title: Biochemical characterization and zinc binding group (ZBGs) inhibition studies on the catalytic domain of MMP7 (cdMMP7).
abstract: Matrix metalloproteinase 7 (MMP7/matrilysin-1) has been implicated in many pathological conditions, such as in cancer and inflammatory diseases; therefore, MMP7 has been targeted for drugs. Success in developing a clinical inhibitor, which exhibits suitable specificity and selectivity, will likely require structural and/or kinetic evaluation of enzyme/inhibitor interactions. To enable these future studies we herein describe the over-expression, purification, and characterization of the catalytic domain of MMP7 (cdMMP7). cdMMP7 was over-expressed in an E. coli over-expression system, and the resulting enzyme was processed into inclusion bodies, which were subsequently solubilized, enabling the enzyme to be re-folded into a catalytically-active form. cdMMP7 was shown to bind 1.8eq of Zn(II), exhibit steady-state kinetic constants of 0.4s-1for kcatand 23μM for Km, and yield CD and fluorescence spectra that are consistent with a properly-folded enzyme. Pre-steady state kinetic studies yielded kinetic mechanisms of cdMMP7, and these mechanisms are similar to those of other MMPs. Inhibition studies on cdMMP7 with four zinc binding group (ZBG) inhibitors showed that maltol, thiomaltol, and allothiomaltol are better inhibitors with lower IC50values and lower Kdvalues against cdMMP7 and cdMMP16 than the commonly-used ZBG inhibitor acetohydroxamic acid. Docking studies suggest that improved inhibitory character may be due to interactions with the S1' substrate binding pocket. Finally, a ZnCo-heterobimetallic analog of cdMMP7 with Co(II) bound in the catalytic site was prepared and characterized. This study describes a well-characterized analog of MMP7 that is available for future inhibitor design efforts.
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA.
authors.forename: Fan
authors.initials: F
authors.lastname: Meng
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA.
authors.forename: Hao
authors.initials: H
authors.lastname: Yang
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA.
authors.forename: Colin
authors.initials: C
authors.lastname: Jack
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA.
authors.forename: Huaqun
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA.
authors.forename: Abraham
authors.initials: A
authors.lastname: Moller
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA.
authors.forename: Devin
authors.initials: D
authors.lastname: Spivey
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA. Electronic address: pagerc@miamioh.edu.
authors.forename: Richard C
authors.initials: RC
authors.lastname: Page
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA. Electronic address: tiernedl@miamioh.edu.
authors.forename: David L
authors.initials: DL
authors.lastname: Tierney
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA. Electronic address: crowdemw@miamioh.edu.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Crowder
mesh_terms: D001665:Binding Sites; D006801:Humans; D020783:Matrix Metalloproteinase 7 / Q000737:chemistry* / Q000235:genetics; D062105:Molecular Docking Simulation; D017510:Protein Folding; D011994:Recombinant Proteins; D015032:Zinc / Q000737:chemistry*
chemical_list: D011994:Recombinant Proteins; C060932:MMP7 protein, human; D020783:Matrix Metalloproteinase 7; D015032:Zinc
keywords: Cobalt(II)-substitution; Matrix metalloproteinases (MMPs); Pre-steady-state kinetic study; Zinc-binding group MMP inhibitors (ZBG-MMPis)