Analysis of the Catalytic and Binding Residues of the Diadenosine Tetraphosphate Pyrophosphohydrolase fromCaenorhabditis elegansby Site-directed Mutagenesis
Journal of Biological Chemistry
The contributions to substrate binding and catalysis of 13 amino acid residues of the Caenorhabditis elegans diadenosine tetraphosphate pyrophosphohydrolase (Ap 4 A hydrolase) predicted from the crystal structure of an enzyme-inhibitor complex have been investigated by site-directed mutagenesis. Sixteen glutathione S-transferase-Ap 4 A 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
... xpected for a Nudix hydrolase, the wild type k cat of 23 s ؊1 was reduced by 10 5 -, 10 3 -, and 30-fold, respectively, by replacement of the conserved P 4 -phosphate-binding catalytic residues Glu 56 , Glu 52 , and Glu 103 by Gln. K m values were not affected, indicating a lack of importance for substrate binding. In contrast, mutating His 31 to Val or Ala and Lys 83 to Met produced 10-and 16-fold increases in K m compared with the wild type value of 8.8 M. These residues stabilize the P 1 -phosphate. H31V and H31A had a normal k cat but K83M showed a 37-fold reduction in k cat . Lys 36 also stabilizes the P 1 -phosphate and a K36M mutant had a 10-fold reduced k cat but a relatively normal K m . Thus both Lys 36 and Lys 83 may play a role in catalysis. The previously suggested roles of Tyr 27 , His 38 , Lys 79 , and Lys 81 in stabilizing the P 2 and P 3 -phosphates were not confirmed by mutagenesis, indicating the absence of phosphate-specific binding contacts in this region. Also, mutating both Tyr 76 and Tyr 121 , 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 P 1 -and P 4 -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.