The Binding of Oxidised Coenzyme to Bovine‐Liver Glutamate Dehydrogenase Studied by Circular‐Difference Spectroscopy

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Abstract
The binding of NAD+ to [bovine] glutamate dehydrogenase may be followed quantitatively by titration, using high-sensitivity circular dichroism (CD) difference spectroscopy. The CD of the bound coenzyme in the binary complex E.cntdot.NAD closely resembles that of bound ADP, although the affinity is much lower, being 350-fold less for NAD+ at 20.degree. C in 0.1 M phosphate, pH 7. A family of CD spectra may be analyzed by unconstrained linear regression assuming only 3 components: free enzyme, free coenzyme and a single binary complex, E.cntdot.NAD. Taking the molar CD of bound ADP as representing the molar CD of the adenine chromophore of bound NAD+, the linear regression shows the formation of a simple 1:1 complex E.cntdot.NAD with Kd = 0.72 mM in a simple binding process without positive or negative cooperativity. NADP+ binding is more than 10-fold weaker than NAD+ binding. From the similarity of the CD of bound ADP and bound NAD+ it is probable that NAD+, in forming a simple binary complex, binds preferentially at the regulatory (adenine nucleotide) binding site (site II). Direct evidence was obtained for the binding of a 2nd molecule of NAD+ to the ternary complex E.cntdot.NAD.cntdot.glutarate. This process occurs with low affinity and is probably also located at the adenine regulatory site. This 2nd-site binding of NAD+ may contribute to the phenomena of non-Michaelis-Menten kinetics and apparent negative homotropic interactions in the binding of NAD+, previously attributed to subunit-subunit cooperative interactions.