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

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 k_cat and K_m values determined after removal of the glutathione S-transferase domain. As expected for a Nudix hydrolase, the wild type k_cat 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. K_m 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 K_m compared with the wild type value of 8.8 μM. These residues stabilize the P¹-phosphate. H31V and H31A had a normal k_cat but K83M showed a 37-fold reduction in k_cat. Lys³⁶ also stabilizes the P¹-phosphate and a K36M mutant had a 10-fold reduced k_cat but a relatively normal K_m. 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 K_m 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.