Analysis of the Catalytic and Binding Residues of the Diadenosine Tetraphosphate Pyrophosphohydrolase fromCaenorhabditis elegansby Site-directed Mutagenesis

Hend M. Abdelghany, Scott Bailey, G. Michael Blackburn, John B. Rafferty, Alexander G. McLennan
2002 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
more » ... 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.
doi:10.1074/jbc.m211983200 pmid:12475970 fatcat:driwvd7mwvf2dpgwjhbrlptbve