Catabolism of bis(5'-nucleosidyl) oligophosphates in Escherichia coli: metal requirements and substrate specificity of homogeneous diadenosine-5',5'''-P1,P4-tetraphosphate pyrophosphohydrolase
- 1 February 1985
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 24 (4) , 914-922
- https://doi.org/10.1021/bi00325a016
Abstract
Diadenosine-5''5''''''-P1,P4-tetraphosphate pyrophosphohydrolase (diadenosinetetraphosphatase) from Escherichia coli strain EM 20031 was 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.degree. C, 50 mM tris(hydroxymethyl)aminomethane, pH 7.8, 50 .mu.M Ap4A, 0.5 .mu.M EDTA and 50 .mu.M CoCl2]. The enzyme is a single polypeptide chain of MW 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 2 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 .mu.M) to the reaction buffer containing 0.5 .mu.M EDTA strongly stimulates Ap4A hydrolysis (stimulation 2500-fold). With 50 .mu.M MnCl2, the stimulation is 900-fold. Ca2+, Fe2+ and Mg2+ have no effect. The Km for Ap4A is 22 .mu.M with Co2+ and 12 .mu.M 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 .mu.M), prior to the assay or within the reaction mixture containing Co2+, causes a marked inhibition of Ap4A hydrolysis. The inhibition is relieved by prolonged incubation of the enzyme with excess EDTA.Keywords
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