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Detection of Functional Matrix Metalloproteinases by Zymography
Published on: November 8, 2010
Biochemical and spectroscopic characterization of the catalytic domain of MMP16 (cdMMP16)
Fan Meng1, Hao Yang1, Mahesh Aitha1
1Department of Chemistry and Biochemistry, Miami University, Oxford, OH, 45056, USA.
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.
Insights
Matrix metalloproteinase 16 (MMP16) is a drug target for cancer and inflammation. This study characterizes the catalytic domain of MMP16, providing a well-defined enzyme for developing novel MMP inhibitors.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Matrix metalloproteinase 16 (MMP16), also known as MT3-MMP, is implicated in cancer and inflammation, making it a significant drug target.
- Developing selective MMP inhibitors (MMPi) requires thorough biochemical characterization of target enzymes like MMP16.
Purpose of the Study:
- To prepare and biochemically characterize the catalytic domain of MMP16 (cdMMP16).
- To provide a well-defined enzyme for future screening of MMP inhibitors.
Main Methods:
- A modified over-expression and purification protocol was employed to obtain recombinant cdMMP16.
- Steady-state kinetics were determined using FS-6 substrate.
- Co(II)-substituted analogs were prepared and analyzed using UV-Vis, 1H NMR, and EXAFS spectroscopies.
Main Results:
- The recombinant cdMMP16 enzyme exhibited kinetic constants of K m = 10.6 ± 0.7 μM and k cat = 1.14 ± 0.02 s(-1).
- The enzyme bound approximately 1.8 equivalents of Zn(II).
- Enzymatic activity was found to be salt concentration-dependent, and autoproteolytic activity was observed under specific conditions.
Conclusions:
- A well-characterized catalytic domain of MMP16 (cdMMP16) has been successfully prepared.
- The characterized cdMMP16 is suitable for future inhibitor screening efforts.
- Observed autoproteolysis may indicate an in vivo regulatory mechanism for MMP16 and other MT-MMPs.
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