A method for the isolation and purification of a reversible transhydrogenase from Azotobacter vinelandii
is described (CHAPTER 3). The purification of the enzyme is hampered by association-dissociation phenomena, resulting in large losses of transhydrogenase activity. The relation between these losses and removal of lipoamide dehydrogenase, pyruvate dehydrogenase complex and a NADPH-ferricyanide diaphorase activity suggests the existence of a multi-enzyme complex, dissociation of this complex resulting in association of the transhydrogenase molecules.
The purified transhydrogenase is a flavoprotein with FAD as prosthetic group. The absorption spectrum of the NADP +
-free enzyme is characterized by a maximum at 442 nm. A minimum molecular weight of 60,000 daltons can be calculated from the experimentally determined extinction coefficient at 442 nm. The oxidized enzyme is stable at elevated temperatures and at high dilution; storage at -20° results in aggregation of the enzyme. The reduced enzyme is thermolabile; inactivation at elevated temperatures can be prevented by addition of FAD. It is difficult to prepare the apoenzyme and incubation with FAD only results in partial restoration of transhydrogenase activity.
Ultracentrifugation and light-scattering studies demonstrate that the transhydrogenase preparations are inhomogeneous. In two types of enzyme preparations three sedimentation coefficients (s 20,w )
are determined: 24, 48 and 88 S, respectively. By addition of NADPH and NADP +
upon sedimentation of the enzyme a relation is demonstrated between the 48 and 88 S components. The sedimentation coefficient of the main component is concentration independent beyond 1 mg/ml. From light-scattering experiments a molecular weight (M z )
of 30-50 million daltons is determined; the calculated length for a rod-like structure is 10,000-15,000 Å
Electron microscopic investigations (CHAPTER 4) confirm the inhomogeneity of the transhydrogenase preparations. Long helical-like structures up to 10,000 Å
are present apart from large amounts of rod-like structures and separate fragments. The diameter of both the threads and rods is 116-120 Å
. No clearcut substructures are observable; possibly they represent projections of sphericle particles; the threads may represent spiral-like structures. Addition of NADP +
to the enzyme solution can result in fragmentation of the thread-like structures, but the dissociation is never complete. Extensive removal of NADP +
results in more homogeneous preparations with structures up to 15,000-18,000 Å
in length. The ratio NADPH/NADP +
seems to be regulatory for the length of the structure. Other nucleotides have no clear-cut effect on the structure of the enzyme.
In some preparations a totally unknown, ladder-like structure is observed. The constituents are very similar to the thread-like structures of the transhydrogenase and the tetramer-like structures, present in pyruvate dehydrogenase complex preparations. Only transhydrogenase and transacetylase activities are detectable in these preparations. This structure supports the idea of a complex between the transhydrogenase and the pyruvate dehydrogenase. The ladder-like structures resist the influence of the different nucleotides.
Ammonium sulfate crystalline suspensions of the enzyme show regular structures, some kind of 'stacked disc' structures; solubilisation results in the appearance of thread-like structures, fragments and rosettes, but no close relation between the different species can be established.
From an examination of the different purification stages it can be concluded that the thread-like transhydrogenase structure is not a purification artefact due to the procedure followed; however, a relation seems to exist between the presence of the thread-like structures, the concentration of the transhydrogenase and the-removal of the pyruvate dehydrogenase complex.
Spectral studies (CHAPTER 5) have shown that 1 mole NADP +
(per mole of flavin) can be easily bound to the oxidized enzyme. This results in a spectral shift in the region 300-500 nm. of the absorption spectrum, a quenching of flavin fluorescence and a shift of the emission maximum; moreover it affects the ORD and CD characteristics.. The dissociation constant (K D )
of this NADP +
complex is ~ 3-4 μM. Changes in the 300-350 nm region of the absorption spectrum at higher NADP +
concentrations point to the possible existence of a second binding site for NADP +
. These shifts are possibly due to either changes in polarity around the flavin, or to interaction between NADP +
and the isoalloxazine ring of the flavin, or to weakening of ribityl side chain interactions; protein conformational changes, however, cannot be excluded.
Anaerobic titrations of the transhydrogenase with NAD(P)H in the presence of NADase show that two moles of reductant are required to reduce one mole of enzyme-bound flavin; this observation is confirmed by titrations with sodium dithionite. The existence of an unknown reducible group X next to the flavin is proposed. On the basis of the differences found in the titrations with dithionite, with NADPH in the absence and presence of NADP +
and with NADH in the absence and presence of NADP +
and NAD +
a reduction scheme is proposed. The titration experiments show that NAD +
and NADP +
interact in a different manner with the different forms of the enzyme, e.g. NADP +
has a higher affinity for the oxidized enzeym, while NAD +
has a higher affinity for the reduced enzyme. Furthermore the level of reduction of the enzyme-bound flavin is determined by the ratio of reduced and oxidized pyridine nucleotides. Titration experiments of the reduced enzyme with NADP +
and photoreduction experiments support the proposals. On the basis of these findings a model is proposed giving an explanation for a specific hydrogen transfer from the 4B site of NADH to the 4B site of NADP +
Kinetic studies (CHAPTER 6) performed with naturally occurring coenzymes and their thio-analogues point to the existence of a ping-pong bi-bi mechanism for the NADH-TNAD +
and NADPH-TNAD +
reactions. The inhibition of NAD +
with respect to NADH in the reaction NADH-TNAD +
and the dependence of the reaction velocity on [NADPH] 2
in the reaction NADPH-TNAD +
in the presence of NADP +
, however, exclude this mechanism. A ternary complex mechanism is proposed, in which the enzyme is first reduced to the 4-equivalent reduced state by 2 moles of donor before 2 moles of acceptor are bound to the reduced enzyme; the possibility of complex formation of the oxidised enzyme with NADP +
is included in the scheme. The kinetic patterns of the NADPH-TNAD +
reaction in the absence and presence of NADP +
can be explained by this mechanism. The explanation of the kinetic patterns of the NADH-TNAD +
reaction can be given by slightly modifying the mechanism in such a way that the sequence of addition of the acceptor molecule and the second donor molecule is changed and assuming irreversible binding of the first acceptor molecule to the reduced enzyme; this latter assumption is justified by the high affinity of NAD +
for the reduced enzyme. The reactions of NADP +
and TNADP +
with NAD(P)H proceed due to the reversible binding of both substrates according to a rapid equilibrium random bi-bi mechanism. The data of the reaction NADH-NADP +
and the NADP +
inhibition patterns of the reaction NADPH-TNADP +
are consistent with this mechanism. The proposed ternary complex or random bi-bi mechanisms are more reasonable for a 4B-4B hydrogen transfer than a ping-pong bi-bi mechanism.
In CHAPTER 7 results are presented from studies on Azotobacter
lipoamide dehydrogenase. The results show that the Azotobacter
enzyme has several properties in common with the pig heart lipoamide dehydrogenase. Gel filtration and ultracentrifugation studies show that the holoenzyme and the apoenzyme have a molecular weight of 102,000 and 52,000 daltons, respectively, supporting the idea of the existence of a monomer-dimer system. The apoenzyme-FAD complex, however, does not show a pronounced DCIP activity; the absorption spectrum points to a more polar environment of the flavin.
Kinetic studies with reduced lipoamide and NAD +
support, as proposed for the pig heart enzyme, an ordered bi-bi mechanism. Assuming this mechanism, kinetic parameters are calculated which are in the same order of magnitude as for the pig heart enzyme. The affinity of the Azotobacter
enzyme for both substrates, however, is much higher.
The apoenzyme prepared according to the acid ammonium sulfate procedure is very stable and can be easily reconstituted with FAD; the recombination process is temperature and FAD dependent. Preparation of the apoenzyme by dialysis against guanidine-HCl, however, results in a totally different structure, as concluded from recombination experiments and from the different characteristics with respect to the holoenzyme and the acid ammonium sulfate treated enzyme.