文摘
The enzyme tyramine 尾-monooxygenase (T尾M) belongs to a small eukaryotic family of physiologically important mononuclear dicopper monooxygenases. The properties of this family include noncoupled mononuclear copper centers 11 脜 apart, with CuM performing C鈥揌 and O2 activation and CuH functioning as an electron storage site [Klinman, J. P. (2006) J. Biol. Chem. 281, 3013鈥?016]. A conserved tyrosine (Y216 in T尾M) is positioned between the copper domains and is associated with CuH (through an interaction with a CuH-coordinating histidine). Mutations at Y216 (to W, I, and A) indicate little or no difference in electron paramagnetic resonance spectra, while X-ray absorption spectroscopy studies show only a very small decrease in distance between CuM and its Met471 ligand in reduced enzyme. High-performance liquid chromatography assays demonstrate that turnover of substrate is complete with Y216W and Y216I, whereas Y216A undergoes a secondary inactivation that is linked to oxidation of ligands at CuM. Steady-state kinetic and isotope effect measurements were investigated. The significantly elevated Km,Tyr for Y216A, together with a very large D(kcat/Km,Tyr) of 12, indicates a major impact on the binding of substrate at the CuM site. The kinetic and isotopic parameters lead to estimated rate constants for C鈥揌 bond cleavage, dissociation of substrate from the CuM site, and, in the case of Y216A, the rate of electron transfer (ET) from CuH to CuM. These studies uncover a rate-limiting ET within the solvent-filled interface and lead to a paradigm shift in our understanding of the mononuclear dicopper monooxygenases.