title: Biochemical and spectroscopic characterization of the catalytic domain of MMP16 (cdMMP16).
abstract: Membrane-bound matrix metalloproteinase 16 (MMP16/MT3-MMP) is considered a drug target due to its role(s) in disease processes such as cancer and inflammation. Biochemical characterization of MMP16 is critical for developing new generation MMP inhibitors (MMPi), which exhibit high efficacies and selectivities. Herein, a modified over-expression and purification protocol was used to prepare the catalytic domain of MMP16 (cdMMP16). The resulting recombinant enzyme exhibited steady-state kinetic constants of K m = 10.6 ± 0.7 μM and k cat = 1.14 ± 0.02 s(-1), when using FS-6 as substrate, and the enzyme bound 1.8 ± 0.1 eq of Zn(II). The enzymatic activity of cdMMP16 is salt concentration-dependent, and cdMMP16 exhibits autoproteolytic activity under certain conditions, which may be related to an in vivo regulatory mechanism of MMP16 and of other membrane-type MMPs (MT-MMPs). Co(II)-substituted analogs (Co2- and ZnCo) of cdMMP16 were prepared and characterized using several spectroscopic techniques, such as UV-Vis, (1)H NMR, and EXAFS spectroscopies. A well-characterized cdMMP16 is now available for future inhibitor screening 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: Mahesh
authors.initials: M
authors.lastname: Aitha
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, OH, 45056, USA.
authors.forename: Sam
authors.initials: S
authors.lastname: George
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, OH, 45056, USA. 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. crowdemw@miamioh.edu.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Crowder
mesh_terms: D055162:Biocatalysis; D006801:Humans; D053513:Matrix Metalloproteinase 16 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D013052:Spectrometry, X-Ray Emission; D013056:Spectrophotometry, Ultraviolet
chemical_list: C507102:MMP16 protein, human; D053513:Matrix Metalloproteinase 16
keywords: Autoproteolysis; Cobalt-substitution; Membrane-bound matrix metalloproteinases (MT-MMPs); Zinc