A double role for a strictly conserved serine: Further insights into the dUTPase catalytic mechanism

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Abstract

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 alpha- and beta-pbosphorus of the substrate. In all reported complexes of dUTPases with the substrate analogue alpha,beta-imido-dUTP center dot Mg, the serine beta-OH is indeed hydrogen bonded to the alpha,beta-bridging nitrogen of the analogue. However, in the complex of the Asp90 -> Asn mutant dUTPase with the true substrate dUTP center dot Mg, the serine P-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 P-OH by hydrogen reduces k(cat) 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) (K-M = 6 x 10(-9) M). We conclude that the serine P-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 P-OH destabilizes the GS by imposing conformational constraints on the enzyme and that formation of the TS depends on a rotation of the serine side chain that not only relieves the constraints but brings the P-OH into a position where it can electrostatically stabilize the TS. This rotation would also allow the P-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 alpha,beta-imido-dUTP center dot Mg, suggesting that the analogue provides the hydrogen in the bond to the serine beta-OH.

Detaljer

Författare
  • Lorena Gonzalez Palmen
  • Kristian Becker
  • Leif Bülow
  • Jan-Olov Kvassman
Enheter & grupper
Forskningsområden

Ämnesklassifikation (UKÄ) – OBLIGATORISK

  • Biokemi och molekylärbiologi
Originalspråkengelska
Sidor (från-till)7863-7874
TidskriftBiochemistry
Volym47
Utgivningsnummer30
StatusPublished - 2008
PublikationskategoriForskning
Peer review utfördJa