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. 1987 May;84(9):2708–2712. doi: 10.1073/pnas.84.9.2708

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
PMCID: PMC304727  PMID: 3106975

Abstract

Myristoyl CoA:protein N-myristoyltransferase (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 Gly-Asn-Ala-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 approximately equal to 500 in the apparent Km 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.

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Selected References

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  1. Aderem A. A., Keum M. M., Pure E., Cohn Z. A. Bacterial lipopolysaccharides, phorbol myristate acetate, and zymosan induce the myristoylation of specific macrophage proteins. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5817–5821. doi: 10.1073/pnas.83.16.5817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aitken A., Cohen P., Santikarn S., Williams D. H., Calder A. G., Smith A., Klee C. B. Identification of the NH2-terminal blocking group of calcineurin B as myristic acid. FEBS Lett. 1982 Dec 27;150(2):314–318. doi: 10.1016/0014-5793(82)80759-x. [DOI] [PubMed] [Google Scholar]
  3. Carr S. A., Biemann K., Shoji S., Parmelee D. C., Titani K. n-Tetradecanoyl is the NH2-terminal blocking group of the catalytic subunit of cyclic AMP-dependent protein kinase from bovine cardiac muscle. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6128–6131. doi: 10.1073/pnas.79.20.6128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Hemmings B. A., Zubenko G. S., Hasilik A., Jones E. W. Mutant defective in processing of an enzyme located in the lysosome-like vacuole of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1981 Jan;78(1):435–439. doi: 10.1073/pnas.78.1.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Henderson L. E., Krutzsch H. C., Oroszlan S. Myristyl amino-terminal acylation of murine retrovirus proteins: an unusual post-translational proteins modification. Proc Natl Acad Sci U S A. 1983 Jan;80(2):339–343. doi: 10.1073/pnas.80.2.339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Itoh H., Kozasa T., Nagata S., Nakamura S., Katada T., Ui M., Iwai S., Ohtsuka E., Kawasaki H., Suzuki K. Molecular cloning and sequence determination of cDNAs for alpha subunits of the guanine nucleotide-binding proteins Gs, Gi, and Go from rat brain. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3776–3780. doi: 10.1073/pnas.83.11.3776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kamps M. P., Buss J. E., Sefton B. M. Rous sarcoma virus transforming protein lacking myristic acid phosphorylates known polypeptide substrates without inducing transformation. Cell. 1986 Apr 11;45(1):105–112. doi: 10.1016/0092-8674(86)90542-8. [DOI] [PubMed] [Google Scholar]
  8. Magee A. I., Courtneidge S. A. Two classes of fatty acid acylated proteins exist in eukaryotic cells. EMBO J. 1985 May;4(5):1137–1144. doi: 10.1002/j.1460-2075.1985.tb03751.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Marchildon G. A., Casnellie J. E., Walsh K. A., Krebs E. G. Covalently bound myristate in a lymphoma tyrosine protein kinase. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7679–7682. doi: 10.1073/pnas.81.24.7679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Marth J. D., Peet R., Krebs E. G., Perlmutter R. M. A lymphocyte-specific protein-tyrosine kinase gene is rearranged and overexpressed in the murine T cell lymphoma LSTRA. Cell. 1985 Dec;43(2 Pt 1):393–404. doi: 10.1016/0092-8674(85)90169-2. [DOI] [PubMed] [Google Scholar]
  11. McIlhinney R. A., Pelly S. J., Chadwick J. K., Cowley G. P. Studies on the attachment of myristic and palmitic acid to cell proteins in human squamous carcinoma cell lines: evidence for two pathways. EMBO J. 1985 May;4(5):1145–1152. doi: 10.1002/j.1460-2075.1985.tb03752.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Medynski D. C., Sullivan K., Smith D., Van Dop C., Chang F. H., Fung B. K., Seeburg P. H., Bourne H. R. Amino acid sequence of the alpha subunit of transducin deduced from the cDNA sequence. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4311–4315. doi: 10.1073/pnas.82.13.4311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Miles E. W. Modification of histidyl residues in proteins by diethylpyrocarbonate. Methods Enzymol. 1977;47:431–442. doi: 10.1016/0076-6879(77)47043-5. [DOI] [PubMed] [Google Scholar]
  14. Olson E. N., Spizz G. Fatty acylation of cellular proteins. Temporal and subcellular differences between palmitate and myristate acylation. J Biol Chem. 1986 Feb 15;261(5):2458–2466. [PubMed] [Google Scholar]
  15. Olson E. N., Towler D. A., Glaser L. Specificity of fatty acid acylation of cellular proteins. J Biol Chem. 1985 Mar 25;260(6):3784–3790. [PubMed] [Google Scholar]
  16. Ozols J., Carr S. A., Strittmatter P. Identification of the NH2-terminal blocking group of NADH-cytochrome b5 reductase as myristic acid and the complete amino acid sequence of the membrane-binding domain. J Biol Chem. 1984 Nov 10;259(21):13349–13354. [PubMed] [Google Scholar]
  17. Pasek M., Goto T., Gilbert W., Zink B., Schaller H., MacKay P., Leadbetter G., Murray K. Hepatitis B virus genes and their expression in E. coli. Nature. 1979 Dec 6;282(5739):575–579. doi: 10.1038/282575a0. [DOI] [PubMed] [Google Scholar]
  18. Pellman D., Garber E. A., Cross F. R., Hanafusa H. Fine structural mapping of a critical NH2-terminal region of p60src. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1623–1627. doi: 10.1073/pnas.82.6.1623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ratner L., Haseltine W., Patarca R., Livak K. J., Starcich B., Josephs S. F., Doran E. R., Rafalski J. A., Whitehorn E. A., Baumeister K. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature. 1985 Jan 24;313(6000):277–284. doi: 10.1038/313277a0. [DOI] [PubMed] [Google Scholar]
  20. Rein A., McClure M. R., Rice N. R., Luftig R. B., Schultz A. M. Myristylation site in Pr65gag is essential for virus particle formation by Moloney murine leukemia virus. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7246–7250. doi: 10.1073/pnas.83.19.7246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schultz A. M., Henderson L. E., Oroszlan S., Garber E. A., Hanafusa H. Amino terminal myristylation of the protein kinase p60src, a retroviral transforming protein. Science. 1985 Jan 25;227(4685):427–429. doi: 10.1126/science.3917576. [DOI] [PubMed] [Google Scholar]
  22. Schultz A. M., Oroszlan S. In vivo modification of retroviral gag gene-encoded polyproteins by myristic acid. J Virol. 1983 May;46(2):355–361. doi: 10.1128/jvi.46.2.355-361.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Semba K., Nishizawa M., Miyajima N., Yoshida M. C., Sukegawa J., Yamanashi Y., Sasaki M., Yamamoto T., Toyoshima K. yes-related protooncogene, syn, belongs to the protein-tyrosine kinase family. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5459–5463. doi: 10.1073/pnas.83.15.5459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sullivan K. A., Liao Y. C., Alborzi A., Beiderman B., Chang F. H., Masters S. B., Levinson A. D., Bourne H. R. Inhibitory and stimulatory G proteins of adenylate cyclase: cDNA and amino acid sequences of the alpha chains. Proc Natl Acad Sci U S A. 1986 Sep;83(18):6687–6691. doi: 10.1073/pnas.83.18.6687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Towler D. A., Eubanks S. R., Towery D. S., Adams S. P., Glaser L. Amino-terminal processing of proteins by N-myristoylation. Substrate specificity of N-myristoyl transferase. J Biol Chem. 1987 Jan 25;262(3):1030–1036. [PubMed] [Google Scholar]
  26. Towler D., Glaser L. Acylation of cellular proteins with endogenously synthesized fatty acids. Biochemistry. 1986 Feb 25;25(4):878–884. doi: 10.1021/bi00352a021. [DOI] [PubMed] [Google Scholar]
  27. Towler D., Glaser L. Protein fatty acid acylation: enzymatic synthesis of an N-myristoylglycyl peptide. Proc Natl Acad Sci U S A. 1986 May;83(9):2812–2816. doi: 10.1073/pnas.83.9.2812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Uhler M. D., Carmichael D. F., Lee D. C., Chrivia J. C., Krebs E. G., McKnight G. S. Isolation of cDNA clones coding for the catalytic subunit of mouse cAMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1300–1304. doi: 10.1073/pnas.83.5.1300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Yatsunami K., Khorana H. G. GTPase of bovine rod outer segments: the amino acid sequence of the alpha subunit as derived from the cDNA sequence. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4316–4320. doi: 10.1073/pnas.82.13.4316. [DOI] [PMC free article] [PubMed] [Google Scholar]

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