MECHANISM OF METAL-INDEPENDENT HYDROXYLATION BY CHROMOBACTERIUM-VIOLACEUM PHENYLALANINE-HYDROXYLASE

Phenylalanine hydroxylase converts phenylalanine to tyrosine utilizing a tetrahydrobiopterin cofactor. Several key mechanistic questions have yet to be resolved, specifically the identity of the hydroxylating species and the role of the non-heme iron which is present in all of the mammalian PAHs. Recently, we have demonstrated that a bacterial PAH from Chromobacterium violaceum does not require any redox active metal for activity [Carr, R. T., and Benkovic, S. J. (1993) Biochemistry 32, 14132-14138]. To identify the function of iron in the mammalian PAH's, we have undertaken a series of experiments to compare the mechanisms of this metal-independent PAH with the iron-dependent PAH from rat liver. Using [4-H-2]phenylalanine as a substrate gave a kinetic isotope effect on hydroxylation of unity for CVPAH which is in agreement with previous values reported for RLPAH. The [4-H-2]phenylalanine underwent an NIH shift upon hydroxylation by CVPAH. The extent of deuterium retention at the 3-position of the tyrosine product was identical within experimental error for both RLPAH and CVPAH using [4-H-2]phenylalanine and [2,3,5,6-H-2]phenylalanine as substrates. This suggests that PAH from either source probably does not directly mediate the NIH shift mechanism. No uncoupled pterin turnover was observed for CVPAH with either L-tyrosine or p-chloro-L-phenylalanine as substrate or tetrahydropterin as cofactor, each of which causes uncoupled turnover with RLPAH. CVPAH readily accepts 4-methylphenylalanine as a substrate giving 4-(hydroxymethyl)phenylalanine as the major product and 3-methyltyrosine as the only other minor product. The ratio of alkyl to aromatic hydroxylation is very close to the ratio previously obtained [Siegmund, H., and Kaufman, S. (1991) J. Biol. Chem. 266, 2903] for this analogue and two deuterated analogues. This suggests that both RLPAH and CVPAH utilize a very similar oxygenating intermediate. We also demonstrate that both metal-free and iron-dependent enzymes hydroxylate cyclohexylalanine in a stereoselective manner.