Premium
Defining the Role of Central Complexes in Activity and Regulation of Glutamate Dehydrogenase
Author(s) -
Brown Sebastian,
Bell Ellis
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a643-b
Subject(s) - kinetic isotope effect , norvaline , chemistry , reaction rate constant , glutamate dehydrogenase , nad+ kinase , deuterium , stereochemistry , cofactor , deamination , photochemistry , enzyme kinetics , kinetics , glutamate receptor , enzyme , amino acid , active site , biochemistry , leucine , physics , receptor , quantum mechanics
Glutamate Dehydrogenase catalyzes the reversible oxidatitive deamination of glutamate and, with lower efficiency, mono‐carboxylic amino acids including norvaline. To investigate the roles that central complexes and abortive complexes play in the overall reaction a combination of stopped flow kinetic studies and binding studies have been employed to allow characterization of the steps on the pathway. Using fluorescence and multiple emission wavelengths to build up emission spectra with excitation at 340nm, the flow of the reaction through the various potential NAD(P)H containing complexes is followed. With norvaline as substrate the rate limiting step is formation of the Enzyme‐NADH‐Products complex and no other complexes are detected. With glutamate there is a clear pre‐steady state burst indicating that either rearrangements of various product complexes or release of reduced cofactor from one or more of the potential NAD(P)H containing complexes is rate limiting. Analysis of the pre‐steady state phase indicates that in most cases the kinetics are best described using a double exponential fit indicating the complex nature of the steps involved in this highly regulated enzyme. Isotope effect experiments using deuterated glutamate indicate that there is a steady state isotope effect but no isotope effect on the rate constant for the pre‐expoential phase of the reaction. This suggests that the overall rate limiting step in the hydride transer phase of the reaction is conformational rather than chemical and that the observed isotope effect on the steady state rate is due to an isotope effect on the equilibrium position of the central complexes of the reaction. This work is supported by NSF Grant MCB 0448905 to EB.