Zhou L, Rosevear P R
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77030, USA.
Biochem Biophys Res Commun. 1995 Nov 13;216(2):648-54. doi: 10.1006/bbrc.1995.2671.
Escherichia coli isoleucyl-tRNA synthetase has been shown to contain two enzyme-bound zinc atoms per polypeptide chain. To investigate the structural and functional significance of the C-terminal enzyme-bound zinc, mutagenesis was used to alter Cys 922 to Ser [IleRS(C922S)] and to replace Cys 922 through Ala 939 with a 33 amino acid peptide unable to bind zinc (AIleRS). Both IleRS(C922S) and AIleRS were found to contain only a single enzyme-bound zinc per polypeptide chain. Substitution of Co2+ for Zn2+ in IleRS(C922S) gave a visible spectrum characteristic of that expected for a single tetrahedrally coordinated enzyme-bound Co2+ atom. Little or no effect on the Km values for ATP or Ile and only a 5 fold reduction of the (kcat/Km)Ile was observed for IleRS(C922S) and AIleRS in the isoleucine-dependent ATP-pyrophosphate exchange reaction. In the tRNA-dependent aminoacylation reaction, Km values for tRNA(Ile) were only slightly affected in the mutant proteins. However, kcat/Km values were decreased approximately 200 and 2500 fold for IleRS(C922S) and AIleRS, respectively. These results suggest that both the C-terminal enzyme-bound zinc and the C-terminal peptide play important roles in aminoacylation of tRNA(Ile).
已证明大肠杆菌异亮氨酰 - tRNA合成酶每条多肽链含有两个与酶结合的锌原子。为了研究C末端与酶结合的锌的结构和功能意义，采用诱变方法将半胱氨酸922突变为丝氨酸[异亮氨酰 - tRNA合成酶（C922S）]，并用一段无法结合锌的33个氨基酸的肽取代半胱氨酸922至丙氨酸939区域（AIleRS）。发现IleRS（C922S）和AIleRS每条多肽链仅含有一个与酶结合的锌。在IleRS（C922S）中用Co2 +替代Zn2 +产生了单个四面体配位的与酶结合的Co2 +原子预期的可见光谱特征。在异亮氨酸依赖性ATP - 焦磷酸交换反应中，对于IleRS（C922S）和AIleRS，观察到对ATP或异亮氨酸的Km值几乎没有影响，并且（kcat / Km）Ile仅降低了5倍。在tRNA依赖性氨酰化反应中，tRNA（Ile）的Km值在突变蛋白中仅受到轻微影响。然而，IleRS（C922S）和AIleRS的kcat / Km值分别降低了约200倍和2500倍。这些结果表明，C末端与酶结合的锌和C末端肽在tRNA（Ile）的氨酰化中都起重要作用。
