Kinetic properties and inhibition of the dimeric dUTPase-dUDPase from Campylobacter jejuni

Juan A. Musso-Buendia, Antonio E. Vidal, Ganasan Kasinthan, Corinne Nguyen, Juana Carrero-Lerida, Luis M. Ruiz-perez, Keith Wilson, Nils Gunnar Johansson, Ian H. Gilbert, Dolores Gonzalez-Pacanowska

Research output: Contribution to journalArticlepeer-review

Abstract

The enzyme deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) catalyses the hydrolysis of dUTP to dUMP and PPi thus controlling the incorporation of uracil into DNA genomes. In Campylobacter jejuni dUTPase exhibits structural properties of dimeric proteins characteristic of protozoa of the Kinetoplastidae family. In the present study we perform a kinetic analysis of Campylobacter dUTPase using the continuous spectrophotometric method and show that the enzyme is highly specific for deoxyuridine nucleotides. The Michaelis-Menten constant for dUTP was 0.66M while the kcat was 12.3s-1. dUDP was also efficiently hydrolysed although the specificity constant, kcat/Km, was five fold lower than for dUTP. The reaction product and the non hydrolysable analogue , imido dUDP are potent inhibitors of the enzyme while several analogues of dUMP with substituents at the 3'- and 5'-positions active against trimeric dUTPases, show poor inhibitory activity. Apparent structural and kinetic differences with other eukaryotic dUTPases suggest that the present enzyme might be exploited as a target for new drugs against campylobacteriosis.

Original languageEnglish
Pages (from-to)111-116
Number of pages6
JournalJOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
Volume24
Issue number1
DOIs
Publication statusPublished - 2009

Keywords

  • duTPase-duDPase
  • inhibition
  • kinetics
  • Campyrobacter jejuni
  • SIMPLEX-VIRUS TYPE-1
  • ESCHERICHIA-COLI
  • LEISHMANIA-MAJOR
  • TRYPANOSOMA-CRUZI
  • MONOMERIC DUTPASE
  • CRYSTAL-STRUCTURE
  • NUCLEOTIDOHYDROLASE
  • SEQUENCE
  • REVEALS
  • ENZYME

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