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A Double Role for a Strictly Conserved Serine: Further Insights into the dUTPase Catalytic Mechanism
Högskolan i Kalmar, Naturvetenskapliga institutionen. (J. Kvassman: The dUTPase project)
Department of Pure and Applied Biochemistry, Lund UniVersity, Lund, Sweden. (L. Bülow)
Department of Pure and Applied Biochemistry, Lund UniVersity, Lund, Sweden. (L. Bülow)
Högskolan i Kalmar, Naturvetenskapliga institutionen. (J. Kvassman: The dUTPase Project)
2008 (Engelska)Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, nr 30, s. 7863-7874Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Ser72 at the active site of the Escherichia coli dUTPase has been mutated to an alanine, and the properties of the mutant have been investigated. The serine is absolutely conserved among the monomeric and trimeric dUTPases (including the bifunctional dCTP deaminase:dUTPases), and it has been proposed to promote catalysis by balancing negative charge at the oxygen that bridges the α- and  β-phosphorus of the substrate. In all reported complexes of dUTPases with the substrate analogue α,β-imido-dUTP·Mg, the serine β-OH is indeed hydrogen bonded to the α,β-bridging nitrogen of the analogue. However, in the complex of the Asp90→Asn mutant dUTPase with the true substrate dUTP·Mg, the serine β-OH points in the opposite direction and may form a hydrogen bond to Asn84 at the bottom of the pyrimidine pocket. Here we show that the replacement of the β-OH by hydrogen reduces kcat from 5.8 to 0.008 s-1 but also k-1, the rate of substrate dissociation, from 6.2 to 0.1 s-1 (KM = 6 × 10-9 M). We conclude that the serine β-OH exercises both ground state (GS) destabilization and transition state (TS) stabilization, effects not usually linked to a single residue. With experimental support, we argue that the β-OH destabilizes the GS by imposing conformational constraints on the enzyme and that formation ofthe TS depends on a rotation of the serine side chain that not only relieves the constraints but brings the β-OH into a position where it can electrostatically stabilize the TS. This rotation would also allow the β-OH to promote both deamination and hydrolysis in the bifunctional deaminases. We find that the E. coli dUTPase does not catalyze the hydrolysis of the α,β-imido-dUTP·Mg, suggesting that the analogue provides the hydrogen in the bond to the serine β-OH.

Ort, förlag, år, upplaga, sidor
American Chemical Society , 2008. Vol. 47, nr 30, s. 7863-7874
Nyckelord [en]
dUTPase, Catalytic mechanism, Transition state stabilization, Ground state destabilization, Conserved serine, Mutant
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
Naturvetenskap, Biokemi
Identifikatorer
URN: urn:nbn:se:hik:diva-900DOI: 10.1021/bi800325jOAI: oai:DiVA.org:hik-900DiVA, id: diva2:114182
Projekt
General and species specific inhibition of dUTP pyrophosphatases, a potential approach in the treatment of cancers and infectious diseasesTillgänglig från: 2008-11-27 Skapad: 2008-11-07 Senast uppdaterad: 2017-12-14Bibliografiskt granskad

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Förlagets fulltexthttp://pubs.acs.org/cgi-bin/abstract.cgi/bichaw/2008/47/i30/abs/bi800325j.html

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Gonzalez Palmén, LorenaKvassman, Jan-Olov

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