ALTERING THE REACTION COORDINATE OF THE ATP SULFURYLASE-GTPASE REACTION

ATP sulfurylase, isolated from Escherichia coli K-12, catalyzes and co uples two reactions: the hydrolysis of GTP and the synthesis of APS (a denosine 5'-phosphosulfate), Its GTPase activity is regulated in respo nse to ligand binding at the APS-fonming active site. In particular, A MP mimics an intermediate-like form of the enzyme that increases the k (cat) for GTP hydrolysis 180-fold. Using equilibrium and pre-steady-st ate methods, we have determined the relative Gibbs energies for many o f the ground and transition states in the GTPase catalytic cycle, in t he presence and absence of AMP. GTP and AMP energetically interact thr oughout the substrate branch of the reaction coordinate; however, once bond breaking occurs, communication between nucleotides ceases. Stopp ed-flow experiments, using the fluorescent nucleotides 2'-deoxy-mant-G TP and -GDP, indicate that the binding of AMP fosters a conformation o f the enzyme that hinders the addition of 2'-deoxy-mant-GTP into the a ctive site without affecting its escaping tendency. These results expl ain the effects of AMP on the equilibrium binding of the 2'-deoxy-mant -GTP. The second-order rate constants for the binding of 2'-deoxy-mant -GTP or -GDP, similar to 1 x 10(-6) M(-1) s(-1), are 2-3 orders of mag nitude less than expected for simple diffusion models, and the binding prepress curves appear biphasic. These findings suggest the presence of an intermediate(s) in the binding reactions. The Gibbs energy chang es that occur in the reaction coordinate upon binding of AMP clearly s how that the catalytic effect of AMP is due primarily to its -3.1 kcal /mol stabilization of the rate-limiting transition state.