Tyramine ␤-monooxygenase (T␤M) catalyzes the synthesis of the neurotransmitter, octopamine, in insects. Kinetic and isotope effect studies have been carried out to determine the kinetic mechanism of T␤M for comparison with the homologous mammalian enzymes, dopamine ␤-monooxygenase and peptidylglycine ␣-hydroxylating monooxygenase. A new and distinctive feature of T␤M 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 T␤M, with substrate inhibition ameliorated at very high ascorbate levels. The rate of ascorbate reduction of the E-Cu(II) form of T␤M 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.The copper hydroxylases are a unique class of enzymes that are found in eukaryotes and play a critical role in the biosynthesis of neurotransmitters and hormones. The most studied enzymes in this family are peptidylglycine ␣-hydroxylating monooxygenase (PHM) 3 and dopamine ␤-monooxygenase (D␤M) (1). PHM catalyzes the conversion of C-terminal glycine-extended peptides to their ␣-hydroxylated products, the first step in the amidation of peptide hormones, required for a range of biological activities (2). D␤M catalyzes the hydroxylation of dopamine to yield norepinephrine and, thus, is vital for the regulation of these neurotransmitters (3-5). More recently, a third member of this enzyme family was identified, tyramine ␤-monooxygenase (6). T␤M is the insect homolog of D␤M, sharing 39% identity and 55% similarity with the mammalian enzyme. T␤M similarly catalyzes the hydroxylation of tyramine at the ␤-carbon position (Scheme 1). Although tyramine plays no role in mammalian physiology, the product of T␤M, octopamine, has been shown to act as a neurotransmitter in invertebrates, regulating physiological functions such as neuromuscular transmission, behavioral development, and ovulation (7-9). One advantage to studies with T␤M is the much more facile expression system for this enzyme in relation to D␤M (10), which makes it possible to pursue structure/function relationships. In this paper, we report a detailed analysis of the kinetic behavior of the wild type T␤M, an essential first step in understanding this complex enzyme system.All three of the copper hydroxylases employ two noncoupled, mononuclear Cu centers for oxygenase activity, termed Cu M and Cu H . Structural information pertaining to the active site of the enzymes is derived primarily from the crystal structure for PHM and extended X-ray absorption fine structure studies with both D␤M and PHM (11-13). Each copper site assumes a unique coordination environment and a distinct mechanistic function. Cu M serves as the site of dioxygen binding and activation, whereas the Cu H site functions as an electron transfer site in the reac...