TAILIEUCHUNG - Báo cáo khoa học: Functional studies of active-site mutants from Drosophila melanogaster deoxyribonucleoside kinase Investigations of the putative catalytic glutamate–arginine pair and of residues responsible for substrate specificity

The catalytic reaction mechanism and binding of substrates was investi-gated for the multisubstrateDrosophila melanogasterdeoxyribonucleoside kinase. Mutation of E52 to D, Q and H plus mutations of R105 to K and H were performed to investigate the proposed catalytic reaction mech-anism, in which E52 acts as an initiating base and R105 is thought to sta-bilize the transition state of the reaction. | ễFEBS Journal Functional studies of active-site mutants from Drosophila melanogaster deoxyribonucleoside kinase Investigations of the putative catalytic glutamate-arginine pair and of residues responsible for substrate specificity Louise Egeblad-Welin1 2 z Yonathan Sonntag Hans Eklund3 and Birgitte Munch-Petersen1 1 Department of Science Systems and Models Roskilde University Denmark 2 Department of Molecular Biosciences Swedish University of AgriculturalSciences Uppsala BiomedicalCentre Sweden 3 Department of Molecular Biology Swedish University of AgriculturalSciences Uppsala BiomedicalCentre Sweden Keywords catalytic mechanism deoxyribonucleoside kinase dTTP enzyme kinetics nucleoside analogues Correspondence B. Munch-Petersen Department of Science Systems and Models Roskilde University Box 260 DK 4000 Roskilde Denmark Fax 45 46743011 Tel 45 46742418 E-mail bmp@ L. Egeblad-Welin Department of Molecular Biosciences Swedish University of Agricultural Sciences Box 575 Biomedical Center S-751 25 Uppsala Sweden Fax 46 18536971 Tel. 46 184714192 E-mail These authors contributed equally to this work Received 2 November 2006 revised 4 January 2007 accepted 16 January 2007 The catalytic reaction mechanism and binding of substrates was investigated for the multisubstrate Drosophila melanogaster deoxyribonucleoside kinase. Mutation of E52 to D Q and H plus mutations of R105 to K and H were performed to investigate the proposed catalytic reaction mechanism in which E52 acts as an initiating base and R105 is thought to stabilize the transition state of the reaction. Mutant enzymes E52D E52H and R105H showed a markedly decreased kcat while the catalytic activity of E52Q and R105K was abolished. The E52D mutant was crystallized with its feedback inhibitor dTTP. The backbone conformation remained unchanged and coordination between D52 and the dTTP-Mg complex was observed. The observed decrease in kcat for E52D was most likely due to an increased

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