Function of the proposed zinc ligand, tyrosine 226, in mouse meprin alpha

Crystallographic evaluation of astacin, a homologue of meprin alpha, indicated that a conserved tyrosine residue in the astacin family was a fifth ligand for zinc binding. The work herein addressed the importance of the conserved tyrosine in zinc binding. The role of the tyrosine 226 in the astacin family protease, mouse meprin alpha, was used as a model. The tyrosine residue at position 226 in meprin alpha was replaced with phenylalanine (Y226F) by site-directed mutagenesis. In another mutant, a histidine, H167, in the conserved zinc binding motif of the meprin alpha protease domain was replaced by alanine (H167A). We investigated the stability and enzyme activity of wild-type and mutant recombinant proteins. We hypothesized that the mutations would affect zinc binding. A purification procedure was developed for the wild-type and mutant Y226F mutant.;Structural evaluation of the wild-type and mutant proteins included assessment of oligomerization and susceptibility to proteolysis. All proteins were synthesized and secreted at comparable levels. Wild-type and Y226F retained the capacity to oligomerize to higher covalently and non-covalently linked structures. Both mutant proteins were more susceptible to extensive degradation by trypsin compared with the wild-type protein. All proteins contained complex glycosylation, although high mannose glycosylation was increased in the mutants. Atomic absorption spectrometry and inductive plasma coupled mass spectrometry indicated that the zinc content in the wild-type and Y226F were similar. Circular dichroism indicated a perturbation of the Y226F alpha helices relative to wild-type. Intrinsic tryptophan fluorescence indicated a structural change in the Y226F mutant protein.;Characterization of catalytic properties included assays with a fluorogenic analog of the peptide, bradykinin (Bk+) and the proteins, azocasein and gelatin. H167A had no detectable activity against the substrates. The kcat/Km for Y226F was 16% of wild-type when Bk+ was used as a substrate. Y226F was not active against azocasein or gelatin. Increasing zinc concentration in the Bk+ assay decreased activity of the enzymes. The pH profiles of wild-type and Y226F were comparable in the pH range of 6.5 to 10.0. Tyrosine, Y226, is important to the structural integrity of mouse meprin alpha, but is not essential for catalytic activity or zinc binding.