Abstract

Two related metallomonooxygenases, peptidylglycine alpha- amindating monooxygenase (PAM) and dopamine-beta-hydroxylase (DbetaM), are of particular neurological importance, since they catalyze final steps in the biosynthetic pathways leading to amidated neuropeptides and catecholamine neurotransmitters. This proposal is focused on investigating the catalytic role of copper and zinc, and the relationship between structure and function. Advanced spectroscopic techniques developed in the previous grant period have already led to a model for the active centers of DbetaM. Thee methods will now be applied to the study of wild-type PAM. Comparisons with DbetaM, whose coordination chemistry and spectroscopy are well- developed, will guide the experimental design. The availability of a high-yield expression system in mammalian CHO cell lines has made possible the preparation of a significant number of PAM site-specific mutants, all of which are secreted as viable proteins by the host cell. Kinetic, spectroscopic, and electrochemical studies on these mutants will be undertaken in order to obtain a better understanding of (1) the identity of the metal-binding side chains, (2) the coordination chemistry and catalytic role of the metal centers, and (3) the mechanistic role of non-coordinated active-site residues.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS027583-09
Application #
2460522
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Baughman, Robert W
Project Start
1989-08-01
Project End
2001-07-31
Budget Start
1997-08-01
Budget End
1998-07-31
Support Year
9
Fiscal Year
1997
Total Cost
Indirect Cost

  Institution

Name
Oregon Graduate Institute Science & Tech
Department
Chemistry
Type
Other Domestic Higher Education
DUNS #
City
Beaverton
State
OR
Country
United States
Zip Code
97006

  Publications

Chauhan, Shefali; Hosseinzadeh, Parisa; Lu, Yi et al. (2016) Stopped-Flow Studies of the Reduction of the Copper Centers Suggest a Bifurcated Electron Transfer Pathway in Peptidylglycine Monooxygenase. Biochemistry 55:2008-21
Park, Ga Young; Lee, Jung Yoon; Himes, Richard A et al. (2014) Copper-peptide complex structure and reactivity when found in conserved His-X(aa)-His sequences. J Am Chem Soc 136:12532-5
Chauhan, Shefali; Kline, Chelsey D; Mayfield, Mary et al. (2014) Binding of copper and silver to single-site variants of peptidylglycine monooxygenase reveals the structure and chemistry of the individual metal centers. Biochemistry 53:1069-80
Osborne, Robert L; Zhu, Hui; Iavarone, Anthony T et al. (2013) Interdomain long-range electron transfer becomes rate-limiting in the Y216A variant of tyramine ýý-monooxygenase. Biochemistry 52:1179-91
Kline, Chelsey D; Mayfield, Mary; Blackburn, Ninian J (2013) HHM motif at the CuH-site of peptidylglycine monooxygenase is a pH-dependent conformational switch. Biochemistry 52:2586-96
Otoikhian, Adenike; Barry, Amanda N; Mayfield, Mary et al. (2012) Lumenal loop M672-P707 of the Menkes protein (ATP7A) transfers copper to peptidylglycine monooxygenase. J Am Chem Soc 134:10458-68
Bauman, Andrew T; Broers, Brenda A; Kline, Chelsey D et al. (2011) A copper-methionine interaction controls the pH-dependent activation of peptidylglycine monooxygenase. Biochemistry 50:10819-28
Hess, Corinna R; Klinman, Judith P; Blackburn, Ninian J (2010) The copper centers of tyramine ?-monooxygenase and its catalytic-site methionine variants: an X-ray absorption study. J Biol Inorg Chem 15:1195-207
Himes, Richard A; Park, Ga Young; Siluvai, Gnana Sutha et al. (2008) Structural studies of copper(I) complexes of amyloid-beta peptide fragments: formation of two-coordinate bis(histidine) complexes. Angew Chem Int Ed Engl 47:9084-7
Himes, Richard A; Park, Ga Young; Barry, Amanda N et al. (2007) Synthesis and X-ray absorption spectroscopy structural studies of Cu(I) complexes of histidylhistidine peptides: the predominance of linear 2-coordinate geometry. J Am Chem Soc 129:5352-3

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