Inactivation of Met471Cys tyramine β-monooxygenase results from site-specific cysteic acid formation.
Tyramine β-monooxygenase (TβM), the insect homologue of dopamine β-monooxygenase, is a neuroregulatory enzyme that catalyzes the β-hydroxylation of tyramine to yield octopamine. Mutation of the methionine (Met) ligand to Cu(M) of TβM, Met471Cys, yielded a form of TβM that is catalytically active but susceptible to inactivation during turnover [Hess, C. R., Wu, Z., Ng, A., Gray, E. E., McGuirl, M. M., and Klinman, J. P. (2008) J. Am. Chem. Soc. 130, 11939-11944]. Further, although the wild-type (WT) enzyme undergoes coordination of Met471 to Cu(M) in its reduced form, the generation of Met471Cys almost completely eliminates this interaction [Hess, C. R., Klinman, J. P., and Blackburn, N. J. (2010) J. Biol. Inorg. Chem. 15, 1195-1207]. The aim of this study is to identify the chemical consequence of the poor ability of Cys to coordinate Cu(M). We show that Met471Cys TβM is ~5-fold more susceptible to inactivation than the WT enzyme in the presence of the cosubstrate/reductant ascorbate and that this process is not facilitated by the substrate tyramine. The resulting 50-fold smaller ratio for turnover to inactivation in the case of Met471Cys prevents full turnover of the substrate under all conditions examined. Liquid chromatography-tandem mass spectrometry analysis of proteolytic digests of inactivated Met471Cys TβM leads to the identification of cysteic acid at position 471. While both Met and Cys side chains are expected to be similarly subject to oxidative damage in proteins, the enhanced reactivity of Met471Cys toward solution oxidants in TβM is attributed to its weaker interaction with Cu(I)(M).
Department of Chemistry, University of California, Berkeley, CA 94720, USA.
Robert L
RL
Osborne
Hui
H
Zhu
Anthony T
AT
Iavarone
Corinna R
CR
Hess
Judith P
JP
Klinman
D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003544:Cysteic Acid / Q000138:chemical synthesis*; D003545:Cysteine / Q000737:chemistry*; D004330:Drosophila; D029721:Drosophila Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D006900:Hydroxylation; D007700:Kinetics; D008715:Methionine / Q000737:chemistry*; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D009154:Mutation; D059748:Proteolysis; D053719:Tandem Mass Spectrometry
D029721:Drosophila Proteins; D003544:Cysteic Acid; D008715:Methionine; D006899:Mixed Function Oxygenases; C527639:tyramine beta-monooxygenase, Drosophila; D003545:Cysteine