Ziak M, Jaussi R, Gehring H, Christen P
Biochemisches Institut, Universität Zürich, Switzerland.
Eur J Biochem. 1990 Jan 26;187(2):329-33. doi: 10.1111/j.1432-1033.1990.tb15309.x.
The active site residue lysine 258 of chicken mitochondrial aspartate aminotransferase was replaced with a histidine residue by means of site-directed mutagenesis. The mutant protein was expressed in Escherichia coli and purified to homogeneity. Addition of 2-oxoglutarate to its pyridoxamine form changed the coenzyme absorption spectrum (lambda max = 330 nm) to that of the pyridoxal form (lambda max = 330/392 nm). The rate of this half-reaction of transamination (kcat = 4.0 x 10(-4)s-1) is five orders of magnitude slower than that of the wild-type enzyme. However, the reverse half-reaction, initiated by addition of aspartate or glutamate to the pyridoxal form of the mutant enzyme, is only three orders of magnitude slower than that of the wild-type enzyme, kmax of the observable rate-limiting elementary step, i.e. the conversion of the external aldimine to the pyridoxamine form, being 7.0 x 10(-2)s-1. Aspartate aminotransferase (Lys258----His) thus represents a pyridoxal-5'-phosphate-dependent enzyme with significant catalytic competence without an active site lysine residue. Apparently, covalent binding of the coenzyme, i.e. the internal aldimine linkage, is not essential for the enzymic transamination reaction, and a histidine residue can to some extent substitute for lysine 258 which is assumed to act as proton donor/acceptor in the aldimine-ketimine tautomerization.
通过定点诱变将鸡线粒体天冬氨酸氨基转移酶的活性位点残基赖氨酸258替换为组氨酸残基。突变蛋白在大肠杆菌中表达并纯化至同质。向其吡哆胺形式中添加2-氧代戊二酸会使辅酶吸收光谱（最大吸收波长=330nm）变为吡哆醛形式的吸收光谱（最大吸收波长=330/392nm）。这种转氨半反应的速率（催化常数=4.0×10⁻⁴s⁻¹）比野生型酶慢五个数量级。然而，由向突变酶的吡哆醛形式中添加天冬氨酸或谷氨酸引发的反向半反应仅比野生型酶慢三个数量级，可观察到的限速基本步骤（即外部醛亚胺向吡哆胺形式的转化）的最大反应速率为7.0×10⁻²s⁻¹。因此，天冬氨酸氨基转移酶（赖氨酸258→组氨酸）代表一种依赖于磷酸吡哆醛的酶，在没有活性位点赖氨酸残基的情况下具有显著的催化能力。显然，辅酶的共价结合，即内部醛亚胺键，对于酶促转氨反应并非必不可少，并且组氨酸残基在一定程度上可以替代赖氨酸258，后者被认为在醛亚胺-酮亚胺互变异构中充当质子供体/受体。
