Carbon-13 and deuterium isotope effects on the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase

Carbon-13 and deuterium isotope effects have been measured on the reaction catalyzed by rabbit muscle glyceraldehyde-3-phosphate dehydrogenase in an effort to locate the rate-limiting steps. With D-glyceraldehyde 3-phosphate as substrate, hydride transfer is a major, but not the only, slow step prior to release of the first product, and the intrinsic primary deuterium and 13C isotope effects on this step are 5-5.5 and 1.034-1.040, and the sum of the commitments to catalysis is approximately 3. The 13C isotope effects on thiohemiacetal formation and thioester phosphorolysis are 1.005 or less. The intrinsic alpha-secondary deuterium isotope effect at C-4 of the nicotinamide ring of NAD is approximately 1.4; this large normal value (the equilibrium isotope effect is 0.89) shows tight coupling of hydrogen motions in the transition state accompanied by tunneling. With D-glyceraldehyde as substrate, the isotope effects are similar, but the sum of commitments is approximately 1.5, so that hydride transfer is more, but still not solely, rate limiting for this slow substrate. The observed 13C and deuterium equilibrium isotope effects on the overall reaction from the hydrated aldehyde are 0.995 and 1.145, while the 13C equilibrium isotope effect for conversion of a thiohemiacetal to a thioester is 0.994, and that for conversion of a thioester to an acyl phosphate is 0.997. Somewhat uncertain values for the 13C equilibrium isotope effects on aldehyde dehydration and formation of a thiohemiacetal are 1.003 and 1.004.