| Abstract: | 1. The reactive analogue oADP produced by periodate oxidation of ADP has been studied as a potential affinity label for the enzyme bovine glutamate dehydrogenase, using circular dichroism (CD) difference spectroscopy to monitor specific binding. 2. The analogue binds stoichiometrically, rapidly and reversibly to the adenine nucleotide binding site with Kd approximately equal to 12 microM (20 degrees C, pH 7) with characteristic intensification of the adenine nucleotide CD at 260 nm. 3. This complex is unstable and decays with a half-life of about 1.5 h; the analogue then becomes attached as a Schiff base to a number of subsidiary sites, including the enzyme active site, with partial inactivation of the enzyme. 4. Depending upon initial concentration of oADP, the enzyme activity is progressively lost during the slow reaction; following borohydride reduction, up to four molecules of analogue are bound/subunit. 5. Protection against loss of enzyme activity is afforded by the coenzyme NAD+ plus glutarate or L-hydroxyglutarate (an effective inhibitor), or by glutarate alone, but not by NAD+ alone. 6. Spectroscopic and protection studies indicate that after the decay of the specific CD signal, the enzyme retains the capacity to bind ADP, but that this is progressively lost in parallel with decay of enzymic activity. 7. The results are consistent with proximity or functional interaction between the adenine nucleotide site and the coenzyme binding portion of the active site. 8. Thus oADP does not act as a true affinity label for the adenine nucleotide binding site, but the reaction subsequent to binding at that site shows some specificity directed towards the active site. |
