Structural and functional studies of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase produced in Escherichia coli. Evidence for an acyl-enzyme intermediate

D. A. Rudnick, C. A. McWherter, S. P. Adams, Ira Ropson, R. J. Duronio, J. I. Gordon

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Abstract

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- 14 C]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 Arg 42 to Thr 220 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 Gly 1 is absolutely necessary for the reaction to proceed forward from the acyl-enzyme reaction intermediate.

Original languageEnglish (US)
Pages (from-to)13370-13378
Number of pages9
JournalJournal of Biological Chemistry
Volume265
Issue number22
StatePublished - Aug 20 1990

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Yeast
Acyl Coenzyme A
Escherichia coli
Saccharomyces cerevisiae
Circular dichroism spectroscopy
Reaction intermediates
Enzymes
Circular Dichroism
Peptides
Quenching
Assays
Apoenzymes
Acyltransferases
Sulfhydryl Reagents
Hydroxylamine
Fluorescence Spectrometry
Fluorescence spectroscopy
Enzyme Assays
Enzyme activity
Isoelectric Focusing

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "Structural and functional studies of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase produced in Escherichia coli. Evidence for an acyl-enzyme intermediate",
abstract = "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- 14 C]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 Arg 42 to Thr 220 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 Gly 1 is absolutely necessary for the reaction to proceed forward from the acyl-enzyme reaction intermediate.",
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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, Ira; Duronio, R. J.; Gordon, J. I.

In: Journal of Biological Chemistry, Vol. 265, No. 22, 20.08.1990, p. 13370-13378.

Research output: Contribution to journalArticle

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T1 - Structural and functional studies of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase produced in Escherichia coli. Evidence for an acyl-enzyme intermediate

AU - Rudnick, D. A.

AU - McWherter, C. A.

AU - Adams, S. P.

AU - Ropson, Ira

AU - Duronio, R. J.

AU - Gordon, J. I.

PY - 1990/8/20

Y1 - 1990/8/20

N2 - 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- 14 C]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 Arg 42 to Thr 220 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 Gly 1 is absolutely necessary for the reaction to proceed forward from the acyl-enzyme reaction intermediate.

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