Metal Ion Dependence of the Matrix Metalloproteinase-1 Mechanism

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• 作者：Hao Yang ; Katherine Makaroff ; Nicholas Paz ; Mahesh Aitha ; Michael W. Crowder ; David L. Tierney
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：June 16, 2015
• 年：2015
• 卷：54
• 期：23
• 页码：3631-3639
• 全文大小：396K
• ISSN：1520-4995

文摘

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鈥搗isible, nuclear magnetic resonance, electron paramagnetic resonance, and extended X-ray absorption fine structure show its presence influences the catalytic metal ion鈥檚 coordination number. The mutants allow us to demonstrate convincingly the preparation of a mixed-metal analogue, Co_CZn_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_CZn_S-MMP-1, and the mixed-metal Co_CZn_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.