| Abstract
| - Peptidylglycine α-hydroxylating monooxygenase (PHM) and dopamine β-monooxygenase(DβM) are homologous copper-containing enzymes that catalyze an oxygen-dependent hydroxylation ofpeptide-extended glycine residues and phenethylamines, respectively. The mechanism whereby theseenzymes activate molecular oxygen and the C−H bond of substrate has been the subject of numerousstudies, and various mechanisms have been put forth. From the magnitude of 18O isotope effects as afunction of substrate structure in DβM, an active site tyrosine had been proposed to function in the reductiveactivation of Cu(II)−OOH to generate a reactive copper−oxo species [Tian et al. (1994) Biochemistry33, 226]. The presence of a tyrosine residue, Y318, in the active site of PHM was subsequently confirmedfrom crystallographic studies [Prigge et al. (1997) Science278, 1300]. We now report extensive kineticand isotope effect studies on the Y318F mutant form of PHM, analyzing the role of this tyrosine in thecatalytic mechanism. It is found that the Y318F mutant has intrinsic hydrogen and 18O isotope effectsthat are within experimental error of the wild-type enzyme and that the mutation causes only a slightreduction in the rate constant for C−H bond cleavage. These findings, together with the recent demonstrationthat C−H activation in PHM is dominated by quantum mechanical tunneling [Francisco et al. (2002) J.Am. Chem. Soc. 124, 8194], necessitate a reexamination of plausible mechanisms for this unique class ofcopper enzymes.
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