Biochemical studies of Saccharomyces cerevisiae myristoyl-coenzyme A:protein N-myristoyltransferase mutants.

The Journal of Biological Chemistry
L ZhangJ I Gordon

Abstract

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: 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 in...Continue Reading

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