5-甲酰基四氢叶酸的甲酰基的氢键结合距离之内。将此残基更改为Leu和Gin，并确定了位点突变体的结构，动力学和光谱特性。两种突变均未显着改变SHMT的结构，其复合物的光谱性质，或Allallreonine和3-phenylserine的逆向羟醛裂解的动力学。但是，这两个突变都阻止了叶酸依赖性的丝氨酸-甘氨酸反应以及亚甲基四氢叶酸转化为5-甲酰基四氢叶酸。这些结果清楚地表明，SHMT催化的叶酸依赖性反应需要Glu75与叶酸的相互作用。而且，我们现在可以提出对丝氨酸裂解的逆醛醇合成机制的有希望的修饰。第一步，四氢叶酸的N5对丝氨酸的C3进行亲核攻击，破坏C2-C3键，形成N5-羟甲基-四氢叶酸和酶结合的甘氨酸阴离子。可能会暂时形成甲醛作为中间体，但不是必需的。该机制解释了在叶酸存在下丝氨酸裂解的速率大大提高，并且避免了亲核置换机制所带来的一些严重困难，其中亲核置换机制涉及C3-OH键的断裂。

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