Yeast PAPS reductase: properties and requirements of the purified enzyme.
The enzymatic mechanism of sulphite formation in Saccharomyces cerevisiae was investigated using a purified 3'-phosphoadenylsulphate (PAPS) reductase and thioredoxin. The functionally active protein (MR 80-85 k) is represented by a dimer which reduces 3'-phosphoadenylyl sulphate to adenosine-3',5'-bisphosphate and free sulphite at a stoichiometry of 1:1. Reduced thioredoxin is required as cosubstrate. Examination of the reaction products showed that free anionic sulphite is formed with no evidence for "bound-sulphite(s)" as intermediate. Vmax of the enriched enzyme was 4-7 nmol sulphite.min-1.mg-1 using the homologous thioredoxin from yeast. The velocity of reaction decreased to 0.4 nmol sulphite.min-1.mg-1 when heterologous thioredoxin (from Escherichia coli) was used instead. The Km of homologous thioredoxin was 0.6.10(-6) M, for the heterologous cosubstrate it increased to 1.4.10(-6) M. The affinity for PAPS remained practically unaffected (Km PAPS: 19.10(-6) M in the homologous, and 21.10(-6) M in the heterologous system). From the kinetic data it is concluded that the enzyme followed an ordered mechanism with thioredoxin as first substrate followed by PAPS as the second. Parallel lines in the reciprocal and a common intersect in the Hanes-plots for thioredoxin were seen as indication of a ping-pong (with respect to thioredoxin) uni-bi (with respect to PAPS) mechanism.
Schwenn
J D
JD
Institut für Biochemie der Pflanzen, Ruhr Universität Bochum, Federal Republic of Germany.
Krone
F A
FA
Husmann
K
K
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013447:Sulfites / Q000378:metabolism; D013879:Thioredoxins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism
D013447:Sulfites; D013879:Thioredoxins; D010088:Oxidoreductases; C052449:3'-phosphoadenylyl-5'-phosphosulfate reductase