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.