Role of copper and catalytic mechanism in the copper monooxygenase, dopamine beta-hydroxylase (D beta H).
Although classically characterized as a Type II copper protein, recent work has shown that D beta H requires two coppers per subunit for optimal activity. A major challenge has been to elucidate the relationship of these copper sites to one another, specifically whether there exists a single binuclear copper site or distinct metal sites catalyzing separate functions. In this paper, copper sites have been investigated by EPR techniques and rapid mixing methods to measure the rates and stoichiometries of copper reduction by ascorbate and subsequent reoxidation by tyramine. As shown, there is little or no evidence by EPR for spin coupling between metal sites in resting enzyme. Both coppers per subunit undergo reduction and reoxidation with single exponential values of 185 s-1 and 80 s-1, respectively. Significantly, the ratio of copper reoxidation to product formation in a single turnover is close to 2:1, implicating both coppers in the catalytic mechanism and ruling out any significant spin coupling in the enzyme-product complex. In contrast to single turnover experiments, rapid kinetics in the presence of excess ascorbate reveal a very low level of E-Cu (II). This result, which appears to conflict with evidence that the dominant enzyme form in the steady state is E-P, leads us to propose a reduction of enzyme at the level of E-P rather than free enzyme. Taken together with steady state kinetic parameters using alternate reductants, the data support separate binding sites for reductants and product/substrate and hence, separate functions for each copper per subunit. A detailed catalytic mechanism for D beta H is discussed in the context of a single metal site catalyzing dopamine hydroxylation.
Klinman
J P
JP
Department of Chemistry, University of California, Berkeley 94720.
Brenner
M
M
D001665:Binding Sites; D003300:Copper / Q000378:metabolism*; D004299:Dopamine beta-Hydroxylase / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D046911:Macromolecular Substances; D008667:Metalloproteins / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding
D046911:Macromolecular Substances; D008667:Metalloproteins; D003300:Copper; D004299:Dopamine beta-Hydroxylase