1b99
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(New page: 200px<br /><applet load="1b99" size="450" color="white" frame="true" align="right" spinBox="true" caption="1b99, resolution 2.7Å" /> '''3'-FLUORO-URIDINE DIP...)
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Revision as of 09:21, 20 November 2007
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3'-FLUORO-URIDINE DIPHOSPHATE BINDING TO NUCLEOSIDE DIPHOSPHATE KINASE
Overview
Nucleoside diphosphate (NDP) kinases display low specificity with respect, to the base moiety of the nucleotides and to the 2'-position of the, ribose, but the 3'-hydroxyl is found to be important for catalysis. We, report in this paper the enzymatic analysis of a series of derivatives of, thymidine diphosphate (TDP) where the 3'-OH group was removed or replaced, by fluorine, azido, and amino groups. With Dictyostelium NDP kinase, kcat, decreases 15-200-fold from 1100 s-1 with TDP, and (kcat/Km)NDP decreases, from 12 x 10(6) to 10(3) to 5 x 10(4) M-1 s-1, depending on the substrate., The poorest substrates are 3'-deoxyTDP and 3'-azido-3'-deoxyTDP, while the, best modified substrates are 2',3'-dehydro-3'-deoxyTDP and, 3'-fluoro-3'-deoxyTDP. In a similar way, 3'-fluoro-2',3'-dideoxyUDP was, found to be a better substrate than 2',3'-dideoxyUDP, but a much poorer, substrate than 2'-deoxyUDP. (kcat/Km)NDP is sensitive to the viscosity of, the solution with TDP as the substrate but not with the modified, substrates. To understand the poor catalytic efficiency of the modified, nucleotides at a structural level, we determined the crystal structure of, Dictyostelium NDP kinase complexed to 3'-fluoro-2',3'-dideoxyUDP at 2.7 A, resolution. Significant differences are noted as compared to the TDP, complex. Substrate-assisted catalysis by the 3'-OH, which is effective in, the NDP kinase reaction, cannot occur with the modified substrate. With, TDP, the beta-phosphate, which is the leaving group when a gamma-phosphate, is transferred to His122, hydrogen bonds to the 3'-hydroxyl group of the, sugar; with 3'-fluoro-2',3'-dideoxyUDP, the beta-phosphate hydrogen bonds, to Asn119 and moves away from the attacking Ndelta of the catalytic, His122. Since all anti-AIDS nucleoside drugs are modified at the, 3'-position, these results are relevant to the role of NDP kinase in their, cellular metabolism.
About this Structure
1B99 is a Single protein structure of sequence from Dictyostelium discoideum with FUP and POP as ligands. Active as Nucleoside-diphosphate kinase, with EC number 2.7.4.6 Full crystallographic information is available from OCA.
Reference
Catalytic mechanism of nucleoside diphosphate kinase investigated using nucleotide analogues, viscosity effects, and X-ray crystallography., Gonin P, Xu Y, Milon L, Dabernat S, Morr M, Kumar R, Lacombe ML, Janin J, Lascu I, Biochemistry. 1999 Jun 1;38(22):7265-72. PMID:10353838
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