Structural and functional studies of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase produced in Escherichia coli. Evidence for an acyl-enzyme intermediate.
Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase has been efficiently expressed in Escherichia coli and subsequently purified to homogeneity using phosphocellulose chromatography. The interactions between apoenzyme and its acyl-CoA and peptide ligands were examined by an isoelectric focusing gel shift assay, circular dichroism, and fluorescence spectroscopy, and a continuous assay of enzyme activity which measures the release of CoA from acyl-CoA using the thiol-specific reagent 5-5'-dithiobis-2-nitrobenzoate. Addition of myristoyl-CoA (without a substrate peptide) results in the formation of a high affinity reaction intermediate which can be operationally defined by the appearance of a more acidic enzyme isoform and by quenching of the tryptophan emission with a maximal difference at 340 nm. Circular dichroism spectroscopy indicates that these changes are accompanied by minimal changes in the enzyme's secondary structure. Incubation of purified NMT with [1-14C] myristoyl-CoA, followed by chymotryptic digestion, denaturing polyacrylamide gel electrophoresis, and treatment with hydroxylamine yielded results that are highly suggestive of a covalent ester-linked acyl-enzyme complex. Edman degradation of chymotryptic peptides has narrowed the site of interaction to a domain spanning Arg42 to Thr220 of the 455 amino acid acyltransferase. An octapeptide containing Gly but not Ala at position 1 is able to reverse the change in pI and reduce the quenching almost entirely. These data suggest a preferred order or ping-pong reaction mechanism with the acyl-CoA substrate binding event occurring first. They also indicate that Gly1 is absolutely necessary for the reaction to proceed forward from the acyl-enzyme reaction intermediate.[1]References
- Structural and functional studies of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase produced in Escherichia coli. Evidence for an acyl-enzyme intermediate. Rudnick, D.A., McWherter, C.A., Adams, S.P., Ropson, I.J., Duronio, R.J., Gordon, J.I. J. Biol. Chem. (1990) [Pubmed]
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