1995 Fiscal Year Final Research Report Summary

The reaction mechanism and base recognition of RNase Rh

Research Project

Project/Area Number	06672154
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	Physical pharmacy
Research Institution	Showa University
Principal Investigator	NAKAMURA Kazuo Showa University, Professor, 薬学部, 教授 (00012675)
Project Period (FY)	1994 – 1995
Keywords	Ribonuclease / X-ray structure analysis / Enzyme-substrate complex
Research Abstract	During the present study we have determined seven crystal structures of RNase RNAP-Rh, a derivative of RNase Rh, and its mutant enzymes (RNAP-Rh, RNAP-Rh+2'-AMP complex, RNAP-Rh+2'-GMP complex, Y57W, Y57W+2'-AMP complex, Y57W+3'-AMP complex and Y57W+2'-GMP complex) . Diffraction data of the crystals were collected using an oscillation camera or a Weissenberg camera. The crystal structures were determined based on the atomic coordinates of RNase Rh, and refined usingthe program X-PLOR.The refinements were done successfully, and all the R-values were coverged to under 0.20. (1) We found out that the base recognition site of this enzyme consists of Trp49, Tyr57 and Asp51. The base of substrate is sandwiched in between the two former residues, and hydrogen-boned to the side chain of Asp51. (2) This enzyme is an adenine preferential RNase, but we could introduce 2'-GMP into the crystals of RNase RNAP-Rh and Y57W by soaking technique. The binding mode of guanine base is similar to that of adenine, but there are some differences between them.