Kinetic isotope effects of peptidylglycine alpha-hydroxylating mono-oxygenase reaction.

Many bioactive polypeptides or neuropeptides possess a C-terminal alpha-amide group as a critical determinant for their optimal bioactivities. The amide functions are introduced by the sequential actions of peptidylglycine alpha-hydroxylating mono-oxygenase (PHM; EC 1.14.17.3) and peptidylamidoglycollate lyase (PAL; EC 4.3.2.5) from their glycine-extended precursors. In the present study we examined the kinetic isotope effects of the frog PHM reaction by competitive and non-competitive approaches. In the competitive approach we employed the double-label tracer method with D-Tyr-[U-14C]Val-Gly, D-Tyr-[3,4-3H]Val-[2,2-2H2]-Gly, and D-Tyr-Val-(R,S)[2-3H]Gly as substrates, and we determined the deuterium and tritium effects on Vmax/Km as 1.625+/-0.041 (mean+/-S. D.) and 2.71+/-0.16 (mean+/-S.D.), respectively. The intrinsic deuterium isotope effect (Dk) on the glycine hydroxylation reaction was estimated to be 6.5-10.0 (mean 8.1) by the method of Northrop [Northrop (1975) Biochemistry 14, 2644-2651]. In the non-competitive approach with N,N-dimethyl-1,4-phenylenediamine as a reductant, however, the deuterium effect on Vmax (DV) was approximately unity, although the deuterium effect on Vmax/Km (DV/K) was comparable to that obtained by the competitive approach. These results indicated that DV was completely masked by the presence of one or more steps much slower than the glycine hydroxylation step and that DV/K was diminished from Dk by a large forward commitment to catalysis. The addition of PAL, however, increased the apparent DV from 1.0 to 1.2, implying that the product release step was greatly accelerated by PAL. These results suggest that the product release is rate-limiting in the overall PHM reaction. The large Dk indicated that the glycine hydroxylation catalysed by PHM might proceed in a stepwise mechanism similar to that proposed for the dopamine beta-hydroxylase reaction [Miller and Klinman (1983) Biochemistry 22, 3091-3096].