AMP nucleosidase: kinetic mechanism and thermodynamics.
The kinetic mechanism of AMP nucleosidase (EC 3.2.2.4; AMP + H2O----adenine + ribose 5-phosphate) from Azotobacter vinelandii is rapid-equilibrium random by initial rate studies of the forward and reverse reactions in the presence of MgATP, the allosteric activator. Inactivation-protection studies have established the binding of adenine to AMP nucleosidase in the absence of ribose 5-phosphate. Product inhibition by adenine suggests a dead-end complex of enzyme, AMP, and adenine. Methanol does not act as a nucleophile to replace H2O in the reaction, and products do not exchange into substrate during AMP hydrolysis. Thus, the reactive complex has the properties of concerted hydrolysis by an enzyme-directed water molecule rather than by formation of a covalent intermediate with ribose 5-phosphate. The Vmax in the forward reaction (AMP hydrolysis) is 300-fold greater than that in the reverse reaction. The Keq for AMP hydrolysis has been experimentally determined to be 170 M and is in reasonable agreement with Keq values of 77 and 36 M calculated from Haldane relationships. The equilibrium for enzyme-bound substrate and products strongly favors the enzyme-product ternary complex ([enzyme-adenine ribose 5-phosphate]/[enzyme-AMP] = 480). The temperature dependence of the kinetic constants gave Arrhenius plots with a distinct break between 20 and 25 degrees C. Above 25 degrees C, AMP binding demonstrates a strong entropic effect consistent with increased order in the Michaelis complex. Below 20 degrees C, binding is tighter and the entropic component is lost, indicating distinct enzyme conformations above and below 25 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
D000225:Adenine / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000032:analysis; D001395:Azotobacter / Q000201:enzymology*; D002250:Carbon Radioisotopes; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D012267:Ribosemonophosphates / Q000494:pharmacology; D013816:Thermodynamics
D002250:Carbon Radioisotopes; D012267:Ribosemonophosphates; D000249:Adenosine Monophosphate; C031626:ribose-5-phosphate; D009699:N-Glycosyl Hydrolases; C019699:AMP nucleosidase; D000225:Adenine