Catabolism of bis(5'-nucleosidyl) oligophosphates in Escherichia coli: metal requirements and substrate specificity of homogeneous diadenosine-5',5'''-P1,P4-tetraphosphate pyrophosphohydrolase

• Published in: Biochemistry (Easton) Vol. 24; no. 4; pp. 914 - 922
• Main Authors: Plateau, Pierre, Fromant, Michel, Brevet, Annie, Gesquiere, Anne, Blanquet, Sylvain
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.02.1985
• Subjects: Acid Anhydride Hydrolases; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Cations, Divalent; Chromatography, High Pressure Liquid; Edetic Acid - pharmacology; Enzymes and enzyme inhibitors; Escherichia coli - enzymology; Fundamental and applied biological sciences. Psychology; Hydrolases; Kinetics; Molecular Weight; Oligonucleotides - metabolism; Phosphoric Diester Hydrolases - isolation & purification; Phosphoric Diester Hydrolases - metabolism; Substrate Specificity; Purification; Enzyme; Escherichia coli; Substrate specificity; Bacteria; Diadenosine tetraphosphatase; Metabolism; Escherichieae; Enterobacteriaceae
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00325a016

Diadenosine-5',5'''-P1,P4-tetraphosphate pyrophosphohydrolase (diadenosinetetraphosphatase) from Escherichia coli strain EM20031 has been purified 5000-fold from 4 kg of wet cells. It produces 2.4 mg of homogeneous enzyme with a yield of 3.1%. The enzyme activity in the reaction of ADP production from Ap4A is 250 s-1 [37 degrees C, 50 mM tris(hydroxymethyl)aminomethane, pH 7.8, 50 microM Ap4A, 0.5 microM ethylenediaminetetraacetic acid (EDTA), and 50 microM CoCl2]. The enzyme is a single polypeptide chain of Mr 33K, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and high-performance gel permeation chromatography. Dinucleoside polyphosphates are substrates provided they contain more than two phosphates (Ap4A, Ap4G, Ap4C, Gp4G, Ap3A, Ap3G, Ap3C, Gp3G, Gp3C, Ap5A, Ap6A, and dAp4dA are substrates; Ap2A, NAD, and NADP are not). Among the products, a nucleoside diphosphate is always formed. ATP, GTP, CTP, UTP, dATP, dGTP, dCTP, and dTTP are not substrates; Ap4 is. Addition of Co2+ (50 microM) to the reaction buffer containing 0.5 microM EDTA strongly stimulates Ap4A hydrolysis (stimulation 2500-fold). With 50 microM MnCl2, the stimulation is 900-fold. Ca2+, Fe2+, and Mg2+ have no effect. The Km for Ap4A is 22 microM with Co2+ and 12 microM with Mn2+. The added metals have similar effects on the hydrolysis of Ap3A into ADP + AMP. However, in the latter case, the stimulation by Co2+ is small, and the maximum stimulation brought by Mn2+ is 9 times that brought by Co2+. Exposure of the enzyme to Zn2+ (5 microM), prior to the assay or within the reaction mixture containing Co2+, causes a marked inhibition of Ap4A hydrolysis.