Kinetic analysis of the effects of mutagenesis of W501 and V432 of the hepatitis C virus NS3 helicase domain on ATPase and strand-separating activity.
作者： Frank Preugschat , Dana P. Danger , Luke H. Carter , Roderick G. Davis , David J. T. Porter
DOI: 10.1021/BI9923860
关键词:
摘要: Two hydrophobic residues, W501 and V432, in the nucleic acid (NA) binding pocket of HCV helicase domain (E) were mutagenized an effort to investigate contributions these residues substrate affinities enzymatic activities. The wild-type [hE(wt)] mutant enzymes [hE(W501F), hE(W501A), hE(V432A)] for NA ATP determined by monitoring changes intrinsic protein fluorescence, fluorescence fluorescently tagged acid, activity. steady-state kinetic parameters hydrolysis (at saturating concentrations NA) similar those hE(wt). hE(W501F), hE(V432A) had strand-separating activities that 136%, 3.8%, 3.1% processivities reduced relative hE(W501F) hE(W501A) primarily due increase rate dissociation E. from E.ATP.NA. processivity was a reduction forward constant strand separation. This result suggested V432 may constitute part "stepping" motor did not display dominant negative phenotype assay with hE(wt) stored presence beta-mercaptoethanol covalently modified at three cysteinyl residues. biological significance potential reactivity on is unknown.
sci-hub.se 参考文章(12)
James A. McSwiggen, David G. Bear, Peter H. von Hippel, Interactions of Escherichia coli transcription termination factor rho with RNA. I. Binding stoichiometries and free energies. Journal of Molecular Biology. ,vol. 199, pp. 609- 622 ,(1988) , 10.1016/0022-2836(88)90305-1
James W. George, Robert M. Brosh, Steven W. Matson, A Dominant Negative Allele of the Escherichia coli uvrD Gene Encoding DNA Helicase II: A Biochemical and Genetic Characterization Journal of Molecular Biology. ,vol. 235, pp. 424- 435 ,(1994) , 10.1006/JMBI.1994.1003
T.M. Lohman, Helicase-catalyzed DNA unwinding. Journal of Biological Chemistry. ,vol. 268, pp. 2269- 2272 ,(1993) , 10.1016/S0021-9258(18)53765-0
Kinlin Chao, Timothy M. Lohman, DNA-induced dimerization of the Escherichia coli Rep helicase Journal of Molecular Biology. ,vol. 221, pp. 1165- 1181 ,(1991) , 10.1016/0022-2836(91)90926-W
J. Fetter, Jonathan Cohen, D. Danger, Joann Sanders-Loehr, E. C. Theil, The influence of conserved tyrosine 30 and tissue-dependent differences in sequence on ferritin function: use of blue and purple Fe(III) species as reporters of ferroxidation Journal of Biological Inorganic Chemistry. ,vol. 2, pp. 652- 661 ,(1997) , 10.1007/S007750050180
Frank Preugschat, Devron R. Averett, Berwyn E. Clarke, David J. T. Porter, A Steady-state and Pre-steady-state Kinetic Analysis of the NTPase Activity Associated with the Hepatitis C Virus NS3 Helicase Domain Journal of Biological Chemistry. ,vol. 271, pp. 24449- 24457 ,(1996) , 10.1074/JBC.271.40.24449
Leah E. Mechanic, Mark C. Hall, Steven W. Matson, Escherichia coli DNA Helicase II Is Active as a Monomer Journal of Biological Chemistry. ,vol. 274, pp. 12488- 12498 ,(1999) , 10.1074/JBC.274.18.12488
Ann D Kwong, Joseph L Kim, Govinda Rao, Dasa Lipovsek, Scott A Raybuck, Hepatitis C virus NS3/4A protease. Antiviral Research. ,vol. 40, pp. 1- 18 ,(1998) , 10.1016/S0166-3542(98)00043-6
B. E. Clarke, A. L. Brown, K. G. Grace, G. Z. Hastings, F. Brown, D. J. Rowlands, M. J. Francis, Presentation and immunogenicity of viral epitopes on the surface of hybrid hepatitis B virus core particles produced in bacteria. Journal of General Virology. ,vol. 71, pp. 1109- 1117 ,(1990) , 10.1099/0022-1317-71-5-1109
Sameer S Velankar, Panos Soultanas, Mark S Dillingham, Hosahalli S Subramanya, Dale B Wigley, Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism. Cell. ,vol. 97, pp. 75- 84 ,(1999) , 10.1016/S0092-8674(00)80716-3