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.

Homogeneous ATP sulfurylase from Penicillium chrysogenum has been reported to have an extremely low activity toward its physiological inorganic substrate, sulfate. This low activity is an artifact resulting from potent product inhibition by 5'-adenylylsulfate (APS) (Ki less than 0.25 microM). Assays based on 35S incorporation from 35SO4(2-) into charcoal-adsorbable [35S]APS are nonlinear with time, even in the presence of a large excess of inorganic pyrophosphatase. However, in the presence of excess APS kinase (along with excess pyrophosphatase), the ATP sulfurylase reaction is linear with time and the enzyme has a specific activity (Vmax) of 6 to 7 units mg protein-1 corresponding to an active site turnover number of at least 400 min-1. Monovalent oxyanions such as NO3-, ClO3-, ClO4-, and FSO3- are competitive with sulfate (or molybdate) and essentially uncompetitive with respect to MgATP. However, thiosulfate (SSO3(2-)), a true sulfate analog and dead-end inhibitor of the enzyme (competitive with sulfate or molybdate), exhibited clear noncompetitive inhibition against MgATP. Furthermore, APS was competitive with both MgATP and molybdate in the molybdolysis assay. These results suggest (a) that the mechanism of the normal forward reaction may be random rather than ordered and (b) that the monovalent oxyanions have a much greater affinity for the E X MgATP complex than for free E. In this respect, FSO3-, ClO4-, etc., are not true sulfate analogs although they might mimic an enzyme-bound species formed when MgATP is at the active site. The nonlinear ATP sulfurylase reaction progress curves (with APS accumulating in the presence of excess pyrophosphatase or PPi accumulating in the presence of excess APS kinase) were analyzed by means of "average velocity" plots based on an integrated rate equation. This new approach is useful for enzymes subject to potent product inhibition over a reaction time course in which the substrate concentrations do not change significantly. The analysis showed that ATP sulfurylase has an intrinsic specific activity of 6 to 7 units mg protein-1. Thus, the apparent stimulation of sulfurylase activity by APS kinase results from the continual removal of inhibitory APS rather than from an association of the two sulfate-activating enzymes to form a "3'-phospho-5'-adenylylsulfate synthetase" complex in which the sulfurylase has an increased catalytic activity. The progress curve analyses suggest that APS is competitive with both MgATP and sulfate, while MgPPi is a mixed-type inhibitor with respect to both substrates. The cumulative data point to a random sequence for the forward reaction with APS release being partially rate limiting.