Purification and characterization of yeast myristoyl CoA: protein N-myristoyltransferase

D. A. Towler, S. P. Adams, S. R. Eubanks, D. S. Towery, E. Jackson-Machelski, L. Glaser, J. I. Gordon

Research output: Contribution to journalArticle

157 Citations (Scopus)

Abstract

Myristoyl CoA:protein N-myristolytransferase (NMT) catalyzes the addition of myristic acid to the amino-terminal glycine residues of a number of eukaryotic proteins. Recently, we developed a cell-free system for analyzing NMT activity and have begun to characterize the substrate specificity of this enzyme by using a series of synthetic peptides. We have now purified NMT from Saccharomyces cerevisiae to apparent homogeneity. The native enzyme is a 55-kDa protein, exhibits no requirement for divalent cation, and appears to contain a histidine residue critical for enzyme activity. A total of 42 synthetic peptides have been used to define structure/activity relationships in NMT substrates. An amino-terminal glycine is required for acylation; substitution with glycine analogues produces peptides that are inactive as substrates or inhibitors of NMT. A broad spectrum of amino acids is permitted at positions 3 and 4, while strict amino acid requirements are exhibited at position 5. Replacement of Ala5 in the peptide Gys-Asn-Ala-Ala-Ala-Arg-Arg with Asp ablates the peptide's myristoyl-accepting activity. A serine at this position results in a decrease by a factor of ≃ 500 in the apparent K(m) in the context of three different sequences. Penta- and hexa-peptides are substrates, but with decreased affinity. These studies establish that structural information important for NMT-ligand interaction exists beyond the first two amino acids in peptide substrates and that the side chains of residue 5 play a critical role in the binding of substrates to this enzyme.

Original languageEnglish (US)
Pages (from-to)2708-2712
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume84
Issue number9
DOIs
StatePublished - Jun 25 1987

Fingerprint

Yeasts
Peptides
Glycine
Enzymes
Amino Acids
Rubiaceae
Proteins
Acylation
Cell-Free System
glycylpeptide N-tetradecanoyltransferase
Divalent Cations
Myristic Acid
Structure-Activity Relationship
Substrate Specificity
Histidine
Serine
Saccharomyces cerevisiae
Ligands

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Purification and characterization of yeast myristoyl CoA : protein N-myristoyltransferase. / Towler, D. A.; Adams, S. P.; Eubanks, S. R.; Towery, D. S.; Jackson-Machelski, E.; Glaser, L.; Gordon, J. I.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 84, No. 9, 25.06.1987, p. 2708-2712.

Research output: Contribution to journalArticle

Towler, D. A. ; Adams, S. P. ; Eubanks, S. R. ; Towery, D. S. ; Jackson-Machelski, E. ; Glaser, L. ; Gordon, J. I. / Purification and characterization of yeast myristoyl CoA : protein N-myristoyltransferase. In: Proceedings of the National Academy of Sciences of the United States of America. 1987 ; Vol. 84, No. 9. pp. 2708-2712.
@article{591c2be6786f4e578f9e52daa1655a5f,
title = "Purification and characterization of yeast myristoyl CoA: protein N-myristoyltransferase",
abstract = "Myristoyl CoA:protein N-myristolytransferase (NMT) catalyzes the addition of myristic acid to the amino-terminal glycine residues of a number of eukaryotic proteins. Recently, we developed a cell-free system for analyzing NMT activity and have begun to characterize the substrate specificity of this enzyme by using a series of synthetic peptides. We have now purified NMT from Saccharomyces cerevisiae to apparent homogeneity. The native enzyme is a 55-kDa protein, exhibits no requirement for divalent cation, and appears to contain a histidine residue critical for enzyme activity. A total of 42 synthetic peptides have been used to define structure/activity relationships in NMT substrates. An amino-terminal glycine is required for acylation; substitution with glycine analogues produces peptides that are inactive as substrates or inhibitors of NMT. A broad spectrum of amino acids is permitted at positions 3 and 4, while strict amino acid requirements are exhibited at position 5. Replacement of Ala5 in the peptide Gys-Asn-Ala-Ala-Ala-Arg-Arg with Asp ablates the peptide's myristoyl-accepting activity. A serine at this position results in a decrease by a factor of ≃ 500 in the apparent K(m) in the context of three different sequences. Penta- and hexa-peptides are substrates, but with decreased affinity. These studies establish that structural information important for NMT-ligand interaction exists beyond the first two amino acids in peptide substrates and that the side chains of residue 5 play a critical role in the binding of substrates to this enzyme.",
author = "Towler, {D. A.} and Adams, {S. P.} and Eubanks, {S. R.} and Towery, {D. S.} and E. Jackson-Machelski and L. Glaser and Gordon, {J. I.}",
year = "1987",
month = "6",
day = "25",
doi = "10.1073/pnas.84.9.2708",
language = "English (US)",
volume = "84",
pages = "2708--2712",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "9",

}

TY - JOUR

T1 - Purification and characterization of yeast myristoyl CoA

T2 - protein N-myristoyltransferase

AU - Towler, D. A.

AU - Adams, S. P.

AU - Eubanks, S. R.

AU - Towery, D. S.

AU - Jackson-Machelski, E.

AU - Glaser, L.

AU - Gordon, J. I.

PY - 1987/6/25

Y1 - 1987/6/25

N2 - Myristoyl CoA:protein N-myristolytransferase (NMT) catalyzes the addition of myristic acid to the amino-terminal glycine residues of a number of eukaryotic proteins. Recently, we developed a cell-free system for analyzing NMT activity and have begun to characterize the substrate specificity of this enzyme by using a series of synthetic peptides. We have now purified NMT from Saccharomyces cerevisiae to apparent homogeneity. The native enzyme is a 55-kDa protein, exhibits no requirement for divalent cation, and appears to contain a histidine residue critical for enzyme activity. A total of 42 synthetic peptides have been used to define structure/activity relationships in NMT substrates. An amino-terminal glycine is required for acylation; substitution with glycine analogues produces peptides that are inactive as substrates or inhibitors of NMT. A broad spectrum of amino acids is permitted at positions 3 and 4, while strict amino acid requirements are exhibited at position 5. Replacement of Ala5 in the peptide Gys-Asn-Ala-Ala-Ala-Arg-Arg with Asp ablates the peptide's myristoyl-accepting activity. A serine at this position results in a decrease by a factor of ≃ 500 in the apparent K(m) in the context of three different sequences. Penta- and hexa-peptides are substrates, but with decreased affinity. These studies establish that structural information important for NMT-ligand interaction exists beyond the first two amino acids in peptide substrates and that the side chains of residue 5 play a critical role in the binding of substrates to this enzyme.

AB - Myristoyl CoA:protein N-myristolytransferase (NMT) catalyzes the addition of myristic acid to the amino-terminal glycine residues of a number of eukaryotic proteins. Recently, we developed a cell-free system for analyzing NMT activity and have begun to characterize the substrate specificity of this enzyme by using a series of synthetic peptides. We have now purified NMT from Saccharomyces cerevisiae to apparent homogeneity. The native enzyme is a 55-kDa protein, exhibits no requirement for divalent cation, and appears to contain a histidine residue critical for enzyme activity. A total of 42 synthetic peptides have been used to define structure/activity relationships in NMT substrates. An amino-terminal glycine is required for acylation; substitution with glycine analogues produces peptides that are inactive as substrates or inhibitors of NMT. A broad spectrum of amino acids is permitted at positions 3 and 4, while strict amino acid requirements are exhibited at position 5. Replacement of Ala5 in the peptide Gys-Asn-Ala-Ala-Ala-Arg-Arg with Asp ablates the peptide's myristoyl-accepting activity. A serine at this position results in a decrease by a factor of ≃ 500 in the apparent K(m) in the context of three different sequences. Penta- and hexa-peptides are substrates, but with decreased affinity. These studies establish that structural information important for NMT-ligand interaction exists beyond the first two amino acids in peptide substrates and that the side chains of residue 5 play a critical role in the binding of substrates to this enzyme.

UR - http://www.scopus.com/inward/record.url?scp=0001016365&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0001016365&partnerID=8YFLogxK

U2 - 10.1073/pnas.84.9.2708

DO - 10.1073/pnas.84.9.2708

M3 - Article

C2 - 3106975

AN - SCOPUS:0001016365

VL - 84

SP - 2708

EP - 2712

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 9

ER -