The kinetic mechanism of AMP nucleosidase (EC 126.96.36.199; 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 H20 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 for KeqAMP 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 °C. Above 25 °C, AMP binding demonstrates a strong entropic effect consistent with increased order in the Michaelis complex. Below 20 °C, binding is tighter and the entropic component is lost, indicating distinct enzyme conformations above and below 25 °C. Similar effects are seen on the energy of activation, which has ΔH‡ of 11.4 kcal/mol at 30 °C and 19.1 kcal/mol at 10 °C. These thermodynamic parameters have been used to construct a reaction coordinate diagram for the enzyme.
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