Thermodynamics and stoicheiometry of the binding of substrate analogues to uricase.
The subunit composition, metal content, substrate-analogue binding and thermal stability of Aspergillus flavus uricase were determined. A. flavus uricase is a tetramer and contains no copper, iron or any other common prosthetic group. Analytical-gel-filtration and equilibrium-dialysis experiments showed one binding site per subunit for urate analogues. The free energy of xanthine binding was -30.5 kJ (-7.3 kcal)/mol of subunit by equilibrium dialysis and -30.1 kJ (-7.2 kcal)/mol of subunit by microcalorimetry. The enthalpy change for xanthine binding was -15.9 kJ (-3.8 kcal)/mol of subunit when determined from the temperature-dependence of the equilibrium constant and -18.0 kJ (-4.3 kcal)/mol of subunit when measured microcalorimetrically. The thermal inactivation rate of A. flavus uricase increases as protein concentration is decreased. This concentration-dependent instability is not due to subunit dissociation.
D001231:Aspergillus flavus / Q000201:enzymology; D003300:Copper / Q000032:analysis; D007501:Iron / Q000032:analysis; D007700:Kinetics; D013379:Substrate Specificity; D013816:Thermodynamics; D014503:Urate Oxidase / Q000378:metabolism*; D019820:Xanthine; D014970:Xanthines / Q000378:metabolism
D014970:Xanthines; D019820:Xanthine; D003300:Copper; D007501:Iron; D014503:Urate Oxidase