Substitution of a lysyl residue for arginine 386 of Escherichia coli aspartate aminotransferase.
Substitution of a lysyl residue for arginine 386 of Escherichia coli aspartate aminotransferase.
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用赖氨酰残基取代大肠杆菌天冬氨酸转氨酶的精氨酸 386。
DOI：
10.1016/s0021-9258(18)60583-6
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发表时间：
1989
期刊：
影响因子：
--
通讯作者：
Yoshimasa Morinon
中科院分区：
文献类型：
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作者：
Y. Inoue;S. Kuramitsu;Katsura Inoue;Hiroyuki KagamiyamaS;Keitaro Hiromie;Sumio Tanasen;Yoshimasa Morinon
与野生型相比，赖氨酸残基取代谷草埃氏菌转氨酶的Arg-386导致invmax值大幅降低（与天门冬氨酸-2-氧葡萄糖酸对相比，天门冬氨酸-2-氧葡萄糖酸对相比，invmax值降低了0.8%，与谷氨酸-草酸对相比，invmax值降低了0.2%）。四组半反应的动力学分析，吡哆醛形式的酶与天冬氨酸或谷氨酸和吡哆胺形式与2-氧戊二酸或草酸酯，使我们能够确定突变对每种底物反应性的独立影响。五碳底物（谷氨酸和2-氧葡萄糖酸酯）反应的一级速率常数（kmax）比四碳底物（天冬氨酸和草酸酯）反应的一级速率常数（kmax）下降更明显，而四碳底物的表观解离常数（Kd）的增加比五碳底物的大。从kmax/Kd值判断，五碳底物反应的总催化效率下降比四碳底物反应更明显。对底物类似物如琥珀酸盐、戊二酸盐、2-甲基天冬氨酸和红-3-羟基天冬氨酸的亲和力也因酶的突变而显著降低。这些发现表明，赖氨酸残基的侧链虽然与精氨酸残基带类似的正电荷，但在结构上不适合在催化过程中与底物的有效结合。
Substitution of a lysyl residue for Arg-386 ofEscherichia coliaspartate aminotransferase resulted in an extensive decrease inVmaxvalues (0.8% with the aspartate-2-oxoglutarate pair and 0.2% with the glutamate-oxalacetate pair, compared with the corresponding values for the wild-type enzyme). Kinetic analysis of the four sets of half-reactions, the pyridoxal form of the enzyme with aspartate or glutamate and the pyridoxamine form with 2-oxoglutarate or oxalacetate, allowed us to define the independent effect of the mutation on the reactivity of each substrate. Decrease in the first order rate constant (kmax) was more pronounced in the reactions with five-carbon substrates (glutamate and 2-oxoglutarate) than in those with four-carbon substrates (aspartate and oxalacetate), while the increase in the apparent dissociation constant (Kd) was greater for four-carbon substrates than for five-carbon substrates. The decrease of overall catalytic efficiency as judged by the values,kmax/Kd, was more pronounced in the reactions with five-carbon substrates than in those with four-carbon substrates. Affinities for substrate analogs such as succinate, glutarate, 2-methylaspartate, and erythro-3-hydroxyaspartate, were also considerably decreased by the mutation of the enzyme. These findings indicate that the side chain of the lysyl residue, although it bears a positive charge similar to that of the arginyl residue, is not structurally adequate for the productive binding of a substrate during catalysis.