Yeast methionine aminopeptidase I - Alteration of substrate specificity bysite-directed mutagenesis

In eukaryotes, two isozymes (I and II) of methionine aminopeptidase (MetAP) catalyze the removal of the initiator methionine if the penultimate residu e has a small radius of gyration (glycine, alanine, serine, threonine, prol ine, valine, and cysteine), Using site-directed mutagenesis, recombinant ye ast MetAP I derivatives that are able to cleave N-terminal methionine from substrates that have larger penultimate residues have been expressed. A Met to Ala change at 329 (Met(206) in Escherichia coli enzyme) produces an ave rage catalytic efficiency Id-fold higher than the native enzyme on normal s ubstrates and cleaves substrates containing penultimate asparagine, glutami ne, isoleucine, leucine, methionine, and phenylalanine, Interestingly, the native enzyme also has significant activity with the asparagine peptide not previously identified as a substrate. Mutation of Gln(356) (Gln(233) in E coli MetAP) to alanine results in a catalytic efficiency about one-third th at of native with normal substrates but which can cleave methionine from su bstrates with penultimate histidine, asparagine, glutamine, leucine, methio nine, phenylalanine, and tryptophan. Mutation of Ser(195) to alanine had no effect on substrate specificity. None of the altered enzymes produced clea ved substrates with a fully charged residue (lysine, arginine, aspartic aci d, or glutamic acid) or tyrosine in the penultimate position.