Mechanism of the Insect Enzyme, Tyramine β-Monooxygenase, Reveals Differences from the Mammalian Enzyme, Dopamine β-Monooxygenase

Corinna R Hess,Michele M Mcguirl,Judith P Klinman

doi:10.1074/jbc.m705911200

Abstract

Tyramine beta-monooxygenase (TbetaM) catalyzes the synthesis of the neurotransmitter, octopamine, in insects. Kinetic and isotope effect studies have been carried out to determine the kinetic mechanism of TbetaM for comparison with the homologous mammalian enzymes, dopamine beta-monooxygenase and peptidylglycine alpha-hydroxylating monooxygenase. A new and distinctive feature of TbetaM is very strong substrate inhibition that is dependent on the level of the co-substrate, O(2), and reductant as well as substrate deuteration. This has led to a model in which tyramine can bind to either the Cu(I) or Cu(II) forms of TbetaM, with substrate inhibition ameliorated at very high ascorbate levels. The rate of ascorbate reduction of the E-Cu(II) form of TbetaM is also reduced at high tyramine, leading us to propose the existence of a binding site for ascorbate to this class of enzymes. These findings may be relevant to the control of octopamine production in insect cells.

Highlights

Ascorbate is the anticipated in vivo reductant as reported for dopamine ␤-monooxygenase (D␤M) and peptidylglycine ␣-hydroxylating monooxygenase (PHM) [2, 19]
All kinetics of Tyramine ␤-monooxygenase (T␤M) were pursued using ascorbate as the reductant. This allows the direct comparison of kinetic data obtained with T␤M with values previously reported in the D␤M and PHM reactions
Reductive Mechanism and Tyramine Inhibition—Based on the present data, we describe a mechanism for T␤M, which exhibits several notable differences from the mechanisms established for the related mammalian enzymes, D␤M and PHM

Summary

Introduction

CuM serves as the site of dioxygen binding and activation, whereas the CuH site functions as an electron transfer site in the reaction mechanism [1, 14]. Loss of a water molecule at CuH accompanies reduction of the copper center, as for the CuM site. The ligands to both copper centers are fully conserved among all three enzymes. A mechanism for substrate hydroxylation by this class of enzymes has been proposed based on extensive kinetic characterization of D␤M and PHM (Scheme 2) [1]. A long range electron transfer from CuH to CuM is required to complete the reaction cycle, leading to formation of the hydroxylated product [17] and reoxidation of both copper centers

Results

Discussion

Conclusion