A study on the NAD(P)+ transhydrogenase from Azotobacter vinelandii

Azotobacter vinelandii transhydrogenase shows a pronounced polymerizing depolymerizing character (Chapter 3 and 5). Several factors seem to influence this phenomenon. With purified enzyme, obtained by a new purification method (Chapter 3), several parameters influencing the association-dissociation behaviour were investigated (Chapter 5).It was found that dissociation of transhydrogenase can be obtained at a minimum concentration of 1 μM of the substrate NADP +. A reversal of this effect is obtained with NADPH depending on the initial concentration of NADP +used. No clear effect of 2'-AMP on the structure is found in contrast to the results obtained with the Pseudomonas enzyme (Louie et al., 1972). Increase of the pH to about pH 9.5 results in a dissociation of the polymerized structures present in purified transhydrogenase as described by Middleditch at al. (1972). A very marked associating effect of divalent metal ions is found (Chapter 3 and 5). Large aggregates are formed, still catalytically active, that can be seen with the electron microscope and even under the phase contrast microscope (Chapter 5).The aggregates thus formed are not soluble, and addition of EDTA does not result in a clear reversal of the effects induced by the metal. Besides an associating effect divalent metal ions also cause some dissociation of the filamentous structures into rozettes, rings and cylinders with a perpendicular striping (Chapter 5).The effect on the highly polymerized structures of thiol reducing agents was investigated. A clear dissociating effect of β-mercaptoethanol was obtained while reduced lipoamide and dithiothreitol are much less effective (Chapter 5). Addition of ammonium sulphate to purified transhydrogenase results initially in dissociation followed by the formation of microcristalline structures (Chapter 5). As the effects of the reagents on the morphological structure are totally different, it must be concluded that these result from a combination of different types of binding (interaction). The dissociating effect of NADP +at low concentration points to a strong effect of this nucleotide m the total structure of the enzyme as a very local interaction (at the catalytical side only) hardly can result in such a drastic change in appearance.As divalent metal ions cause a pronounced association this leads to the conclusion that probably charge neutralization of negative groups within the protein causes this phenomenon. On the other hand decrease of positive charges (upon increasing the pH) results in dissociation of the enzyme.The role of thiol reducing agents also remains obscure as only β-mercaptoethanol has a pronounced effect whereas a more powerfull reducing agent as dithiothreitol has much less effect. This probably must be attributed either to a difference in steric hindrance or the differences in hydrophobicity of these agents. The significance of the different association-dissociation steps for the catalytic mechanism is not clear at this time. In the catalytic reaction it is found that divalent metal ions have a pronounced effect on the kinetic patterns (Chapter 3).It is discussed that these effects must be due to modification of the enzyme structure rather than of the substrate structure.A remarkable inhibition by anions is also found in the reduction of NADP +or S-NAD +by NADH. From the kinetic inhibition picture obtained with phosphate and sulphate a pingpong mechanism might be favoured but the inhibition by nitrate points to the existance of a rapid ternary complex mechanism (Chapter 3).Spectral studies of Van den Broek et al. (1971) showed a pronounced binding of NADP +to the oxidized enzyme and an absence of 2'-AMP binding. It is however found (Chapter 6) that the spectral effects of NADP +and 2'-AMP strongly depend on the presence of phosphate. The fluorescence emission quenching by NADP +is much stronger in the presence of phosphate than in the absence and concomitantly the dissociation constant becomes lower in the presence of phosphate than in the absence. From the effect on the fluorescence emission it can be derived that there are two binding sites for NADP +. Also a more pronounced shift of the emission peak towards shorter wavelengths is observed in the presence of phosphate. No effects of 2'-AMP on the emission characteristics are found unless phosphate is present, while the quenching effect induced by phosphate. is reversed by 2'-AMP.By studying the absorption difference spectrum also binding of phosphate can be observed; addition of 2'-AMP reverses the spectral effects initially induced by phosphate (Chapter 6). The chemical reduction of the enzyme was reported to require two moles of reductant per mole of enzyme bound flavin (Van den Broek et al., 1971). Reexamination of these experiments show however that 1 mole of reductant suffices to get complete reduction of the enzyme bound flavin. (Chapter 6).An interesting effect of the ionic strength on the equilibrium constant of the transhydrogenase catalyzed reaction was found (Chapter 4). The shift of this constant in relation to the ionic strength and hence the chemical nature of the products and substrates involved is discussed. It is concluded that the folded form of the pyridine nucleotides is responsible for the slope as obtained in the log K vs.VI plot. The conformation of the pyridine nucleotides was studied with N.M.R. (Chapter 4), in the absence and presence of neutral salt.No definite conclusions can be drawn from the data obtained but both electrostatics interactions and folding do attribute to the proton shifts obtained.