Purification and steady-state kinetics of adenosine 5'-pyrophosphate sulphurylase from baker's yeast.
ADP sulphurylase (EC 2.7.7.5) was purified by chromatography on Sephadex G-200 and DEAE-cellulose. The enzyme was assayed by measuring the incorporation of [32P]Pi into ADP in the presence of the substrate for the reverse reaction, adenosine 5'-sulphatophosphate. In the concentration ranges investigated, by using initial-velocity, product-inhibition and isotope-exchange studies, the data were consistent with a Ping Pong reaction mechanism, with Km for adenosine 5'-sulphatophosphate of 1.20 +/- 0.08 mM and a Km for Pi of 4.95 +/- 0.15 mM. Competitive substrate inhibition by Pi (Ki = 11.7 +/- 0.3 mM) was found. ADP sulphurylase catalyses a sulphate-independent Pi-ADP exchange reaction, the kinetics of which are consistent with the kinetics of the overall reaction, inconsistent with the assay of Burnell & Anderson [(1973) Biochem. J. 133, 417-428], which is based on a sulphate-dependent Pi-ADP exchange reaction.
D000244:Adenosine Diphosphate; D007700:Kinetics; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D010710:Phosphates / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013431:Sulfates / Q000494:pharmacology
D010710:Phosphates; D013431:Sulfates; D000244:Adenosine Diphosphate; D009713:Nucleotidyltransferases