Effects of pH and temperature on the reaction of milk xanthine oxidase with 1-methylxanthine

The reaction of milk Xanthine Oxidase (XO) with 1-methylxanthine has been i nvestigated by steady state and stopped flow transient kinetic studies to u nderstand the effect of a methyl group substituent on the purine ring. The pH dependence of the steady state kinetic parameter (V-max/K-m) shows a bel l-shaped curve implying at least two ionisable groups are involved in the b inding of XO with 1-methylxanthine, the higher pK(a) 7.7 corresponding to i onisation of the substrate and the lower one 6.2 to the enzyme (possibly Gl u-1261 by analogy to Glu-869 of aldehyde oxidoreductase from Desulfovibrio gigas). The temperature dependence of the steady state and transient kineti c studies suggests the existence of at least one molecular intermediate dur ing breakdown of the enzyme-substrate complex. The thermodynamic parameters of the microscopic rate constants were determined from the temperature dep endence studies.