Biochemical studies of Saccharomyces cerevisiae myristoylcoenzyme A:Protein AT-myristoyltransferase mutants

Saccharomyces cerevisiae inyristoyl-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: Asn¹⁰², Ala²⁰², Cys²¹⁷, Ser³²⁸, Val³⁹⁵, Asn⁴⁰⁴, Leu⁴²⁰, and Asn⁴²⁶. Ala²⁰² → Thr, Cys²¹⁷ → Arg, Ser³²⁸ → Pro, Asn⁴⁰⁴ → Tyr, and Asn⁴²⁶ → 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, Cys²¹⁷ → Arg produces 3- and 6-fold increases in K_i for SC-58272 at 24 and 37 °C but no change in K_i for S-(2-oxo)pentadecyl-CoA, indicating that the substitution selectively affects Nmt1p's peptide binding site. Asn⁴²⁶ → He 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). Ala²⁰² → Thr, which confers the most severe ts phenotype, provides an example of a substitution that affects both sites, producing 3- and6-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 °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 ≤50% of total cellular Arf1p.