STRUCTURE (1.3 ANGSTROM) AND CHARGE STATES OF A RIBONUCLEASE-A URIDINE VANADATE COMPLEX - IMPLICATIONS FOR THE PHOSPHATE ESTER HYDROLYSIS MECHANISM

A joint X-ray crystallographic (1.3 Angstrom resolution) and ab initio quantum mechanical analysis of a uridine vanadate-ribonuclease A comp lex (W-RNase A) is undertaken to probe specific aspects of the microsc opic mechanism by which ribonuclease functions to catalyze the hydroly sis of its natural substrate, phosphate esters. Comparison of the stru ctural features of the vanadate portion from the final X-ray refinemen t with the oxy-vanadate model compounds determined computationally pro vides direct evidence of the likely protonation state of the UV inhibi tor bound in the active site. Specifically, the UV bound in the active site of UV-RNase A is found to be monoanionic, and the most likely so urce of this proton is from the active site residue His12. Together wi th the structural data, these results strongly suggest that even thoug h His12 may act as the catalytic base in the first step of ale mechani sm, transphosphorylation, and the catalytic acid in the second step, h ydrolysis, it must also play an additional, although perhaps secondary , role in stabilizing the pentacoordinate phosphorane structure throug h proton transfer. On the basis of its close proximity to critical van adate oxygen in the UV, and data obtained from a previous computationa l study, Lys41 is Likely to play a more intimate role in the catalytic mechanism than previously proposed, potentially acting as the catalyt ic base in certain cases. Two possible detailed microscopic mechanisms are presented.