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.
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
D A
DA
Rudnick
C A
CA
McWherter
S P
SP
Adams
I J
IJ
Ropson
R J
RJ
Duronio
J I
JI
Gordon
D000217:Acyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D011994:Recombinant Proteins; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase