Title
Biochemical characterization and zinc binding group (ZBGs) inhibition studies on the catalytic domain of MMP7 (cdMMP7)
Author
Fan Meng, Hao Yang, Colin Jack, Huaqun Zhang, Abraham Moller, Devin Spivey, Richard C. Page, David L. Tierney, Michael W. Crowder
Year
2016
Journal
Journal of Inorganic Biochemistry
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 1.8 eq of Zn(II), exhibit steady-state kinetic constants of 0.4 s− 1 for kcatand 23 μM 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 IC50 values and lower Kd values 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.
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, Biochemistry