Document Detail


Interdomain long-range electron transfer becomes rate-limiting in the Y216A variant of tyramine β-monooxygenase.
MedLine Citation:
PMID:  23320946     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The enzyme tyramine β-monooxygenase (TβM) belongs to a small eukaryotic family of physiologically important mononuclear dicopper monooxygenases. The properties of this family include noncoupled mononuclear copper centers ~11 Å apart, with Cu(M) performing C-H and O(2) activation and Cu(H) functioning as an electron storage site [Klinman, J. P. (2006) J. Biol. Chem. 281, 3013-3016]. A conserved tyrosine (Y216 in TβM) is positioned between the copper domains and is associated with Cu(H) (through an interaction with a Cu(H)-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 Cu(M) 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 Cu(M). Steady-state kinetic and isotope effect measurements were investigated. The significantly elevated K(m,Tyr) for Y216A, together with a very large (D)(k(cat)/K(m,Tyr)) of ~12, indicates a major impact on the binding of substrate at the Cu(M) site. The kinetic and isotopic parameters lead to estimated rate constants for C-H bond cleavage, dissociation of substrate from the Cu(M) site, and, in the case of Y216A, the rate of electron transfer (ET) from Cu(H) to Cu(M). 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.
Authors:
Robert L Osborne; Hui Zhu; Anthony T Iavarone; Ninian J Blackburn; Judith P Klinman
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2013-02-06
Journal Detail:
Title:  Biochemistry     Volume:  52     ISSN:  1520-4995     ISO Abbreviation:  Biochemistry     Publication Date:  2013 Feb 
Date Detail:
Created Date:  2013-02-19     Completed Date:  2013-04-26     Revised Date:  2014-03-19    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1179-91     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Alanine
Animals
Catalytic Domain
Copper / metabolism*
Drosophila Proteins / chemistry*,  genetics*,  metabolism*
Electron Spin Resonance Spectroscopy
Electron Transport
Enzyme Activation
Hydrophobic and Hydrophilic Interactions
Kinetics
Mixed Function Oxygenases / chemistry*,  genetics*,  metabolism*
Mutation
Octopamine / chemistry,  metabolism
Oxidation-Reduction
Tyramine / metabolism
X-Ray Absorption Spectroscopy
Grant Support
ID/Acronym/Agency:
1S10RR022393-01/RR/NCRR NIH HHS; GM025765/GM/NIGMS NIH HHS; GM082095/GM/NIGMS NIH HHS; NS027583/NS/NINDS NIH HHS; R01 GM025765/GM/NIGMS NIH HHS; R01 NS027583/NS/NINDS NIH HHS
Chemical
Reg. No./Substance:
0/Drosophila Proteins; 14O50WS8JD/Octopamine; 789U1901C5/Copper; EC 1.-/Mixed Function Oxygenases; EC 1.14.99.-/tyramine beta-monooxygenase, Drosophila; OF5P57N2ZX/Alanine; X8ZC7V0OX3/Tyramine
Comments/Corrections

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