Altering the reaction coordinate of the ATP sulfurylase-GTPase reaction.
ATP sulfurylase, isolated from Escherichia coli K-12, catalyzes and couples two reactions: the hydrolysis of GTP and the synthesis of APS (adenosine 5'-phosphosulfate). Its GTPase activity is regulated in response to ligand binding at the APS-forming active site. In particular, AMP mimics an intermediate-like form of the enzyme that increases the k(cat) for GTP hydrolysis 180-fold. Using equilibrium and pre-steady-state methods, we have determined the relative Gibbs energies for many of the ground and transition states in the GTPase catalytic cycle, in the presence and absence of AMP. GTP and AMP energetically interact throughout the substrate branch of the reaction coordinate; however, once bond breaking occurs, communication between nucleotides ceases. Stopped-flow experiments, using the fluorescent nucleotides 2'-deoxy-mant-GTP and -GDP, indicate that the binding of AMP fosters a conformation of the enzyme that hinders the addition of 2'-deoxy-mant-GTP into the active site without affecting its escaping tendency. These results explain 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, approximately 1 x 10(-6) M(-1) s(-1), are 2-3 orders of magnitude less than expected for simple diffusion models, and the binding progress curves appear biphasic. These findings suggest the presence of an intermediate(s) in the binding reactions. The Gibbs energy changes that occur in the reaction coordinate upon binding of AMP clearly show that the catalytic effect of AMP is due primarily to its -3.1 kcal/mol stabilization of the rate-limiting transition state.
Yang
M
M
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Leyh
T S
TS
D000249:Adenosine Monophosphate / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D007700:Kinetics; D013050:Spectrometry, Fluorescence; D013430:Sulfate Adenylyltransferase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013816:Thermodynamics
D006165:Guanylyl Imidodiphosphate; D000249:Adenosine Monophosphate; D006160:Guanosine Triphosphate; D013430:Sulfate Adenylyltransferase; D020558:GTP Phosphohydrolases