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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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The Purification and Characterization of a Bacillus stearothermophilus Methionine Aminopeptidase (MetAP)

  • Chung, Jae-Min (Department of Biochemistry and Molecular Biology, Hanyang University) ;
  • Chung, Il-Yup (Department of Biochemistry and Molecular Biology, Hanyang University) ;
  • Lee, Young-Seek (Department of Biochemistry and Molecular Biology, Hanyang University)
  • Published : 2002.03.31
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Abstract

Methionine aminopeptidase (MetAP) catalyzes the removal of an amino-terminal methionine from a newly synthesized polypeptide. The enzyme was purified to homogeneity from Bacillus stearothermophilus (KCTC 1752) by a procedure that involves heat precipitation and four sequential chromatographs (including DEAE-Sepharose ion exchange, hydroxylapatite, Ultrogel AcA 54 gel filtration, and Reactive red 120 dye affinity chromatography). The apparent molecular masses of the enzyme were 81,300 Da and 41,000 Da, as determined by gel filtration chromatography and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), respectively. This indicates that the enzyme is comprised of two identical subunits. The MetAP specifically hydrolyzed the N-terminal residue of Met-Ala-Ser that was used as a substrate, and exhibited a strong preference for Met-Ala-Ser over Leu-Gly-Gly, Leu-Ser-Phe, and Leu-Leu-Tyr. The enzyme has an optimal pH at 8.0, an optimal temperature at $80^{\circ}C$, and pI at 4.1. The enzyme was heat-stable, as its activity remained unaltered when incubated at $80^{\circ}C$ for 45 min. The Km and Vmax values of the enzyme were 3.0mM and 1.7 mmol/min/mg, respectively. The B. stearothernmophilus MetAP was completely inactivated by EDTA and required $Co^{2+}$ ion(s) for activation, suggesting the metal dependence of this enzyme.

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References

  1. Artin, S. M. and Bradshaw, R. A. (1988) Cotranslational processing and protein turnover in eukaryotic cells. Biochemistry 27, 7979-7984. https://doi.org/10.1021/bi00421a001
  2. Artin, S. M., Kendall, R. L., Hall, L., Weaver, L. H., Stewart, A. E., Matthews, B. W. and Bradshaw, A. (1995) Eukaryotic methionyl aminopeptidases: two classes of cobalt-dependent enzymes. Proc. Natl. Acad. Sci. USA 92, 7714-7718. https://doi.org/10.1073/pnas.92.17.7714
  3. Bachmair, A., Finley, D. and Varshavsky, A. (1985) In vivo half-life of a protein is a function of its amino-teoninal residue. Science 234, 179-186. https://doi.org/10.1126/science.3018930
  4. Bazan, J. F., Weaver, L. H., Roderick, S. L., Huber, R. and Matthews, B. W. (1994) Sequence and structure comparison suggest that methionine aminopeptidase, prolidase, aminopeptidase P. and creatinase share a common fold. Proc. Natl. Acad. Sci. USA 91, 2473-2477. https://doi.org/10.1073/pnas.91.7.2473
  5. Ben-Bassat, A., Bauer, K., Chang, S. Y., Myambo, K., Boosman, A. and Chang, S. (1987) Processing of the initiation methionine from proteins: properties of the Escherichia coli methionine aminopeptidase and its gene structure. J. Bacteriol. 169,751-757. https://doi.org/10.1128/jb.169.2.751-757.1987
  6. Bradshaw, R. A., Brickey, W. W. and Walker, K. W. (1998) N-terminal processing: the methionine aminopeptidase and N alpha-acetyl transferase families. Trends Biochem. Sci. 23, 263-267. https://doi.org/10.1016/S0968-0004(98)01227-4
  7. Cha, M. H., Yong, W. M., Lee, S. M., Lee, Y. S. and Chung, I. Y. (2000) The biochemical and molecular characterization of recombinant Bacillus subtilis tripeptidase (pepT) as a zinc-dependent metalloenzyme. Mol. Cells 10, 423-431.
  8. Chang, S. Y., McGary, E. C. and Chang, S. (1989) Methionine aminopeptidase gene of Escherichia coli is essential for cell growth. J. Bacterial. 171, 4071-4072. https://doi.org/10.1128/jb.171.7.4071-4072.1989
  9. Chang, Y. -H., Teichert, U. and Smith, J. A. (1990) Purification and characterization of a methionine aminopeptidase from Saccharomyces cerevisiae. J. BioI. Chem. 265, 19892-19897.
  10. Chang, Y. -H., Teichert, U. and Smith, J. A. (1992) Molecular cloning, sequencing, deletion, and overexpression of a methionine anlinopeptidase gene from Saccharomyces cerevisiae. J. BioI. Chem. 267, 8007-8011.
  11. D'souza, V. M. and Holz, R. C. (1999) The methionyl aminopeptidase from Escherichia coli can function as an iron (II) enzyme. Biochemistry 38, 11079-11085. https://doi.org/10.1021/bi990872h
  12. Rinta,C., Persson, B., Jomvall, H. and von Heijne, G. (1986) Sequence determinants of cytosolic N-terminal protein processing. Eur. J. Biochem. 154, 193-196. https://doi.org/10.1111/j.1432-1033.1986.tb09378.x
  13. Kendall, R. L. and Bradshaw, R. A. (1992) Isolation and characterization of the methionine aminopeptidase from porcine liver responsible for the co-translational processing of proteins. J. Biol. Chem. 267, 20667-20673.
  14. Laemmli, U. K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. https://doi.org/10.1038/227680a0
  15. Li, X. and Chang, Y. -H. (1995) Amino-terminal protein processing in Saccharomyces cerevisiae is an essential function that requires two distinct methionine aminopeptidases. Proc. Natl. Acad. Sci. USA 92, 12357-12361. https://doi.org/10.1073/pnas.92.26.12357
  16. Li, X. and Chang, Y. -H. (1996) Evidence that the human homologue of a rat initiation factor-2 associated protein (p67) is a methionine aminopeptidase. Biochem. Biophys. Res. Commun. 227, 152-159. https://doi.org/10.1006/bbrc.1996.1482
  17. Liu, S., Widom, J., Kemp, C. W., Crews, C. M. and Clardy, J. (1998) Structure of human methionine aminopeptidase-2 complexed with fumagillin. Science 282, 1324-1327. https://doi.org/10.1126/science.282.5392.1324
  18. Lowther, W. T. and Matthews, B. W. (2000) Structure and function of the methionine aminopeptidases. Biochim. Biophys. Acta. 1477, 157-167. https://doi.org/10.1016/S0167-4838(99)00271-X
  19. Lowther, W. T., Orville, A. M., Madden, D. T., Lim, S., Rich, D. H. and Matthews, B. W. (1999) Escherichia coli methionine aminopeptidase: implications of crystallographic analyses of the native, mutant, and inhibited enzymes for the mechanism of catalysis. Biochemistry 38, 7678-7688. https://doi.org/10.1021/bi990684r
  20. Miller, C. G., Kukral, A. M., Miller, J. L. and Movva, N. R. (1989) PepM is an essential gene in Salmonella typhimurium. J. Bacterial. 171, 5215-5217. https://doi.org/10.1128/jb.171.9.5215-5217.1989
  21. Miller, C. G., Strauch, K. L., Kukral, A. M., Miller, J. L., Wingfield, P. T., Mazzei, G. J., Weden, R. C., Graber, P. and Movva, N. R. (1987) N-terminal methionine-specific peptidase in Salmonella typhimurium. Proc. Natl. Acad. Sci. USA 84, 2718-2722. https://doi.org/10.1073/pnas.84.9.2718
  22. Moerschell, R. P., Hosokawa, Y., Tsunasawa, S. and Sherman, F. (1990) The specificities of yeast methionine aminopeptidase and acetylation of amino-terminal methionine in vivo. Processing of altered iso-1-cytochromes c created by oligonucleotide transformation. J. Biol. Chem. 265, 19638-19643.
  23. Movva, N. R., Semon, D., Meyer, C., Kawashima, E., Wmgfield, P., Miller, J. L. and Miller, C. G. (1990) Cloning and nucleotide sequence of the Salmonella typhimurium pepM gene. Mol. Gen. Genet. 223, 345-348.
  24. Park, Y. S., Cha, M. H., Yong, H. M., Kim, H. J., Chung, I. Y. and Lee, Y. S. (1999) The purification and characterization of Bacillus subtilis tripeptidase (PepT). J. Biochem. Mol. Biol. 32, 239-246.
  25. Roderick, S. L. and Matthews, B. W. (1993) Structure of the cobalt-dependent methionine aminopeptidase from Escherichia coli: a new type of proteolytic enzyme. Biochemistry 32, 3907-3912. https://doi.org/10.1021/bi00066a009
  26. Simitsopoulou, M., Vafopoulou, A., Choli-Papadopoulou, T. and Alichanidis, E. (1997) Purification and partial characterization of a tripeptidase from Pediococcus pentosaceus K9.2. Appl. Environ. Microbiol. 63, 4872-4876.
  27. Simpson, R. J., Neuberger, M. R. and Liu, T. Y. (1976) Complete amino acid analysis of proteins from a single hydrolysate. J. BioI. Chem. 251, 1936-1940.
  28. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J. and Klenk, D. C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76-85. https://doi.org/10.1016/0003-2697(85)90442-7
  29. Tahirov, T. H., Oki, H., Tsukihara, T., Ogasahara, K., Yutani, K., Ogata, K., Izu, Y., Tsunasawa, S. and Kato, I. (1998) Crystal structure of methionine aminopeptidase from hyperthermophile, Pyrococcus furiosus. J. Mol. Biol. 284, 101-124. https://doi.org/10.1006/jmbi.1998.2146
  30. Taylor, A. (1993) Aminopeptidase: towards a mechanism of action. Trends Biochem Sci. 18, 167-172.
  31. Tsunasawa, S., lzu, Y., Miyagi, M. and Kato, I. (1997) Methionine aminopeptidase from the hyperthermophilic Archaeon Pyrococcus furiosus: molecular cloning and overexpression in Escherichia coli of the gene, and characteristics of the enzyme. J. Biochem. 122, 843-850. https://doi.org/10.1093/oxfordjournals.jbchem.a021831
  32. Walker, K. W. and Bradshaw, R. A. (1998) Yeast methionine aminopeptidase I can utilize either$Zn^{2+} \; or \; Co^{2+}$as a cofactor: a case of mistaken identity? Protein Sci. 7, 2684-2687. https://doi.org/10.1002/pro.5560071224

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