Vern L. Schramm Department of Biochemistry, Albert Einstein College of Medicine, Bronx NY, 10461 vern@aecom.yu.edu Computational chemistry and enzyme-based chemical clocks indicated that local catalytic site motions on the fsec time scale modulate transition state (TS) formation in human purine nucleoside phosphorylase (PNP). Fsec protein motions in PNP were modulated by synthesis of human PNP with replacement of H, C and N with 2H, 13C and 15N to generate a vibrationally altered enzyme. Heavy PNP showed changes only in the on-enzyme chemistry rate when analyzed by steady-state kinetics, TS analysis and pre-steady-state kinetics.  Transition path sampling suggested that perturbation of local modes decreased the coordinated local fsec motions to locate the PNP TS. Similar results were found with HIV-1 protease, but indications of slower motions were also recognized. Are all enzyme barrier-crossing events altered by heavy enzymes? No. Earlier computational chemistry studies on dihydrofolate reductase (DHFR) suggested no contributions of fsec protein motions to catalysis. Heavy DHFR showed no change in on-enzyme chemistry. Heavy proteins offer a fundamental new tool for investigation of protein motion in catalysis.

Protein dynamics, transition state structure and barrier crossing

247th National Meeting (2014)

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