Linked Life Data

18597482

Source:http://linkedlifedata.com/resource/pubmed/id/18597482

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
30
pubmed:dateCreated
2008-7-23
pubmed:abstractText
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-phosphorus of the substrate. In all reported complexes of dUTPases with the substrate analogue alpha,beta-imido-dUTP.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.Mg, the serine beta-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 beta-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 beta-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 beta-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 beta-OH into a position where it can electrostatically stabilize the TS. This rotation would also allow the beta-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.Mg, suggesting that the analogue provides the hydrogen in the bond to the serine beta-OH.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jul
pubmed:issn
1520-4995
pubmed:author
pubmed:issnType
Electronic
pubmed:day
29
pubmed:volume
47
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
7863-74
pubmed:meshHeading
pubmed:year
2008
pubmed:articleTitle
A double role for a strictly conserved serine: further insights into the dUTPase catalytic mechanism.
pubmed:affiliation
Department of Chemistry and Biomedical Sciences, School of Pure and Applied Natural Sciences, University of Kalmar, Kalmar, Sweden.
pubmed:publicationType
Journal Article