Kinetic and Computational Investigations into the Origin of Metal‐Ion Specificity in Matrix Metalloproteinase‐1

Abstract

Matrix Metalloproteinases (MMPs) are a family of Zn‐dependent proteases responsible for cleaving peptide bonds. MMPs have essential roles in cell proliferation, differentiation, immune responses, among many others. Human MMPs have been implicated in a number of diseases including cancer and arthritis and as such have been the focus of studies related to their roles. We are interested in exploring the high specificity for the Zn2+ ion by MMP‐1 using kinetic assays and computational modeling. Human MMP‐1 contains a classical HEXXHXXGXXH Zn‐binding domain. Both the Zn2+ and second sphere Glu219 residue are involved in the first step of catalysis – deprotonating a Zn‐coordinated water. Interestingly, the Volvox species has a proteinase (VMP3) with a modified QEXXHXXGXXH metal‐binding domain that displays enhanced activity with Cu2+ and is much less active with Zn2+. In our study, a variety of metal‐substituted samples of MMP‐1 were prepared using dialysis to remove the Zn2+ ion. Kinetic assays using a FRET substrate demonstrate less than 5% activity in all cases when Cu2+ is substituted for Zn2+. To further explore these results, we developed a series of computational models of the MMP‐1 active site substituting either Zn2+ or Cu2+ into the active site. Density Functional Theory (DFT) as implemented in the ORCA program was used to develop these models, simulating the protein environment using either solvation models or by directly including second sphere residues as part of the quantum mechanics (QM) calculation. Models were validated by comparison to experimental results, including the kinetic data and spectroscopy data in the case of the Cu2+ models. Our work shows two effects at play. First, the first sphere geometry in MMP‐1 is primed to support a tetrahedral coordination environment, which is preferred by Zn2+ whereas Cu2+ prefers a square planar coordination environment. Second, the positioning of the Zn2+‐OH2 unit in the active site positions the water for deprotonation by Glu219. In the Cu2+ models, the distortion of the Cu2+‐OH2 unit due to the less stable coordination species increases the energy associated with proton transfer. This work provides a clear example of how first and second sphere active site residues are finely tuned to maximize enzyme activity.Support or Funding InformationWentworth Institute of Technology