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Defining the Role of Productive and Abortive Complexes in Glutamate Dehydrogenase

Glutamate Dehydrogenase catalyzes the reversible oxidatitive deamination of L Glutamate and, with significantly lower efficiency, a number of mono‐carboxylic amino acids including Norvaline. With Glutamate initial rate kinetic studies show negative cooperativity, atributed to subunit interactions in the homo‐hexameric enzyme. With Norvaline as substrate no cooperative interactions are observed. To investigate the roles that central complexes and abortive complexes play in the overall reaction stiopped flow kinetic studies have been employed to allow characterization of the steps on the pathway with either glutamate or norvaline as substrate. Using fluorescence to characterize the various complexes containing reduced cofactor [NAD(P)H] and using multiple emmission wavelengths to build up emmission spectra with excitation at 340nm the flow of the reaction through the various potential NAD(P)H containing complexes can be followed. With norvaline as substrate the rate limiting step is the initial 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 the various product complexes or release of reduced cofactor from one or more of the potential NAD(P)H containing complexes is rate limiting. By combining stopped flow kinetcs and characterization of the fluorescence properties of the various possible complexes we are able to quantitate the amounts of some of the product complexes as well as follow their interconversion. 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 steps involved in the turnover of this highly regulated enzyme.