MUTATION OF THE ACTIVE-SITE GLUTAMIC-ACID OF HUMAN GELATINASE-A - EFFECTS ON LATENCY, CATALYSIS, AND THE BINDING OF TISSUE INHIBITOR OF METALLOPROTEINASES-1

Human gelatinase A, a member of the matrix metalloproteinase family, i s secreted from cells as the M(r) 72 000 latent precursor, progelatina se A. The autolytic removal of an N-terminal propeptide generates the M(r) 66 000 active form. Mutants of recombinant progelatinase A, alter ed such that the proposed active site glutamic acid residue (E(375)) w as replaced by either an aspartic acid (proE(375)-->D), an alanine (pr oE(375)-->A), or a glutamine (proE(375)-->Q), were purified from mediu m conditioned by transfected NS0 mouse myeloma cells. Like wild-type p rogelatinase A, the mutant proenzymes were inactive and could bind tis sue inhibitor of metalloproteinases (TIMP)-2 but not TIMP-1 to their C -terminal domains. Their rates of autolytic processing induced by the organomercurial (4-aminophenyl)mercuric acetate, however, were markedl y slower and, of the three M(r) 66 000 forms so produced, only E(375)- ->D displayed any proteolytic activity against either a synthetic subs trate (k(cat)/K-m = 10% that of the wild-type enzyme) or denatured typ e I collagen (specific activity = 0.9% that of the wild-type enzyme). ProE(375)-->A and proE(375)-->Q could be more rapidly processed to the ir M(r) 66 000 forms by incubation with a deletion mutant of gelatinas e A that has full catalytic activity but lacks the C-terminal domain [ Delta(418-631)gelatinase A]. These two M(r) 66 000 forms displayed low activity on a gelatin zymogram (approximately 0.01% that of the wild- type enzyme) but, like E(375)-->D, were able to bind TIMP-1 with an af finity equal to that of the activated wild-type enzyme. These results confirm the importance of E(375) in catalysis but indicate that this r esidue is not involved in either the maintenance of proenzyme latency or the binding of TIMP-1 to the active site.