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Dealwis Chris G.; Chen Liqing; Brennan Catherine; Mandecki Wlodek; Abad-Zapatero Cele 《Protein engineering, design & selection : PEDS》1995,8(9):865-871
The substitution of aspartate at position 153 in Escherichiacoli alkaline phosphatase by glycine results in a mutant enzymewith 5-fold higher catalytic activity (kcat but no change inKm at pH 8.0 in 50 mM Tris-HCl. The increased kcat is achievedby a faster release of the phosphate product as a result ofthe lower phosphate affinity. The mutation also affects Mg2+binding, resulting in an enzyme with lower metal affinity. The3-D X-ray structure of the D153G mutant has been refined at2.5 Å to a crystallographic Rfactor of 16.2%. An analysisof this structure has revealed that the decreased phosphateaffinity is caused by an apparent increase in flexibility ofthe guanidinium side chain of Argl66 involved in phosphate binding.The mutation of Aspl53 to Gly also affects the position of thewater ligands of Mg2+, and the loop Glnl52Thrl55 is shiftedby 0.3 Å away from the active site. The weaker Mg2+ bindingof the mutant compared with the wild type is caused by an alteredcoordination sphere in the proximity of the Mg2+ ion, and alsoby the loss of an electrostatic interaction (Mg2+.COO-Aspl53)in the mutant Its ligands W454 and W455 and hydroxyl of Thrl55,involved in the octahedral coordination of the Mg2+ ion, arefurther apart in the mutant compared with the wild-type 相似文献
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Dianqing Sun Dr. Hai Xu Dr. Sanath R. Wijerathna Chris Dealwis Dr. Richard E. Lee Dr. 《ChemMedChem》2009,4(10):1649-1656
Analysis of the recently solved X‐ray crystal structures of Saccharomyces cerevisiae ribonucleotide reductase I (ScRnr1) in complex with effectors and substrates led to the discovery of a conserved water molecule located at the active site that interacted with the 2′‐hydroxy group of the nucleoside ribose. In this study 2′‐(2‐hydroxyethyl)‐2′‐deoxyadenosine 1 and the 5′‐diphosphate derivative 2 were designed and synthesized to see if the conserved water molecule could be displaced by a hydroxymethylene group, to generate novel RNR inhibitors as potential antitumor agents. Herein we report the synthesis of analogues 1 and 2 , and the co‐crystal structure of adenosine diphosphate analogue 2 bound to ScRnr1, which shows the conserved water molecule is displaced as hypothesized. 相似文献
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