COMPARATIVE SEQUENCE SPECIFICITIES OF HUMAN 72-KDA AND 92-KDA GELATINASES (TYPE-IV COLLAGENASES) AND PUMP (MATRILYSIN)

The sequence specificities of human 72-kDa fibroblast gelatinase (type IV collagenase), human 92-kDa neutrophil gelatinase (type IV collagen ase), and putative metalloproteinase (PUMP or matrilysin) have been ex amined by measuring the rate of hydrolysis of over 50 synthetic oligop eptides covering the P4 through P4' subsites of the substrate. The pep tides investigated in this paper were those employed in our previous s tudy which systematically examined the sequence specificity of human f ibroblast and neutrophil collagenases [Netzel-Arnett et al. (1991) J. Biol. Chem. 266, 6747]. The initial rate of hydrolysis of the P1-P1' b ond of each peptide has been measured under first-order conditions ([S 0] << K(M)), and k(cat)/K(M) values have been calculated from the init ial rates. The specificities of these five metalloproteinases are simi lar, but distinct, with the largest differences occurring at subsites P1, P1', and P3'. The specificities of the two gelatinases are the mos t similar to each other. They tolerate only small amino acids such as Gly and Ala in subsite P1. In contrast, larger residues such as Met, P ro, Gln, and Glu are also accommodated well by PUMP. All five enzymes prefer hydrophobic, aliphatic residues in subsite P1'. PUMP exhibits a stronger preference for Leu in this subsite than is shown by the othe r enzymes. The P3' subsite specificities of the gelatinases and collag enases are very similar but different from those of PUMP which particu larly prefers Met in this position. The specificity data from this stu dy allow the design of optimized substrates and selective inhibitors f or these metalloproteinases.