Kinetic isotope effects of peptidylglycine α-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-C-14]Val-Gly, D-Tyr-[3,4-H-3]Val-[2,2-H-2(2)]-Gly, and D-Tyr-Val-(R,S)[2-H-3]Gly as substrates, and we determined the deuterium and tritium effects on V-max/K-m as 1.625+/-0.041 (mean+/-S.D.) and 2.71+/-0.16 (mean+/-S.D.), respectively. The intrinsic deuterium isotope effect ((D)k) 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 V-max (V-D) was approximately unity, although the deuterium effect on V-max/K-m (V-D/K) was comparable to that obtained by the competitive approach. These results indicated that V-D was completely masked by the presence of one or more steps much slower than the glycine hydroxylation step and that V-D/K was diminished from Dk by a large forward commitment to catalysis. The addition of PAL, however, increased the apparent V-D 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 (D)k 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].