Stathopoulos C, Jacquin-Becker C, Becker H D, Li T, Ambrogelly A, Longman R, Söll D
Department of Molecular Biophysics and Biochemistry, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8114, USA.
Biochemistry. 2001 Jan 9;40(1):46-52. doi: 10.1021/bi002108x.
The protein translation apparatus of Methanococcus jannaschii possesses the unusual enzyme prolyl-cysteinyl-tRNA synthetase (ProCysRS), a single enzyme that attaches two different amino acids, proline and cysteine, to their cognate tRNA species. Measurement of the ATP-PP(i) exchange reaction revealed that amino acid activation, the first reaction step, differs for the two amino acids. While Pro-AMP can be formed in the absence of tRNA, Cys-AMP synthesis is tRNA-dependent. Studies with purified tRNAs indicate that tRNA(Cys) promotes cysteine activation. The k(cat) values of wild-type ProCysRS for tRNA prolylation (0.09 s(-1)) and cysteinylation (0.02 s(-1)) demonstrate that both aminoacyl-tRNAs are synthesized with comparable rates, the cysteinyl-tRNA synthetase activity being only 4.5-fold lower than prolyl-tRNA synthetase activity. Kinetic analysis of ProCysRS mutant enzymes, generated by site-directed mutagenesis, shows glutamate at position 103 to be critical for proline binding, and proline at position 100 to be involved in cysteine binding. The proximity in ProCysRS of amino acid residues affecting binding of either cysteine or proline strongly suggests that structural elements of the two amino acid binding sites overlap.
詹氏甲烷球菌的蛋白质翻译装置拥有一种不同寻常的酶——脯氨酰 - 半胱氨酰 - tRNA合成酶（ProCysRS），这是一种能将两种不同氨基酸——脯氨酸和半胱氨酸，连接到其相应tRNA种类上的单一酶。对ATP-PP(i)交换反应的测定表明，氨基酸活化这一第一个反应步骤，对于这两种氨基酸而言是不同的。虽然在没有tRNA的情况下可以形成Pro-AMP，但Cys-AMP的合成是依赖于tRNA的。对纯化tRNA的研究表明，tRNA(Cys)促进半胱氨酸的活化。野生型ProCysRS对tRNA脯氨酰化（0.09 s(-1)）和半胱氨酰化（0.02 s(-1)）的k(cat)值表明，两种氨酰 - tRNA都是以相当的速率合成的，半胱氨酰 - tRNA合成酶活性仅比脯氨酰 - tRNA合成酶活性低4.5倍。通过定点诱变产生的ProCysRS突变酶的动力学分析表明，103位的谷氨酸对于脯氨酸结合至关重要，而100位的脯氨酸参与半胱氨酸结合。ProCysRS中影响半胱氨酸或脯氨酸结合的氨基酸残基的接近程度强烈表明，两个氨基酸结合位点的结构元件重叠。
