Purification and properties of the enzyme ATP-sulfurylase and its relation to vitamin A

Abstract

1. 1. The enzyme ATP-sulfurylase which catalyzes the formation of adenosine 5′-phosphosulfate (APS) was purified about 1000-fold over supernatant fraction by ammonium sulfate and acid precipitation and by elution from Sephadex G-200, agarose and hydroxyapatite columns. 2. 2. The molecular weight of the enzyme was between 800 000 and 900 000; its optimum pH, at 8.0; its K m with respect to ATP, 1.6 · 10 −3M; K m (sulfate), 1.0 · 10 −4M; K m (APS), 2.5 · 10 −4 M; K m (PP 1), 3.7 · 10 −5M. There was inhibition at high PP 1 and APS concentrations and at low concentrations of ADP. Cu 2+ and Co 2+ were strongly inhibitory. p-Chloromercuribenzoate (PCMB) and EDTA also inhibited: the former inhibition was completely relieved by glutatione; the latter, by Mg 2+. The enzyme was irreversibly inactivated by deoxycholate. It was unstable in Tris-HCl buffer and decayed to about 15% activity in 24 h whether measured by ATP generation or molybdolysis. It was completely stabilized by phosphate but not by barbiturate or sulfate ions. 3. 3. The mechanism of the reaction was explored by a study of the reaction kinetics and the following was observed: (a) the rate of exchange of 32PP i into ATP was greater in presence than in absence of sulfate and greater (even in absence of SO 4 2− than the overall reaction of APS synthesis; (b) there was no exchange of 35SO 4 2− into APS in presence or absence of PP 1; and (c) the rate of reaction of APS with P i at low APS concentrations was independent and at high concentrations was dependent on PP i concentration. We conclude that the reaction proceeds through intermediate formation of an AMP-enzyme complex. 4. 4. 14C-Labeled vitamin A was administered to vitamin A-deficient rats, and ATP-sulfurylase was isolated after 3 days. Although radioactivity remained associated with the enzyme over some of the purification steps, highly active enzyme which had lost all radioactivity was obtained by fractionation on agarose columns. Similar results were obtained when the labeled vitamin was homogenized with rat embryo livers or injected into newborn rats. We conclude that neither vitamin A nor a metabolite of the vitamin is an integral or necessary part of the enzyme and suggest that since the enzyme is unstable, particularly under conditions of low phosphate concentration, vitamin A may help to stabilize the enzyme.