The substrate specificity of saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase: Polar probes of the enzyme's myristoyl-CoA recognition site

Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is a monomeric enzyme that is essential for vegetative growth. Nmtlp catalyzes the co-translational transfer of myristate from CoA to the ammo-terminal Gly of cellular proteins in an ordered Bi Bi reaction mechanism that initially involves binding of myristoyl-CoA to the apoenzyme. Forty one fatty acid analogs were synthesized to define features in the acyl chain of myristoyl-CoA which are important determinants of its recognition by Nmt1p's acyl-CoA binding site as well as to help us deduce the structure of the binding site itself. These analogs included dicarboxylic acids, ω-nitrocarboxylic acids, analogs equivalent in length to C13:0-C15:0 which contain electronegative halogens at their ω-termini, hydroxytetradecanoic acids with hydrogen replaced by OH from C3 to C13, and azidophenyl-containing fatty acids with the linear azide unit attached either meta or para to phenyl and with variations in the length of their methylene chains. These compounds were converted to their CoA derivatives using Pseudomonas acyl-CoA synthetase and then surveyed as substrates for purified Nmtlp in an in vitro assay system that included an octapeptide derived from residues 1-8 of the human immunodeficiency virus Pr55^gag polyprotein precursor. The results suggest that the myristoyl-CoA binding site contains a conical-shaped "receptor" that interacts with the ω-terminus of the bound acyl chain of acyl-CoAs. The acuteness of this cone determines the enzyme's capacity to accommodate steric bulk at the ω-terminus as well as Nmtlp's sensitivity to the distance between the eclipsed C5-C6 bond of a bound acyl chain and its ω-terminus. The activity profile of the various analog-CoAs also indicates that the enzyme's myristoyl-CoA binding site can accommodate fatty acid analogs with marked increases in polarity at their ω-terminus (compared to C14:0) as long as their chain length is equivalent to that of myristate.