- Directed evolution of sulfotransferases and paraoxonases by ancestral libraries
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Large libraries of randomly mutated genes are applied in directed evolution experiments in order to obtain sufficient variability. These libraries, however, contain mostly inactive variants, and the very low frequency of improved variants can only be isol
- Alcolombri, Uria,Elias, Mikael,Tawfik, Dan S.
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Read Online
- METHOD FOR PRODUCING P-NITROPHENYL SULFATE
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PROBLEM TO BE SOLVED: To provide a method for producing a p-nitrophenyl sulfate (PNPS salt) such as potassium p-nitrophenyl sulfate (PNPS-K). SOLUTION: A p-nitrophenyl sulfate is produced by reacting p-nitrophenol (PNP) with sulfur trioxide complex in a medium having a low capacity of dissolving p-nitrophenyl sulfate such as tetrahydrofuran, and neutralizing the reaction product with tert-butoxy salt. SELECTED DRAWING: None COPYRIGHT: (C)2017,JPOandINPIT
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Paragraph 0043; 0046
(2017/12/27)
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- Probing the origin of the compromised catalysis of E. coli alkaline phosphatase in its promiscuous sulfatase reaction
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The catalytic promiscuity of E. coli alkaline phosphatase (AP) and many other enzymes provides a unique opportunity to dissect the origin of enzymatic rate enhancements via a comparative approach. Here, we use kinetic isotope effects (KIEs) to explore the origin of the 109-fold greater catalytic proficiency by AP for phosphate monoester hydrolysis relative to sulfate monoester hydrolysis. The primary 18O KIEs for the leaving group oxygen atoms in the AP-catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP) and p-nitrophenylsulfate (pNPS) decrease relative to the values observed for nonenzymatic hydrolysis reactions. Prior linear free energy relationship results suggest that the transition states for AP-catalyzed reactions of phosphate and sulfate esters are "loose" and indistinguishable from that in solution, suggesting that the decreased primary KIEs do not reflect a change in the nature of the transition state but rather a strong interaction of the leaving group oxygen atom with an active site Zn2+ ion. Furthermore, the primary KIEs for the two reactions are identical within error, suggesting that the differential catalysis of these reactions cannot be attributed to differential stabilization of the leaving group. In contrast, AP perturbs the KIE for the nonbridging oxygen atoms in the reaction of pNPP but not pNPS, suggesting a differential interaction with the transferred group in the transition state. These and prior results are consistent with a strong electrostatic interaction between the active site bimetallo Zn2+ cluster and one of the nonbridging oxygen atoms on the transferred group. We suggest that the lower charge density of this oxygen atom on a transferred sulfuryl group accounts for a large fraction of the decreased stabilization of the transition state for its reaction relative to phosphoryl transfer.
- Catrina, Irina,O'Brien, Patrick J.,Purcell, Jamie,Nikolic-Hughes, Ivana,Zalatan, Jesse G.,Hengge, Alvan C.,Herschlag, Daniel
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p. 5760 - 5765
(2008/02/04)
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- Ground state structures of sulfate monoesters and sulfamates reveal similar reaction coordinates for sulfuryl and sulfamyl transfer
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Structure/reactivity and structure/structure correlations of 5 sulfate monoesters and 11 sulfamate esters determined by low temperature X-ray crystallography reveal similar ground state deformations that suggest similar reaction coordinates for sulfuryl and sulfamyl group transfer. The Royal Society of Chemistry 2006.
- Denehy, Emma,White, Jonathan M.,Williams, Spencer J.
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p. 314 - 316
(2008/02/08)
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