The effects of x-irradiation in solution on the kinetic, sedimentation and fluorescent properties of glutamic acid dehydrogenase

Abstract

A mathematical statement for the effects of irradiation on the kinetic parameters K (Michaelis constant) and V (maximum velocity) of enzymes is provided. Irradiation of glutamic acid dehydrogenase, in solution, increases K and decreases V for the substrates glutamate and a-oxoglutarate. V is decreased and K is unchanged for the coenzymes diphosphopyridine nucleo-tide and reduced diphosphopyridine nucleotide (DPNH). The increase in K for glutamate is not associated with a change in the relative inhibitory power of dicarboxylic analogues of glutamate. Irradiation reduces the main protein peak seen in the ultracentrifuge and produces slower sedimenting material. This is consistent with a splitting of the large kinetic molecular unit into sub-units. The decrease in the main component is coincident with a decrease in enzymic activity. Irradiation of the enzyme reduces its ability to enhance the fluorescence of DPNH. This is observed whether wavelength of the exciting light is 365 m[mu] or 280 m[mu]. The change parallels a decrease in the maximum velocity, V. The increase in K for glutamate is not accompanied by a decrease in the augmented DPNH fluorescence observed on the addition of glutamate to enzyme-DPNH mixtures. The decrease in maximum enzyme velocity is not paralleled by a marked fall in intrinsic protein fluorescence. It is suggested that the primary action of the aqueous radicals formed on irradiation leads to modification in the tertiary structure of the enzyme with resulting loss in maximum velocity.