Human gelatinase A, a member of the matrix metalloproteinase family, is secreted from cells as the M(r) 72,000 latent precursor, progelatinase A. The autolytic removal of an N-terminal propeptide generates the M(r) 66,000 active form. Mutants of recombinant progelatinase A, altered such that the proposed active site glutamic acid residue (E375) was replaced by either an aspartic acid (proE375-->D), an alanine (proE375-->A) or a glutamine (proE375-->Q), were purified from medium conditioned by transfected NS0 mouse myeloma cells. Like wild-type progelatinase A, the mutant proenzymes were inactive and could bind tissue 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 markedly slower and, of the three M(r) 66,000 forms so produced, only E375-->D displayed any proteolytic activity against either a synthetic substrate (kcat/Km = 10% that of the wild-type enzyme) or denatured type I collagen (specific activity = 0.9% that of the wild-type enzyme). ProE375-->A and proE375-->Q could be more rapidly processed to their M(r) 66,000 forms by incubation with a deletion mutant of gelatinase 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 E375-->D were able to bind TIMP-1 with an affinity equal to that of the activated wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)