PUB - Publikationen an der Universität Bielefeld

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-NH2, 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 1.46 x 10(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 0.87 x 10(4) M-1 . s(-1) for DeltaTM-MT1-MMP based on the value for cdMT1-MMP of 5.12 x 10(4) M-1. s(-1); however, the actual value was determined to be 2.5-fold higher, i.e. 2.18 x 10(4) M-1. s(-1). These results suggest that cdMT1-MMP is catalytically more efficient towards small peptide substrates than ATM-MT1-MMP and the haernopexin 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 haernopexin domain does not significantly modify the enzyme active-site structure.