Catalytic- and ecto-domains of membrane type 1-matrix metalloproteinase have similar inhibition profiles but distinct endopeptidase activities.
Membrane type 1-matrix metalloproteinase (MT1-MMP/MMP-14) is a major collagenolytic enzyme that plays a vital role in development and morphogenesis. To elucidate further the structure-function relationship between the human MT1-MMP active site and the influence of the haemopexin domain on catalysis, substrate specificity and inhibition kinetics of the cdMT1-MMP (catalytic domain of MT1-MMP) and the ecto domain DeltaTM-MT1-MMP (transmembrane-domain-deleted MT1-MMP) were compared. For substrate 1 [Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH(2), where Mca stands for (7-methoxycoumarin-4-yl)acetyl- and Dpa for N -3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl], the activation energy E (a) was determined to be 11.2 and 12.2 kcal/mol (1 cal=4.184 J) for cdMT1-MMP and DeltaTM-MT1-MMP respectively, which is consistent with k (cat)/ K (M) values of 7.37 and 1.46x10(4) M(-1).s(-1). The k (cat)/ K (M) values for a series of similar single-stranded peptide substrates were determined and found to correlate with a slope of 0.17 for the two enzyme forms. A triple-helical peptide substrate was predicted to have a k (cat)/ K (M) of 0.87x10(4) M(-1).s(-1) for DeltaTM-MT1-MMP based on the value for cdMT1-MMP of 5.12x10(4) M(-1).s(-1); however, the actual value was determined to be 2.5-fold higher, i.e. 2.18x10(4) M(-1).s(-1). These results suggest that cdMT1-MMP is catalytically more efficient towards small peptide substrates than DeltaTM-MT1-MMP and the haemopexin domain of MT1-MMP facilitates the hydrolysis of triple-helical substrates. Diastereomeric inhibitor pairs were utilized to probe further binding similarities at the active site. Ratios of K (i) values for the inhibitor pairs were found to correlate between the enzyme forms with a slope of 1.03, suggesting that the haemopexin domain does not significantly modify the enzyme active-site structure.
Hurst
Douglas R
DR
Department of Chemistry and Biochemistry and Institute of Molecular Biophysics, 203 Dittmer Laboratory of Chemistry Building, Florida State University, Tallahassee, FL 32306, U.S.A.
Schwartz
Martin A
MA
Ghaffari
Mohammad A
MA
Jin
Yonghao
Y
Tschesche
Harald
H
Fields
Gregg B
GB
Sang
Qing-Xiang Amy
QX
D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D020134:Catalytic Domain / Q000187:drug effects / Q000502:physiology; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000037:antagonists & inhibitors / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000502:physiology; D013379:Substrate Specificity / Q000187:drug effects / Q000502:physiology; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000494:pharmacology*
D010455:Peptides; D019715:Tissue Inhibitor of Metalloproteinase-1; D010450:Endopeptidases; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases