The mechanism of substrate and coenzyme binding to clostridial glutamate dehydrogenase during oxidative deamination.
The binding of NAD+ and L-Glutamate to glutamate dehydrogenase (GDH) from Clostridium symbiosum has been investigated by stopped-flow fluorescence spectroscopy. The formation of the binary complexes produces little change in the protein fluorescence but formation of the ternary complex results in quenching of its fluorescence with a maximum value of 40%. This finding, coupled with the finding that a step prior to hydride transfer but subsequent to ternary complex formation is rate limiting, has enabled us to monitor the kinetics of ternary complex formation in detail. The ternary complex can be formed via the GDH-NAD+ or the GDH-L-Glu binary complexes, but the route via the GDH-NAD+ binary complex is the preferred pathway. The equilibrium and rate constants for the formation of the two binary complexes and the ternary complex formed via the two possible pathways have been determined. These studies have revealed an interaction between the coenzyme-binding site and the substrate-binding site, which lead to a decrease in the binding constant for the second substrate binding to the enzyme. The free energy coupling between the binary and ternary complexes is about 2.4-2.8 kJ.mol-1. We propose that there is a further isomerisation of the ternary complex, which is rate limiting for the steady-state turnover of the enzyme. Formation of this complex is characterised by an increased negative interaction, with a free energy coupling between these complexes of 6.3-11.6 kJ.mol-1.
Basso
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Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, England.
Engel
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Walmsley
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D001665:Binding Sites; D003013:Clostridium / Q000201:enzymology*; D003641:Deamination; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D007700:Kinetics; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction
D005971:Glutamates; D009243:NAD; D018698:Glutamic Acid; D005969:Glutamate Dehydrogenase