Zareen Neela, Hopkinson Angela, Levinger Louis
York College of The City University of New York, Jamaica, 11451, USA.
RNA. 2006 Jun;12(6):1104-15. doi: 10.1261/rna.4206. Epub 2006 Apr 17.
tRNase Z, which can endonucleolytically remove pre-tRNA 3'-end trailers, possesses the signature His domain (HxHxDH; Motif II) of the beta-lactamase family of metal-dependent hydrolases. Motif II combines with Motifs III-V on its carboxy side to coordinate two divalent metal ions, constituting the catalytic core. The PxKxRN loop and Motif I on the amino side of Motif II have been suggested to modulate tRNase Z activity, including the anti-determinant effect of CCA in mature tRNA. Ala walks through these two homology blocks reveal residues in which the substitutions unexpectedly reduce catalytic efficiency. While substitutions in Motif II can drastically affect k(cat) without affecting k(M), five- to 15-fold increases in k(M) are observed with substitutions in several conserved residues in the PxKxRN loop and Motif I. These increases in k(M) suggest a model for substrate binding. Expressed tRNase Z processes mature tRNA with CCA at the 3' end approximately 80 times less efficiently than a pre-tRNA possessing natural sequence of the 3'-end trailer, due to reduced k(cat) with no effect on k(M), showing the CCA anti-determinant to be a characteristic of this enzyme.
tRNase Z能够通过内切核酸酶作用去除前体tRNA的3'末端拖尾序列，它具有金属依赖性水解酶β-内酰胺酶家族的标志性His结构域（HxHxDH；基序II）。基序II与其羧基侧的基序III-V结合以配位两个二价金属离子，构成催化核心。有人提出，基序II氨基侧的PxKxRN环和基序I可调节tRNase Z的活性，包括成熟tRNA中CCA的反决定簇效应。对这两个同源结构域进行丙氨酸扫描，发现某些位点的取代意外地降低了催化效率。虽然基序II中的取代可显著影响催化常数（kcat）而不影响米氏常数（kM），但在PxKxRN环和基序I中的几个保守残基发生取代时，米氏常数增加了5至15倍。这些米氏常数的增加提示了一种底物结合模型。由于催化常数降低而对米氏常数无影响，表达的tRNase Z处理3'末端带有CCA的成熟tRNA的效率比具有3'末端拖尾天然序列的前体tRNA低约80倍，这表明CCA反决定簇是该酶的一个特征。
