Metalloproteinases are active in hormone processing, digestion and in many situations requiring remodeling of the extracellular matrix, .g. normal morphogenesis, wound healing and uterine resorption. Long bone formation, which depends on bone morphogenetic factors, e.g. BMP-1, also likely involves metalloproteinase action. We recently identified a zinc protease, astacin, by recognizing a potential catalytic zinc binding site signature sequence, HExxHxxGxxH. This signature differs from that seen in thermolysin and carboxypeptidase A and an x-ray structure of astacin now confirms our prediction that the 3 histidines are ligands to the zinc. The signature occurs in 60 different proteins which can be subdivided by homology into the astacin, snake venom, g-negative bacterium and matrix metalloproteinase (MMP) families. Recent x-ray crystallographic structures of a member of each of these sub-families also confirms the presence of this zinc binding site. The astacin family consists of a group of proteins that have a 200 amino acid domain that is homologous to astacin. These include human, mouse and Xenopus laevis BMP-1, the mouse and rat kidney and human intestinal brush border metalloendopeptidases, the protein encoded by the Drosophila dorsal-ventral patterning gene tolloid and the proteins encoded by one avian, two sea urchin and three fish embryogenesis genes. The smallest member of the MMP family, pump-1, contains the basic features for both latent and active enzyme forms of this family. It has the propeptide domain that contains a single Cys believed to be involved in zymogen activation and the central domain that contains the zinc signature. It has both the catalytic zinc binding site and a second, possible structural zinc site. Several reactive amino acids are conserved in both the astacin family and the MMP family. The present study addresses the chemical properties of two of the immediate family members, astacin and BMP-1 and two of the extended family members of the MMPs pump-1 and the catalytic domain of stromelysin-1, strom-1. A combination of kinetic and spectroscopic approaches that allow direct visualization of ES complexes and site-directed mutagenic changes of the enzymes will examine a) the substrate specificity, b) the characteristics of the mechanism, c) reveal the amino acids that are critical for substrate binding and catalysis, d) activation of latent forms. The results of this study will be used to design assays based on specific substrates and inhibitors for the detection of these enzymes under physiological and early developmental cellular conditions.
