Adenosine 5'-triphosphate sulphurylase from Saccharomyces cerevisiae.
1. ATP sulphurylase from Saccharomyces cerevisiae was purified 140-fold by using heat treatment, DEAE-cellulose chromatography and Sepharose 6B gel filtration. 2. The enzyme was stable at -15 degrees C, optimum reaction velocity was between pH7.0 and 9.0, and the activation energy was 62kJ/mol (14.7kcal/mol). 3. The substrate was shown to be the MgATP(2-) complex, free ATP being inhibitory. 4. Double-reciprocal plots from initial-velocity studies were intersecting and the K(m) of each substrate was determined at infinite concentration of the other (K(m) MgATP(2-), 0.07mm; MoO(4) (2-), 0.17mm). 5. Radio-isotopic exchange between the substrate pairs, adenosine 5'-[(35)S]sulphatophosphate and SO(4) (2-), (35)SO(4) (2-) and adenosine 5'-sulphatophosphate, occurred only in the presence of either MgATP(2-) or PP(i). This suggests, along with the initial-velocity data, a sequential reaction mechanism in which both substrates bind before any product is released. 6. The enzyme reaction was specific for ATP and was not inhibited by l-cysteine, l-methionine, SO(3) (2-), S(2)O(3) (2-) (all 2mm) nor by p-chloromercuribenzoate (1mm). 7. Competitive inhibition of the enzyme with respect to MoO(4) (2-) was produced by SO(4) (2-) (K(i)=2.0mm) and non-competitive inhibition by sulphide (K(i)=3.4mm). 8. Adenosine 5'-sulphatophosphate inhibited strongly and concentrations as low as 0.02mm altered the normal hyperbolic velocity-substrate curves with both MgATP(2-) and MoO(4) (2-) to sigmoidal forms.
D000255:Adenosine Triphosphate; D002247:Carbon Isotopes; D002729:Chloromercuribenzoates; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004590:Electrophoresis, Paper; D004592:Electrophoresis, Starch Gel; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008274:Magnesium; D008982:Molybdenum; D009713:Nucleotidyltransferases / Q000302:isolation & purification*; D011134:Polysaccharides; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013431:Sulfates / Q000378:metabolism; D013460:Sulfur Isotopes
D002247:Carbon Isotopes; D002729:Chloromercuribenzoates; D011134:Polysaccharides; D013431:Sulfates; D013460:Sulfur Isotopes; D008982:Molybdenum; D000255:Adenosine Triphosphate; D009713:Nucleotidyltransferases; D008274:Magnesium