Cloning of the ATP sulphurylase gene of Schizosaccharomyces pombe by functional complementation

The ATP sulphurylase gene of Schizosaccharomyces pombe has been cloned by complementation of cysteine auxotrophy of a selenate-resistant mutant, which supposedly had a defect in ATP sulphurylase. A sulphate nonutilizing (cysteine auxotrophic) and selenate-resistant mutant of S. pombe was transformed with a wild-type S. pombe genomic library and sulphate-utilizing clones were isolated. The open reading frame encoding the ATP sulphurylase enzyme was found to be responsible for the restoration of sulphate assimilation. Transformants became as sensitive for selenate as the wild-type strain and produced a comparable amount of ATP sulphurylase as the prototrophic strains. The cloned ATP sulphurylase gene (sua1) proved to be an efficient selection marker in an ARS vector, when different isogenic or nonisogenic S. pombe selenate-resistant mutants were used as cloning hosts. Complementation of sua1– mutations by sua1-bearing multicopy vectors functions as a useful dual positive and negative selection marker. The cloned sua1 gene also complemented the met3 (ATP sulphurylase deficient) mutation in Saccharomyces cerevisiae .

Kinetic mechanism of adenosine 5′-phosphosulphate kinase from rat chondrosarcoma

Biosynthesis of the activated sulphate donor adenosine 3′-phosphate 5′-phosphosulphate (PAPS) involves the sequential action of two enzyme activities. ATP-sulphurylase catalyses the formation of APS (adenosine 5′-phosphosulphate) from ATP and free sulphate, and APS is then phosphorylated by APS kinase to produce PAPS. Initial-velocity patterns for rat chondrosarcoma APS kinase indicate a single-displacement formal mechanism with KmAPS 76 nM and KmATP = 24 microM. Inhibition studies using analogues of substrates and products were carried out to determine the reaction mechanism. An analogue of PAPS, adenosine 3′-phosphate 5′-[beta-methylene]phosphosulphate, exhibited competitive inhibition with APS and non-competitive inhibition with ATP. An analogue of APS, adenosine 5′-[beta-methylene]phosphosulphate was also competitive with APS and non-competitive with ATP. Adenosine 5′-[beta gamma-imido]triphosphate showed competitive inhibition with respect to ATP and produced mixed-type inhibition, with a pronounced intercept effect and a small slope effect, with respect to APS. These results are in accord with the formulation of the predominant pathway as a steady-state ordered mechanism with APS as the leading substrate and PAPS as the final product released.

Kinetic mechanism of ATP-sulphurylase from rat chondrosarcoma

ATP-sulphurylase catalyses the production of adenosine 5′-phosphosulphate (APS) from ATP and free sulphate with the release of PPi. APS kinase phosphorylates the APS intermediate to produce adenosine 3′-phosphate 5′-phosphosulphate (PAPS). The kinetic mechanism of rat chondrosarcoma ATP-sulphurylase was investigated by steady-state methods in the physiologically forward direction as well as the reverse direction. The sulphurylase activity was coupled to APS kinase activity in order to overcome the thermodynamic constraints of the sulphurylase reaction in the forward direction. Double-reciprocal initial-velocity plots for the forward sulphurylase intersect to the left of the ordinate for this reaction. KmATP and Kmsulphate were found to be 200 and 97 microM respectively. Chlorate, a competitive inhibitor with respect to sulphate, showed uncompetitive inhibition with respect to ATP with an apparent Ki of 1.97 mM. Steady-state data from experiments in the physiologically reverse direction also yielded double-reciprocal initial-velocity patterns that intersect to the left of the ordinate axis, with a KmAPS of 39 microM and a Kmpyrophosphate of 18 microM. The results of steady-state experiments in which Mg2+ was varied indicated that the true substrate is the MgPPi complex. An analogue of APS, adenosine 5′-[beta-methylene]phosphosulphate, was a linear inhibitor competitive with APS and non-competitive with respect to MgPPi. The simplest formal mechanism that agrees with all the data is an ordered steady-state single displacement with MgATP as the leading substrate in the forward direction and APS as the leading substrate in the reverse direction.

Effect of humic substances on photosynthetic sulphate assimilation in maize seedlings

Maize plants were grown in nutrient solution containing 0, 15, 30, 50 mg organic carbon L−1 of humic substances (HS) (M.W. < 12 KDa) extracted from a cambisol. After 14 d of growth the second and third leaves were collected and analyzed for chlorophyll and protein content and for the activities of ATP sulphurylase (ATP-s) and O-acetylserine sulphydrylase (OAS-s) to evaluate the effect of HS on maize sulphate assimilation pathway. Humic substances induced significant decreases in chlorophyll content, whereas the protein level slightly increased up to 30 mg L−1 of HS. ATP-s and OAS-s were positively affected by HS. The stimulations were different according to the enzyme and leaf investigated. Our results suggest that HS might play an important role in the efficiency of maize plants to assimilate sulphate. Key words: ATP-sulphurylase, chlorophyll, humic substances, maize, O-acetylserine sulphydrilase