Primary 14C and alpha secondary 3H substrate kinetic isotope effects for some phosphoribosyltransferases.

Abstract

Summary of kinetic isotope effects on reactions of PRibPP FIG. 5. The observed combined, x, and primary, +, kinetic isotope effects for spontaneous hydrolysis of PRibPP at different percentages of completion of reaction for the tritiated component. The computer tit of the observed pri- mary effects to Equation 1 is drawn at the bottom and gave k,2C/k,4C equal to 1.02 2 0.027. The computer tit of the observed combined effects to Equation 2 is drawn at the top and gave k iH, ,+/k3,,, ,+ equal to 1.39 ? 0.026. Reaction Percentage of completion of re- action Type effectd Observed isotope 0% reaction isotope Estimated deute- effect rium PRibPP % Rib-5-P % PRibPP ATP-PRTU PRibATP PRibATP ATEPRT” PRibPP 20 10 PRibPP HGPRTh IMP 27 5 PRibPP 5 OMP 24 24 a Adenosine triphosphate phosphoribosyltransferase. * Hypoxanthine (guanine) phosphoribosyltransferase. c Orotate phosphoribosyltransferase. ” C, combined effect; l”, primary effect; 2”, secondary effect, of reaction for the reverse direction ATP phosphoribosyltrans- ferase reaction, that is, for the conversion of doubly labeled PRibATP to PRibPP. No effects were observed (Table II). In the hypoxanthine (guanine) phosphoribosyltransferase reaction, a primary ‘Y! kinetic isotope effect of 1.05 and a combined effect of 1.24 corresponding to an (Y secondary “H effect of 1.30 were obtained (Table II). In the orotate phospho- ribosyltransferase reaction, a primary “C! effect of 1.03 and a combined effect of 1.14 corresponding to an 01 secondary “H effect of 1.17 were obtained (Table II). Thus, all three enzy- matic reactions studied here as well as the spontaneous hydrolysis of PRibPP exhibit significant cy secondary :‘H ki- netic isotope effects. 1.27 1.08 C 1.39 1” 1.02 2” 1.41 C 1.10 1” 1.01 2” 1.11 C 0.99 0.99 1” 1.00 1.00 2” 0.99 C 1.20 1.24 1” 1.05 1.05 2” 1.30 1.20 C 1.12 1.14 1” 1.03 1.03 2” 1.17 1.12 1.00