title: Kinetic studies on the binding of 1,N6-etheno-NAD+ to glutamate dehydrogenase from Clostridium symbiosum.
abstract: The mechanism of the binding of reduced coenzyme (NAD+) to clostridial glutamate dehydrogenase (GDH) was determined by transient kinetics. The fluorescent 1,N6-ethenoadenine analogue of NAD+ (epsilonNAD+) was used as a probe of nucleotide binary and ternary complex formation because the binding of NAD+ is optically silent. The kinetics of epsilonNAD+ binding were consistent with a 3-step binding process. The enzyme was found to oscillate between two conformational forms, termed E1 and E2, in the presence and absence of L-glutamate. However, L-glutamate shifted the equilibrium from 96.8% to 99% of the enzyme in the E1 form. The rapid-equilibrium binding of epsilonNAD+ to the E2 form was rate limited by a slow isomerisation of the ternary complex as the binary complex became saturated with epsilonNAD+. The L-glutamate binary complex had a greater affinity for the coenzyme (Kd = 11 microM) than the free enzyme (Km = 39 microM), 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 Kd for epsilonNAD+ of 6.8 microM. Consequently, there is stabilization of successive complexes on the reaction pathway.
authors.affiliation: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, UK.
authors.forename: L A
authors.initials: LA
authors.lastname: Basso
authors.forename: P C
authors.initials: PC
authors.lastname: Engel
authors.forename: A R
authors.initials: AR
authors.lastname: Walmsley
mesh_terms: D003013:Clostridium / Q000201:enzymology*; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D007536:Isomerism; D007700:Kinetics; D008433:Mathematics; D009243:NAD / Q000031:analogs & derivatives* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D009243:NAD; C011038:nicotinamide 1,N(6)-ethenoadenine dinucleotide; D018698:Glutamic Acid; D005969:Glutamate Dehydrogenase