Relaxation kinetic studies of coenzyme binding to glutamate dehydrogenase from beef liver

Fluorescence temperature-jump experiments were performed to study the binding of coenzyme to glutamate dehydrogenase from beef liver in 0.1 M sodium phosphate buffer, pH 7.4, T = 25.0°C. 1. 1) NADPH shows a single second-order relaxation process, indicating coenzyme binding to one site per enzyme subunit. The dissociation constant of the reaction was calculated from the rate constants (k 21 = 92 sec −1, k 12 = 2.13 .10 6 M −1 sec −1) to be 43 μM. No deviation from a straight line was observed over the whole concentration range. 2. 2) With NADH two well separated second-order processes are detected. 3. 3) The observed concentration dependence of these two relaxation processes is consistent with a reaction scheme assuming two completely independent but non-equivalent NADH binding sites per enzyme subunit. 4. 4) From the rate constants of the faster process (k 21 = 235 sec −1 k 12 = 3.83 .10 6 M −1 sec −1) and of the slower process (k 32 = 4.2 sec −1, k 23 = 0.18 .10 6 M −1 sec −1) the dissociation constants are calculated to be K 21 = k 21 k 12 = 61 μM and K 32 = k 32 k 23 = 23 μM , respectively. 5. 5) Small amounts of ADP (