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Biochemical and spectroscopic characterization of the catalytic domain of MMP16 (cdMMP16)

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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, 1H NMR, and EXAFS spectroscopies. A well-characterized cdMMP16 is now available for future inhibitor screening efforts.

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Abbreviations

DNP:

2,4-Dinitrophenyl

EDTA:

Ethylenediaminetetraacetic acid

EPR:

Electron paramagnetic resonance

EXAFS:

Extended X-ray absorption fine structure

Hepes:

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

IPTG:

Isopropyl-β-d-thiogalactoside

MALDI-TOF:

Matrix-assisted laser desorption/ionization-time of flight

MCA:

(7-Methoxycoumarin-4-yl) acetic acid

MMP:

Matrix metalloproteinases

MT-MMP:

Membrane-type matrix metalloproteiniases

NMR:

Nuclear magnetic resonance

OP:

Phenanthroline

SDS:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Tris:

Tris(hydroxymethyl)aminomethane

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Acknowledgments

This work was supported by the National Institutes of Health (P30-EB-009998 to the Center for Synchrotron Biosciences from the NIBIB, which supports beamline X3B at the NSLS) and the National Science Foundation (CHE-1151658 to MWC and DLT). The authors thank Theresa Ramelot for assistance in obtaining 1H NMR spectra on the enzymes.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Miami University, Oxford, OH, 45056, USA

    Fan Meng, Hao Yang, Mahesh Aitha, Sam George, David L. Tierney & Michael W. Crowder

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  1. Fan Meng
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  2. Hao Yang
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  3. Mahesh Aitha
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  4. Sam George
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  5. David L. Tierney
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Correspondence to David L. Tierney or Michael W. Crowder.

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Meng, F., Yang, H., Aitha, M. et al. Biochemical and spectroscopic characterization of the catalytic domain of MMP16 (cdMMP16). J Biol Inorg Chem 21, 523–535 (2016). https://doi.org/10.1007/s00775-016-1362-y

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