Kinetic studies on liver xanthinedehydrogenase. I. Activity with purine substrates

Abstract

Kinetical Studies on Liver Xanthindehydro- genase. — I. Activities with Purine Substrates

The initial reaction velocities observed in the xanthine-xanthine dehydrogenase- NAD system, according to the theoretical prediction method of Cleland, points to the existence of an irreversible step between the respective times of addition of both substrates to the enzyme. The limiting values found for the Michaelis constants are: Km (xanthine) = 2,4 X 10-5 M and Km (NAD) = 4,6 X 10"5 M.

On extending the initial velocity measurements to concentrations of xanthine or hypoxanthine surpassing 1 X 10-4 M, it isapparent that liver xanthine dehydrogena- se undergoes inhibition by its own subs­ trates. Such an inhibition, when analyzed by Dalziel’s method, proves to be of a non competitivo type: although high con­ centrations of NAD do attenuate it, the inhibition does not become completely nullified.

Adenine, purine and xanthine are dehydrogenated by liver xanthine dehydrogenase at the following ratio of reaction ve­ locities: adenine, 1; purine, 8; xanthine, 80. These permit purine to be considered as a middling substrato of xanthin dehydrogenase, while adenine cannot really be classed among the substrates of the enzy­ me. On studying the maximum velocities of transformation of purine, hypoxanthi­ne, xanthine and of equimolar mixtures of two of them, it is observed that they fulfil the kinetic relationship which indicates that the Chemical changes undergone by these substrates takes place at the same active centre of the enzyme.

Intraperitoneal administration of puri­ne, adenine or xanthine did not give rise to any variation of the liver xanthine de-hydrogenase level.

Our experimental results suggest that any circumstantial accumulation of puric substrates in the liver could contribute to the physiological regulation of uricogenesis.