title: Metal Ion Dependence of the Matrix Metalloproteinase-1 Mechanism.
abstract: Matrix metalloproteinase-1 (MMP-1) plays crucial roles in disease-related physiologies and pathological processes in the human body. We report here solution studies of MMP-1, including characterization of a series of mutants designed to bind metal in either the catalytic site or the structural site (but not both). Circular dichroism and fluorescence spectroscopy of the mutants demonstrate the importance of the structural Zn(II) in maintaining both secondary and tertiary structure, while UV-visible, nuclear magnetic resonance, electron paramagnetic resonance, and extended X-ray absorption fine structure show its presence influences the catalytic metal ion's coordination number. The mutants allow us to demonstrate convincingly the preparation of a mixed-metal analogue, Co(C)Zn(S)-MMP-1, with Zn(II) in the structural site and Co(II) in the catalytic site. Stopped-flow fluorescence of the native form, Zn(C)Zn(S)-MMP-1, and the mixed-metal Co(C)Zn(S)-MMP-1 analogue shows that the internal fluorescence of a nearby Trp residue is modulated with catalysis and can be used to monitor reactivity under a number of conditions, opening the door to substrate profiling.
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, United States.
authors.forename: Hao
authors.initials: H
authors.lastname: Yang
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, United States.
authors.forename: Katherine
authors.initials: K
authors.lastname: Makaroff
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, United States.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Paz
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, United States.
authors.forename: Mahesh
authors.initials: M
authors.lastname: Aitha
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, United States.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Crowder
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, United States.
authors.forename: David L
authors.initials: DL
authors.lastname: Tierney
mesh_terms: D001051:Apoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D055162:Biocatalysis; D020134:Catalytic Domain; D002942:Circular Dichroism; D003035:Cobalt / Q000378:metabolism*; D006801:Humans; D007501:Iron / Q000378:metabolism*; D020781:Matrix Metalloproteinase 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D015032:Zinc / Q000378:metabolism*
chemical_list: D001051:Apoenzymes; D050505:Mutant Proteins; D011994:Recombinant Proteins; D003035:Cobalt; D014364:Tryptophan; D007501:Iron; C522360:MMP1 protein, human; D020781:Matrix Metalloproteinase 1; D015032:Zinc