Transition-state analysis of the DNA repair enzyme MutY.
The transition state (TS) structure of MutY-catalyzed DNA hydrolysis was solved using multiple kinetic isotope effect (KIE) measurements. MutY is a base excision repair enzyme which cleaves adenine from 8-oxo-G:A mismatches in vivo, and also from G:A mismatches in vitro. TS analysis of G:A-DNA hydrolysis revealed a stepwise S(N)1 (D(N)*A(N)(double dagger)) mechanism proceeding through a highly reactive oxacarbenium ion intermediate which would have a lifetime in solution of <10(-10) s. C-N bond cleavage is reversible; the N-glycoside bond breaks and reforms repeatedly before irreversible water attack on the oxacarbenium ion. KIEs demonstrated that MutY uses general acid catalysis by protonating N7. It enforces a 3'-exo sugar ring conformation and other sugar ring distortions to stabilize the oxacarbenium ion. Combining the experimental TS structure with the previously reported crystal structure of an abortive Michaelis complex elucidates the step-by-step catalytic sequence.
McCann
Joe A B
JA
Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main Street West, Hamilton, ON, L8S 4M1, Canada.
Berti
Paul J
PJ
D002244:Carbon / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D018360:Crystallography, X-Ray; D004247:DNA / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D045647:DNA Glycosylases / Q000032:analysis / Q000378:metabolism*; D004260:DNA Repair; D006027:Glycosides / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D007554:Isotopes / Q000737:chemistry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009584:Nitrogen / Q000737:chemistry / Q000378:metabolism; D011522:Protons; D012997:Solvents / Q000737:chemistry
D006027:Glycosides; D007554:Isotopes; D011522:Protons; D012997:Solvents; D002244:Carbon; D004247:DNA; D045647:DNA Glycosylases; C061738:mutY adenine glycosylase; D009584:Nitrogen