Formation of transient complexes in the glutamate dehydrogenase catalyzed reaction.
The reaction of glutamate dehydrogenase and glutamate (gl) with NAD+ and NADP+ has been studied with stopped-flow techniques. The enzyme was in all experiments present in excess of the coenzyme. The results indicate that the ternary complex (E-NAD(P)H-kg) is present as an intermediate in the formation of the stable complex (E-NAD(P)H-gl). The identification of the complexes is based on their absorption spectra. The binding of the coenzyme to (E-gl) is the rate-limiting step in the formation of (E-NAD(P)H-kg) while the dissociation of alpha-ketoglutarate (kg) from this complex is the rate-limiting step in the formation of (E-NAD(P)H-gl). The Km for glutamate was 20-25 mM in the first reaction and 3 mM in the formation of the stable complex. The Km values were independent of the coenzyme. The reaction rates with NAD+ were approximately 50% greater than those with NADP+. Furthermore, high glutamate concentration inhibited the formation of (E-NADH-kg) while no substrate inhibition was found with NADP+ as coenzyme. ADP enhanced while GTP reduced the rate of (E-NAD(P)H-gl) formation. The rate of formation of (E-NAD(P)H-kg) was inhibited by ADP, while it increased at high glutamate concentration when small amounts of GTP were added. The results show that the higher activity found with NAD+ compared to NADP+ under steady-state assay conditions do not necessarily involve binding of NAD+ to the ADP activating site of the enzyme. Moreover, the substrate inhibition found at high glutamate concentration under steady-state assay condition is not due to the formation of (E-NAD(P)H-gl) as this complex is formed with Km of 3 mM glutamate, and the substrate inhibition is only significant at 20-30 times this concentration.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D005971:Glutamates; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009243:NAD; D009249:NADP; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors
D005971:Glutamates; D009243:NAD; D009249:NADP; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D005969:Glutamate Dehydrogenase