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Volumn 121, Issue 37, 1999, Pages 8415-8426

The kinetic characterization of Escherichia coli MurG using synthetic substrate analogues

Author keywords

[No Author keywords available]

Indexed keywords

BACTERIAL ENZYME; MEMBRANE ENZYME;

EID: 0033595530     PISSN: 00027863     EISSN: None     Source Type: Journal    
DOI: 10.1021/ja991556t     Document Type: Article
Times cited : (79)

References (95)
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    • note
    • It is obvious that the biotin-capture assay described can also be adapted for a scintillation proximity assay. We have found that MurG will transfer radiolabeled GlcNAc to biotinylated substrate 1b that is already bound to the avidin resin, raising the possibility of developing a continuous scintillation proximity assay.
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    • note
    • The murg gene was subcloned into the pET3a vector from a pET15b: murg plasmid generously supplied by Ms. Sunita Midha of Transcell Technologies. The sequence of this gene was found to be identical to that previously reported (ref 15a,b) except for the presence of an additional ATG codon at the 5′ end, which was introduced during construction of the pET15b:murg plasmid. The N-terminal sequence of the expressed protein is MMSGQG....We chose not to remove the additional methionine because it appears to increase the level of expression of the protein.
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    • Crouvoisier et al. have reported the purification of His-tagged MurG to greater than 80% using immobilized metal ion affinity chromatography. See: Crouvoisier, M.; Mengin-Lecreulx, D.; van Heijenoort, J. FEBS Lett. 1999, 449, 289-292.
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    • We have not yet been able to determine the precise mechanism. UDP is a noncompetitive inhibitor of the acceptor substrate and a competitive inhibitor of the UDP-GlcNAc donor, implying that both UDP and UDP-GlcNAc bind to the free form of the enzyme at the same site. This rules out a compulsory-ordered mechanism in which the acceptor substrate binds first. None of the acceptor analogues reported here are both competitive inhibitors of 1b and incapable of reacting, and we cannot complete the mechanistic analysis until we obtain suitable inhibitors. See: (a) Fromm, H. J. Methods Enzymol. 1979, 63, 467-486. Both types of mechanisms have been observed in other glycosyltransferases. See: (b) Qiao, L.; Murray, B. W.; Shimazaki, M.; Schultz, J.; Wong, C.-H. J. Am. Chem. Soc. 1996, 118, 7653-7662. (c) Palcic, M. M.; Heerze, L. D.; Srivastava, O. P.; Hindsgaul, O. J. Biol. Chem. 1989, 264, 17174-17181. Additional compounds are being made to probe the mechanism further.
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    • We have not yet been able to determine the precise mechanism. UDP is a noncompetitive inhibitor of the acceptor substrate and a competitive inhibitor of the UDP-GlcNAc donor, implying that both UDP and UDP-GlcNAc bind to the free form of the enzyme at the same site. This rules out a compulsory-ordered mechanism in which the acceptor substrate binds first. None of the acceptor analogues reported here are both competitive inhibitors of 1b and incapable of reacting, and we cannot complete the mechanistic analysis until we obtain suitable inhibitors. See: (a) Fromm, H. J. Methods Enzymol. 1979, 63, 467-486. Both types of mechanisms have been observed in other glycosyltransferases. See: (b) Qiao, L.; Murray, B. W.; Shimazaki, M.; Schultz, J.; Wong, C.-H. J. Am. Chem. Soc. 1996, 118, 7653-7662. (c) Palcic, M. M.; Heerze, L. D.; Srivastava, O. P.; Hindsgaul, O. J. Biol. Chem. 1989, 264, 17174-17181. Additional compounds are being made to probe the mechanism further.
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    • note
    • cat values, which are based on a molecular weight of 38 kDa for the active enzyme and an assumption of full activity based on the nominal concentration, could be underestimated. The enzyme is purified as a dimer with a molecular weight of ∼76 kDa; furthermore, control experiments have shown that it loses activity rapidly upon exposure to new surfaces - as upon dilution and transfer.
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    • note
    • UDP-MurNAc-pentapeptide is being evaluated as an alternative acceptor.
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    • Interfacial catalysis has been extensively studied with phospholipases, which are very sensitive to the presence of membranes. See, for example: Jain, M. K.; Gelb, M. H.; Rogers, J.; Berg, O. G. Methods Enzymol. 1995, 249, 567-614.
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    • 2O are sharp and show no signs of the broadening that would indicate aggregation. Therefore, the better activity of the lipid-linked substrates is not believed to be due to the presence of micellular structures in the enzymatic reactions.
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    • 52e,f Mechanistic studies have been reported for some glycosyltransferases. See, e.g.: (a) Murray, B. W.; Wittmann, V.; Burkart, M. D.; Hung, S.-C.; Wong, C.-H. Biochemistry, 1997, 36, 823-831. (b) Kim, S. C.; Singh, A. N.; Raushel, F. M. Arch. Biochem. Biophys. 1988, 267, 54-59. (c) Bruner, M.; Horenstein B. A. Biochemistry, 1998, 37, 289-297.
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