A COMPARATIVE-ANALYSIS OF THE KINETIC MECHANISM AND PEPTIDE SUBSTRATE-SPECIFICITY OF HUMAN AND SACCHAROMYCES-CEREVISIAE MYRISTOYL-COA - PROTEIN N-MYRISTOYLTRANSFERASE

Human myristoyl-CoA:protein N-myristoyltransferase (hNmt) catalyzes th e transfer of myristate from CoA to the amino-terminal Gly residue of a number of cellular proteins involved in signal transduction pathways , to structural and nonstructural proteins encoded by retroviruses, he padnaviruses, picornaviruses, and reoviruses, as well as to several tr ansforming tyrosine kinases. hNmt has been purified 230-fold from an e rythroleukemia cell line. The monomeric enzyme has no associated methi onyl aminopeptidase activity. To determine the enzyme's kinetic mechan ism, we examined the effect of covariation of subsaturating concentrat ions of myristoyl-CoA and peptide substrate on initial velocity. Doubl e-reciprocal plots excluded a double displacement (ping-pong) mechanis m. Product inhibition studies indicated that CoA was a noncompetitive inhibitor against myristoyl-CoA and a mixed-type inhibitor against pep tide substrates. Together these results are consistent with a sequenti al ordered mechanism where, in a typical catalytic cycle, myristoyl-Co A binds to apoenzyme before peptide followed by release of the CoA and then myristoylpeptide products. This kinetic mechanism is identical t o that described for Saccharomyces cerevisiae N-myristoyltransferase ( Nmt1p) and emphasizes the impact that regulation of myristoyl-CoA pool size and accessibility may have in modulating protein N-myristoylatio n in these two species. Comparative studies of the peptide substrate s pecificities of hNmt and Nmt1p using a panel of 12 octapeptides reveal ed distinct differences in their tolerance for amino acid substitution s at positions 3, 4, 7, and 8 of parental peptides derived from the am ino-terminal sequences of known N-myristoyl-proteins. This finding con trasts with our recent observation that the acyl-CoA substrate specifi cities of hNmt and Nmt1p are highly conserved and suggests that these differences in peptide recognition provide an opportunity to develop s pecies-specific enzyme inhibitors.