Analysis of the catalytic and binding residues of the diadenosine tetraphosphate pyrophosphohydrolase from Caenorhabditis elegans by site-directed mutagenesis

Hend M. Abdelghany, Scott Bailey, G. Michael Blackburn, John B. Rafferty, Alexander G. McLennan

Research output: Contribution to journalArticlepeer-review

Abstract

The contributions to substrate binding and catalysis of 13 amino acid residues of the Caenorhabditis elegans diadenosine tetraphosphate pyrophosphohydrolase (Ap4A hydrolase) predicted from the crystal structure of an enzyme-inhibitor complex have been investigated by site-directed mutagenesis. Sixteen glutathione S-transferase-Ap4A hydrolase fusion proteins were expressed and their kcat and Km values determined after removal of the glutathione S-transferase domain. As expected for a Nudix hydrolase, the wild type kcat of 23 s-1 was reduced by 105-, 103-, and 30-fold, respectively, by replacement of the conserved P4-phosphate-binding catalytic residues Glu56, Glu52, and Glu103 by Gln. Km values were not affected, indicating a lack of importance for substrate binding. In contrast, mutating His31 to Val or Ala and Lys83 to Met produced 10- and 16-fold increases in Km compared with the wild type value of 8.8 μM. These residues stabilize the P1-phosphate. H31V and H31A had a normal kcat but K83M showed a 37-fold reduction in kcat. Lys36 also stabilizes the P1-phosphate and a K36M mutant had a 10-fold reduced kcat but a relatively normal Km. Thus both Lys36 and Lys83 may play a role in catalysis. The previously suggested roles of Tyr27, His38, Lys79, and Lys81 in stabilizing the P2 and P3-phosphates were not confirmed by mutagenesis, indicating the absence of phosphate-specific binding contacts in this region. Also, mutating both Tyr76 and Tyr121, which clamp one substrate adenosine moiety between them in the crystal structure, to Ala only increased Km 4-fold. It is concluded that interactions with the P1- and P4-phosphates are minimum and sufficient requirements for substrate binding by this class of enzyme, indicating that it may have a much wider substrate range then previously believed.

Original languageEnglish (US)
Pages (from-to)4435-4439
Number of pages5
JournalJournal of Biological Chemistry
Volume278
Issue number7
DOIs
StatePublished - Feb 14 2003
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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