Biochemical studies of Saccharomyces cerevisiae myristoyl-coenzyme A Protein N-myristoyltransferase mutants

Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is an essential 455-residue, monomeric enzyme that catalyzes the transfer of myristate from myristoyl-CoA to the NH2-terminal Gly residue of cellular proteins, Nmt1p has an ordered Bi Bi reaction mechanism with binding of myristoyl-CoA occurring before binding of peptide substrates, To define residues important for function, the polymerase chain reaction was used to generate random mutations in the NMT1 gene, A colony color sectoring assay was used to screen a library of 52,000 transformants for nmt1 alleles encoding enzymes with reduced activity, nmt1 alleles were identified that produced temperature sensitive (ts) growth arrest due to substitutions affecting eight residues conserved in orthologous Nmts: Asn(102), Ala(202), Cys(217), Ser(328), Val(395), Asn(404), Leu(420), and Asn(426). Ala(202) --> Thr, Cys(217) --> Arg, Ser(328) --> Pro, Asn(404) --> Tyr, and Asn(426) --> Ile produced the most severe ts phenotype, Their effects on the functional properties of the enzyme's myristoyl CoA and peptide binding sites were defined by purifying each mutant from Escherichia coli and conducting in vitro kinetic analyses with acyl-CoA and peptide substrates and with two competitive inhibitors: S-(2-oxo)pentadecyl-CoA, a nonhydrolyzable myristoyl-CoA analog, and SC-58272, a peptidomimetic derived from the NH2-terminal sequence of an Nmt1p substrate (ADP-ribosylation factor-2, Arf2p), None of the substitutions affect the enzyme's acyl chain length selectivity, When compared with wild type Nmt1p, Cys(217) --> Arg produces 3- and 6-fold increases in K-i for SC-58272 at 24 and 37 degrees C but no change in K-i for S-(2-oxo)pentadecyl-CoA, indicating that the substitution selectively affects Nmt1p's peptide binding site. Asn(426) --> Ile selectively perturbs the myristoyl-CoA binding site, resulting in the most pronounced reduction in affinity for S-(2-oxo)pentadecyl-CoA (12- and 20-fold), Ala(202) --> Thr, which confers the most severe ts phenotype, provides an example of a substitution that affects both sites, producing 3- and 6-fold increases in the K-i for S-(2-oxo)pentadecyl-CoA and 6- and 9-fold increases in the K-i for SC-58272 at 24 and 37 degrees C. An N-myristoylation-dependent change in the electrophoretic mobility of Arf1p was used to assay the effects of the mutants on cellular levels of protein N-myristoylation under a variety of growth conditions. The ts growth arrest produced by nmt1 alleles correlates with a reduction in myristoyl-Arf1p to less than or equal to 50% of total cellular Arf1p.