ATP sulfurylase from Penicillium chrysogenum: Measurements of the true specific activity of an enzyme subject to potent product: Inhibition and a reassessment of the kinetic mechanism☆

摘要: Abstract Homogeneous ATP sulfurylase from Penicillium chrysogenum has been reported to have an extremely low activity toward its physiological inorganic substrate, sulfate. This is artifact resulting potent product inhibition by 5′-adenylylsulfate (APS) ( K i 35 S incorporation SO 4 2− into charcoal-adsorbable [ S]APS are nonlinear with time, even in the presence of a large excess pyrophosphatase. However, APS kinase (along pyrophosphatase), reaction linear time and enzyme specific V max ) 6 7 units mg protein −1 corresponding active site turnover number at least 400 min . Monovalent oxyanions such as NO 3 − , ClO FSO competitive sulfate (or molybdate) essentially uncompetitive respect MgATP. thiosulfate (SSO ), true analog dead-end inhibitor (competitive or molybdate), exhibited clear noncompetitive against Furthermore, was both MgATP molybdate molybdolysis assay. These results suggest (a) that mechanism normal forward may be random rather than ordered (b) monovalent much greater affinity for E · complex free E. In this respect, etc., not analogs although they might mimic enzyme-bound species formed when site. The progress curves (with accumulating pyrophosphatase PP kinase) were analyzed means “average velocity” plots based on integrated rate equation. new approach useful enzymes subject over course which substrate concentrations do change significantly. analysis showed intrinsic Thus, apparent stimulation continual removal inhibitory association two sulfate-activating form “3′-phospho-5′-adenylylsulfate synthetase” increased catalytic activity. curve analyses sulfate, while Mg mixed-type substrates. cumulative data point sequence release being partially limiting.