Purification and properties of ATP-sulphurylase from Nitrobacter agilis

Abstract

1. 1. As in other bacteria, SO 4 2− in Nitrobacter agilis is first activated by ATP, before it is reduced. Both 35S-labelled adenosine 5′-phosphosulphate ([ 35S]APS) and 35S-labelled 3′-phosphoadenosine 5′-phosphosulfate ([ 35S]PAPS) are produced from 35SO 4 2− and ATP in cell-free extracts of this bacterium. The enzymes catalysing these reactions are found in the supernatant fractions left after centrifuging cell extracts at 144 000 × g for 4 h . 2. 2. In addition to the 35S radioassay technique, the bioluminescence method of the firely Photinus pyralis (luciferin-luciferase) was used to follow ATP production from APS and PP 1 during the purification of the enzyme. 3. 3. ATP-sulphurylase ATP- sulphate adenyltransferase, EC 2.7.7.4), which catalyzes the formation of APS, was purified about 820-fold by DEAE-cellulose, DEAE-Sephadex and Sephadex G-200 column chromatography, followed by the sucrose density gradient. A starch-gel electrophoresis of the purified enzyme produced a single protein band. 4. 4. K m values for the purified enzyme are as follows: ATP, 1.4 · 10 −3 M ; APS, 2.5 · 10 −5 M; PP i, 1.2 · 10 −4 ; MgCl 2 3.5 · 10 −4 M . 5. 5. The molecular weight of the enzyme is about 700 000 and the sedimentation coefficient is 12.8 S. The pH optimum is 7.4. 6. 6. The enzyme was inhibited by group IV anions. The following thiol reagents stimulated activity: GSH, l-cysteine, β-mercaptoethanol and Cleland's reagent (dithiothreitol). p- Chloromercuribenzoate was a strong inhibitor and this effect was reversed by thiol compounds.