The binding of 1, N6-etheno-nad to bovine liver glutamate dehydrogenase

Abstract

The binding of 1, N 6-etheno-NAD (ϵNAD) to bovine liver glutamate dehydrogenase ( l-glutamate;NAD(P) + oxidoreductase (deaminating), EC 1.4.1.3) saturated with glutarate has been investigated at pH 7.0, 0.05 M phosphate buffer at 20°C, by fluorescence titrations. ϵNAD binds to the protein in a simple fashion: one molecule of coenzyme per enzyme polypeptide chain in the range of enzyme concentrations investigated (from above 50 to a few micromoles of enzyme polypeptide chains/liter). The fluorescence enhancement factor, Q, of bound ϵNAD relative to free ϵNAD is independent of the saturation degree, as deduced from the constant value of the long fluorescence decay lifetime (about 21 ns), and is about 17, as deduced from F max/ F 0 ratio values obtained after extrapolation from double reciprocal plots of 1/Δ F vs. 1/[glutamate dehydrogenase]. This value for the Q factor is also independent of enzyme concentration, as well as of the presence of either GTP or ADP. At low enzyme concentrations (below 20 μM polypeptide chains/liter), the dissociation constant of ϵNAD increases progressively from a plateau value of about 50 μM to about 100 μM at infinite dilution. This is interpreted as being due to a minor affinity of glutamate dehydrogenase hexamers, with respect to higher aggregation states of the enzyme, towards ϵNAD. As expected, GTP and ADP change the affinity of glutamate dehydrogenase towards ϵNAD in an opposite manner: GTP strongly increases it, whereas ADP strongly decreases it ( K d app around 6 μM with saturating GTP and around 300 μM with saturating ADP). Furthermore, in the case of GTP, both GTP and ϵNAD bind to glutamate dehydrogenase with positive cooperativity, with a Hill coefficient of approx. 1.8 for both and a K d app ≈ 30 μM for the binding of GTP to glutamate dehydrogenase saturated with ϵNAD and glutarate. The value of the Q factor remains the same, even in the presence of the effectors (again from lifetime measurements), as well as the number of ϵNAD binding sites per enzyme polypeptide chain. These results are interpreted in terms of independent active sites, in the case without effectors. With ADP the binding appears to be simple, but no careful investigation has been attempted at low enzyme concentrations because of the low saturation degree achievable, whereas with GTP the cooperativity can be explained as due to a shift towards hexamers from higher aggregation states.