title: Recognition of beta beta'-substituted and alpha beta,alpha'beta'-disubstituted phosphonate analogues of bis(5'-adenosyl) tetraphosphate by the bis(5'-nucleosidyl)-tetraphosphate pyrophosphohydrolases from Artemia embryos and Escherichia coli.
abstract: A total of 13 phosphonate analogues of bis(5'-adenosyl) tetraphosphate (AppppA) have been tested as substrates and inhibitors of the asymmetrically cleaving bis(5'-nucleosidyl) tetraphosphatase (NppppNase) from Artemia and the symmetrically cleaving NppppNase from Escherichia coli. With the Artemia enzyme, the substrate efficiency of beta beta'-substituted compounds decreased with decreasing substituent electronegativity (O greater than CF2 greater than CHF greater than CCl2 greater than CHCl greater than CH2) such that AppCF2ppA and AppCH2ppA were hydrolyzed at 70% and 2.5% of the rate of AppppA, respectively. These compounds were competitive inhibitors of this enzyme with Ki values that generally also decreased with electronegativity from 12 microM for AppCF2ppA to 0.4 microM for AppCH2ppA (Km for AppppA = 33 microM). AppCH = CHppA and AppCH2CH2ppA were neither effective substrates nor inhibitors of the Artemia enzyme. Alpha beta,alpha'beta'-Disubstituted analogues were generally less effective inhibitors with Ki values ranging from 23 microM (ApCH2ppCH2pA) to greater than 1.5 mM (ApCH2CH2ppCH2CH2pA). However, they displayed a low and unexpected rate of symmetrical cleavage by the Artemia enzyme: e.g., ApCHFppCHFpA yielded ApCHFp at 3% of the rate of AppppA breakdown. Both sets of analogues were also competitive inhibitors of the E. coli NppppNase with Ki values ranging from 7 microM (AppCH2ppA) to 250 microM (ApCH2CH2ppCH2CH2pA) (Km for AppppA = 28 microM). The only alpha beta,alpha'beta'-disubstituted analogue to be hydrolyzed by the E. coli enzyme was ApCF2ppCF2pA at 0.2% of the rate of AppppA; however, several of the beta beta'-substituted compounds showed a limited degree of asymmetrical cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, University of Liverpool, U.K.
authors.forename: A G
authors.initials: AG
authors.lastname: McLennan
authors.forename: G E
authors.initials: GE
authors.lastname: Taylor
authors.forename: M
authors.initials: M
authors.lastname: Prescott
authors.forename: G M
authors.initials: GM
authors.lastname: Blackburn
mesh_terms: D017766:Acid Anhydride Hydrolases; D000818:Animals; D001156:Artemia / Q000196:embryology / Q000201:enzymology*; D001665:Binding Sites; D015226:Dinucleoside Phosphates / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology*; D063065:Organophosphonates / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011485:Protein Binding; D013379:Substrate Specificity
chemical_list: D015226:Dinucleoside Phosphates; D063065:Organophosphonates; C020733:diadenosine tetraphosphate; D010727:Phosphoric Diester Hydrolases; D017766:Acid Anhydride Hydrolases; C036849:bis(5'-nucleosyl)tetraphosphatase (symmetrical)