The binding of oxidised coenzyme to bovine-liver glutamate dehydrogenase studied by circular-difference spectroscopy.
1. The binding of NAD+ to glutamate dehydrogenase may be followed quantitatively by titration, using high-sensitivity circular dichroism (CD) difference spectroscopy. 2. The CD of the bound coenzyme in the binary complex E . NAD closely resembles that of bound ADP, although the affinity is much lower, being 350-fold less for NAD+ at 20 degrees C in 0.1 M phosphate, pH 7. 3. A family of CD spectra may be analysed by unconstrained linear regression assuming only three components: free enzyme, free coenzyme, and a single binary complex, E . NAD. 4. 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 . NAD with Kd = 0.72 mM in a simple binding process without positive or negative cooperativity. 5. NADP+ binding is more than 10-fold weaker than NAD+ binding. 6. 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). 7. Direct evidence has been obtained for the binding of a second molecule of NAD+ to the ternary complex E . NAD . glutarate. This process occurs with low affinity and is probably also located at the adenine regulatory site. 8. This second-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.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002942:Circular Dichroism; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D005977:Glutarates / Q000378:metabolism; D009243:NAD / Q000378:metabolism*; D012044:Regression Analysis
D005977:Glutarates; D009243:NAD; D000244:Adenosine Diphosphate; D005969:Glutamate Dehydrogenase