Kinetic studies on the binding of 1,N-6-etheno-NAD(+) to glutamate dehydrogenase from Clostridium symbiosum

The mechanism of the binding of reduced coenzyme (NAD(+)) to clostridial glutamate dehydrogenase (GDH) was determined by transient kinetics. The fluorescent 1,N-6-ethenoadenine analogue of NAD(+) (epsilon NAD(+)) was used as a probe of nucleotide binary and ternary complex formation because the binding of NAD(+) is optically silent. The kinetics of epsilon NAD(+) binding were consistent with a 3-step binding process. The enzyme was found to oscillate between two conformational forms, termed E-1 and E-2, in the presence and absence of L-glutamate. However, L-glutamate shifted the equilibrium from 96.8% to 99% of the enzyme in the E-1 form. The rapid-equilibrium binding of epsilon NAD(+) to the E-2 form was rate limited by a slow isomerisation of the ternary complex as the binary complex became saturated with epsilon NAD(+). The L-glutamate binary complex had a greater affinity for the coenzyme (K-d = 11 mu M) than the free enzyme (K-m = 39 mu M) indicative of a positive interaction of the substrate and coenzyme binding sites. Steady-state studies were also indicative of a positive interaction in the formation of the catalytic complex, with this complex having a K-d for epsilon NAD(+) of 6.8 mu M. Consequently, there is stabilization of successive complexes on the reaction pathway. (C) 1997 Elsevier Science B.V.