Cleavage of bovine collagen I by neutrophil collagenase MMP-8 - Effect of pH on the catalytic properties as compared to synthetic substrates

The enzymatic processing of bovine collagen I by neutrophil collagenase (MM P-8) has been monitored at 37 degrees C, envisaging the occurrence of multi ple intermediate steps, following the initial cleavage, which leads to the formation of 1/4 and 3/4 fragments. Further, the first cleavage event has b een investigated at 37 degrees C as a function of pH, and catalytic paramet ers have been obtained through a global analysis of steady-state kinetic da ta, such as to get an overall consistent picture of k(cat)/K-m, k(cat), and K-m. These data have been compared with those obtained from the catalysis by MMP-8 of two synthetic fluorogenic substrates under the same experimenta l conditions. The overall behavior can be accounted for by the existence of five protonating groups, which vary to a different extent their pK(a) valu es for the three substrates investigated. The main observation concerns the fact the two of these residues, which play a relevant role in the enzymati c activity of MMP-8, are relatively far from the primary recognition site, and they are coming into action only for large macromolecular substrates, s uch as bovine collagen I. This finding opens the question of appropriate te sting for inhibitors of the enzymatic action of MMP-8, which must take into account, and also of these relevant interactions occurring only with natur al substrates.