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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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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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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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73
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0344033223
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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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76
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77
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0344033222
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note
-
UDP-MurNAc-pentapeptide is being evaluated as an alternative acceptor.
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78
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0031013904
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Sequence similarities between MurG homologues and monogalactosyldiacylglycerol synthases have been noted previously. See: Shimojima, M.; Ohta, H.; Iwamatsu, A.; Masuda, T.; Shioi, Y.; Takamiya, K. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 333-337.
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Radominska, A.7
Lester, R.8
Siest, G.9
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80
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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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81
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0344033221
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note
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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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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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A crystal structure of a monomeric prokaryotic glycosyltransferase (SpsA) was recently reported. See: Charnock, S. J.; Davies, G. J. Biochemistry 1999, 38, 6380-6385.
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