title: Analysis of the catalytic and binding residues of the diadenosine tetraphosphate pyrophosphohydrolase from Caenorhabditis elegans by site-directed mutagenesis.
abstract: 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 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 microm. 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.
authors.affiliation: School of Biological Sciences, Biosciences Building, University of Liverpool, P. O. Box 147, Liverpool L69 7ZB, United Kingdom.
authors.forename: Hend M
authors.initials: HM
authors.lastname: Abdelghany
authors.forename: Scott
authors.initials: S
authors.lastname: Bailey
authors.forename: G Michael
authors.initials: GM
authors.lastname: Blackburn
authors.forename: John B
authors.initials: JB
authors.lastname: Rafferty
authors.forename: Alexander G
authors.initials: AG
authors.lastname: McLennan
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D017173:Caenorhabditis elegans / Q000201:enzymology*; D020134:Catalytic Domain / Q000235:genetics; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity
chemical_list: D017766:Acid Anhydride Hydrolases; C020023:bis(5'-nucleosyl)tetraphosphatase (asymmetrical)