Serine hydroxymethyltransferase: Role of Glu75 and evidence that serine is cleaved by a retroaldol mechanism
Serine hydroxymethyltransferase: Role of Glu75 and evidence that serine is cleaved by a retroaldol mechanism
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DOI：
10.1021/bi049791y
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发表时间：
2004-06-08
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：
Szebenyi, DME;Musayev, FN;Schirch, V
丝氨酸羟甲基转移酶（SHMT）以四氢叶酸为一碳载体，催化丝氨酸和甘氨酸的可逆相互转化。SHMT还催化别苏氨酸和3-苯基丝氨酸的叶酸非依赖性逆醛醇裂解以及5，10-亚甲基四氢叶酸向5-甲酰基四氢叶酸的不可逆转化。SHMT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-羟基氨基酸裂解为甘氨酸和醛通过逆醛醇机理发生。然而，丝氨酸的叶酸依赖性裂解可以通过与甲醛作为酶结合的中间体的相同的逆醛醇机制或通过亲核置换机制来描述，其中四氢叶酸的N5置换丝氨酸的C3羟基，形成共价中间体。Glu 75的SHMT是明确参与的反应机制，它是在氢键的丝氨酸的羟基基团和5-甲酰四氢叶酸在这些物种与SHMT的复合物的甲酰基基团的距离。该残基被改变为Leu和Gln，并确定了位点突变体的结构、动力学和光谱性质。无论是突变显着改变了SHMT的结构，其复合物的光谱特性，或retroaldol切割别苏氨酸和3-苯基丝氨酸的动力学。然而，这两个突变阻断了叶酸依赖性丝氨酸-甘氨酸反应和亚甲基四氢叶酸转化为5-甲酰四氢叶酸。这些结果清楚地表明Glu 75与叶酸的相互作用是由SHMT催化的叶酸依赖性反应所必需的。此外，我们现在可以提出一个有前途的修改后醛醇丝氨酸裂解机制。作为第一步，四氢叶酸的N5对丝氨酸的C3进行亲核攻击，破坏C2-C3键以形成N5-羟亚甲基-四氢叶酸和酶结合的甘氨酸阴离子。作为中间体的甲醛的瞬时形成是可能的，但不是必需的。这一机制解释了在叶酸存在下丝氨酸裂解速率大大提高的原因，并避免了涉及C3-OH键断裂的亲核置换机制所带来的一些严重困难。
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 Gln, 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.