The real-time resolution of proton-related transient-state steps in an enzymatic reaction. The early steps in the oxidative deamination reaction of bovine liver glutamate dehydrogenase

• Published in: The Journal of biological chemistry 1993-01, Vol.268 (1), p.21-28
• Main Authors: NARINDER SINGH, MANISCALCO, S. J, FISHER, H. F
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Binding Sites; Biological and medical sciences; Cattle; Clostridium - enzymology; Deamination; enzymatic activity; Enzymes - metabolism; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; glutamate dehydrogenase; Glutamate Dehydrogenase - metabolism; Glutamates - metabolism; Glutamic Acid; Hydrogen-Ion Concentration; Kinetics; liver; Liver - enzymology; Lysine - metabolism; Macromolecular Substances; Mathematics; mechanisms; Models, Molecular; Models, Theoretical; Oxidation-Reduction; Protein Conformation; protons; reaction; Time Factors
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)54109-0

We introduce a novel transient-state kinetic approach which can resolve proton and product time courses into a series of individual steps that comprise the reaction path. We have applied this approach to the oxidative deamination reaction catalyzed by bovine liver glutamate dehydrogenase, measuring both the product (NADPH) and proton time courses at various pH values. The global treatment (over all pH values) resolves the very early portion of this reaction quantitatively and provides a continuous time course for each of the six protonic species. We propose the following mechanism: L-glutamate binds to an open conformation of the enzyme-NADP complex, forming salt bridges between its alpha- and gamma-carboxyl groups and the protonated forms of enzyme lysine residues 114 and 90, respectively. In this position, the alpha-H atom of the substrate is too far from the nicotinamide ring for hydride transfer to occur. In the next step, three events occur in a concerted manner: lysine 126 loses a proton and acquires a single water molecule; the active site cleft closes; bulk water is expelled; the substrate and coenzyme are forced closer together and remain in a nonaqueous environment during the ensuing chemical events, returning to an open conformation only in time to allow the product release steps to occur. Thus, substrate binding accomplishes a number of important tasks which are themselves an integral part of the catalytic mechanism. Combining the novel transient state approach developed here with steady-state kinetic information can produce a detailed mechanistic resolution of otherwise hidden steps.