Gorshkova I I, Datsiĩ I I, Lavrik O I, Nevinskiĩ G A
Biokhimiia. 1981 Apr;46(4):699-707.
The effect of modification of Phe-RSase from E. coli MRE-600 by pyridoxal-5'-phosphate and 2', 3'-dialdehyde derivative of ATP and L-phenylalanynyl-5'-adenylate obtained by periodate oxidation on the enzyme interaction with substrates was investigated. It was shown that modification of Phe-RSase by pyridoxal-5'-phosphate and 2', 3'-dialdehyde derivative of ATP leads to a decrease of the aminoacylation rate without changing the rate of the ATP-[32P]-pyrophosphate exchange reaction. The substrate analogs L-phenylalanynol and L-phenyl-alanynyladenylate increase the degree of Phe-RSase inactivation in the aminoacylation reaction. tRNAphe strongly protects the enzyme against inactivation. ATP, both in the absence (in case of modification with pyridoxal-5'-phosphate) and in- the presence of Mg2+ and phenylalanine (in case of modification with o-ATP) exhibits a pronounced protective effect. L-Phe does not protect the enzyme against the inactivation by pyridoxal-5'-phosphate or o-ATP. The dissociation constant of the Phe-RSase[14C]-Phe-tRNAphe complex increases 2.5 -- 5-fold after the enzyme modification by pyridoxal-5'-phosphate, while the Km value for tRNAphe decreases approximately two times in the aminoacylation reaction. There are no changes in the Km values for amino acid and ATP and the Hill coefficients for all substrates tested. Modification of Phe-RSase by pyridoxal-5'-phosphate leads to a decrease of stability of the aminoacyladenylate -- enzyme complex. Oxidized L-phenylalanynyladenylate does not produce enzyme inactivation either by aminoacylation or in the isotropic ATP-PP iota exchange reaction. It is assumed that Phe-RSase from E. coli MRE-600 contains some lysine residues essential for binding and aminoacylation of tRNA, which do not occur in the ATP-binding subsite and aminoacyladenylate formation center.
研究了用磷酸吡哆醛、ATP的2', 3'-二醛衍生物以及高碘酸盐氧化得到的L-苯丙氨酰-5'-腺苷酸对大肠杆菌MRE-600苯丙氨酰-tRNA合成酶（Phe-RSase）进行修饰后，该酶与底物相互作用的影响。结果表明，用磷酸吡哆醛和ATP的2', 3'-二醛衍生物修饰Phe-RSase会导致氨酰化速率降低，而ATP-[32P]-焦磷酸交换反应速率不变。底物类似物L-苯丙氨醇和L-苯丙氨酰腺苷酸会增加Phe-RSase在氨酰化反应中的失活程度。tRNAphe能强烈保护该酶不被失活。ATP在不存在（在用磷酸吡哆醛修饰的情况下）以及存在Mg2+和苯丙氨酸（在用o-ATP修饰的情况下）时都表现出明显的保护作用。L-苯丙氨酸不能保护该酶不被磷酸吡哆醛或o-ATP失活。用磷酸吡哆醛修饰Phe-RSase后，Phe-RSase-[14C]-Phe-tRNAphe复合物的解离常数增加2.5至5倍，而在氨酰化反应中tRNAphe的Km值降低约两倍。所测试的所有底物的氨基酸和ATP的Km值以及希尔系数均无变化。用磷酸吡哆醛修饰Phe-RSase会导致氨酰腺苷酸 - 酶复合物的稳定性降低。氧化的L-苯丙氨酰腺苷酸在氨酰化反应或各向同性的ATP-PPi交换反应中均不会使酶失活。据推测，大肠杆菌MRE-600的Phe-RSase含有一些对于tRNA的结合和氨酰化至关重要的赖氨酸残基，这些残基不存在于ATP结合亚位点和氨酰腺苷酸形成中心。
