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 GST-Ap₄A hydrolase fusion proteins were expressed and their kcat and Km values determined after removal of the GST 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 to the wild type value of 8.8 M. These residues stabilise the P1 phosphate. H31V and H31A had a normal kcat but K83M showed a 37-fold reduction in kcat. Lys36 also stabilises 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 stabilising 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.