The isolation and some alternatives for purification of PDC from Azotobacter vinelandii
are described (CHAPTER 3). Ultimate extent and recovery seem to be limited by the lability of the enzyme: sensitivity to shearing forces. Moreover, sedimentation-velocity runs and light-scattering experiments show dissociation of the complex at low protein concentrations (< 0.3 mg.ml -1
The maximal specific activity achieved (10 units.mg -1
) is much lower than that reported for pure PDC from E. coli
. PTA-activity was found to be closely associated with PDC from Azotobacter.
The question, whether or not this PTA may be called part of the complex or that its presence in the complex is due to a purification-artefact, could not be answered unequivocally. Each attempt to improve the specific activity by removing the PTA also leads to dissociation of LTA and lipoamide dehydrogenase from the complex, with concomitant loss of activity. It was shown, that connection of PDC and PTA may be meaningful, for in the presence of a non-rate limiting amount of acetate kinase substrate-bound phosphorylation could be demonstrated, due to combined actions.
After correction for light-scattering the absorption spectrum of pure PDC shows a maximum at 455 mn and shoulders at about 430 and 485 nm, exactly as for pure lipoamide dehydrogenase. The flavin content of most PDC preparations varied between 1.6 and 1.8 nmoles FAD per mg protein, pointing at a minimal molecular weight of 550,000-620,000 daltons. Data from light-scattering studies and sedimentation-diffusion experiments indicate, that the actual molecular weight of the pure active multi-enzyme complex is 1,000,000-1,200,000 daltons. From molecular weight and S20,w
(19.5 S), a frictional ratio of 1.5 could be calculated, thus a more open structure than a solid sphere is possible. In electron micrographs, the typical structures of PDC as observed with the E. coli
complex, are not present. In preparations, that in addition to PDC- and PTA activity contain some pyridine nucleotide transhydrogenase, tetrad-like structures (12-14 nm on a side) are visible.
The overall activity of the complex (CHAPTER 4) was shown to be dependent on the Mg 2+
.TPP concentration (K M
= 25-50 μM). The value K M
= 0. 1 mM as found for Mg 2+
does not reflect affinity between enzyme and Mg 2+
. AMP and sulphate enhance the affinity of the enzyme for Mg 2+
.TPP; CoASAc inhibits competitively ( K I
= 10 μM).
The kinetics of the overall reaction with different pyruvate concentrations show a sigmoidal response ( S0.5
= 1.9 mM under the conditions used). AMP and sulphate stimulate the activity at low pyruvate concentrations. In contrast, CoASAc behaves as a strong competitive inhibitor with respect to Mg 2+
.TPP-pyruvate ( I0.5
= 8 pM), which inhibition is reversed by AMP and sulphate. The HILL-coefficients (h),
apparently for the pyruvate-binding, as calculated from the plots were above 2 (h
= 2.6-2.7) and not influenced by the presence of the effectors. The influence of effectors was also exerted on the ferricyanide-linked (partial)PDH reaction. However cooperativity seems to be nearly completely absent in this reaction (h
The rate dependence of the overall reaction on the pyruvate concentration studied at different pH's allowed analysis of pS 0.5
, log V
versus pH profiles. Due to ionizations, two p K
's (at pH's 6.7 and 8.0) in the free enzyme and two p K
(at pH's 6.7 and 8.0) in the enzyme-Mg 2+
-TPP-pyruvate complex ( ES
-complex) are present; the slopes of the straight-line sections in the curves are approximately integrals (1,0 and -1). Protonation of PDC causes an increase in affinity between enzyme and pyruvate; AMP and sulphate also enhance the affinity of the PDC for pyruvate in de whole pH-range studied. Deprotonation of the ES
-complex upon increasing the pH, which leads to diminished activity, is largely prevented by AMP and sulphate by shifting p K2ES
(8.0) towards a higher value. These phenomena are also present in the ferricyanide-linked PDH reaction. The HILL-coefficient for the overall reaction depends in a similar way on the pH as log V
, viz.; at least partially, h
is determined by ionizations within the subunits of the ES
Apart from the allosteric control, which was based on measurements of initial (overall) reaction rates, the active proportion of PDC from Azotobacter
is controlled by the [CoASAc]/[CoA] ratio in a second line. Due to PTA activity, in the presence of phosphate CoASAc is reversibly converted into acetylphosphate and CoA, thus abolishing part of the inhibitory action of CoASAc.
A K M
for NAD +
in the overall reaction of 0.1 mM is found (in good correspondence with the value found for pure lipoamide dehydrogenase from Azotobacter).
Phosphate inhibits non-competitively, NADH inhibits competitively (K I
= 40 μM) with respect to NAD +
. In the presence of phosphate however the affinity of the enzyme for NADH increases (K I
= 20 μM).
The activity of PDC from Azotobacter
was shown not to be modified by phosphorylation and dephosphorylation, but instead controlled by the energy charge of the adenylate pool (especially in the presence of CoASAc) over a large range according to a R-system; ATP being inhibitory at high charge.
In CHAPTER 5 the rapid irreversible inactivation of PDC by sulfhydryl reagents is described. Under anaerobic conditions, sulfhydryl groups (originating from the protein-bound oxidized lipoyl moieties) are generated during reaction with either pyruvate plus Mg 2+
.TPP in the absence of CoA or with NAD(P)H; it was shown that lipoamide dehydrogenase from Azotobacter,
either unbound or complexed, forms a two-equivalent-reduced enzyme with excess NADPH.
The sulfhydryl groups developed are blocked by NEM, iodoacetic acid and bromopyruvate. The overall activity disappears completely within a reaction period of 10 min., without affecting the partial activities of the complex at comparable rates. To accomplish blocking of the LTA-bound lipoyl moieties by NEM via the reduced flavoprotein, the addition of this reagent prior to NAD(P)H turned out to be important. Reduction of the flavin leads to a rapid conformational change within the PDC, resulting in less accessibility of the lipoyl moieties for the action of NEM.
Instead of NEM, maleimide spin label can be selectively introduced in the complex in a either pyruvate plus Mg 2+
.TPP- or a NAD(P)H-dependent process. Mainly a semi-mobile species is visible in the EPR spectrum (τ c
= 0.5 nsec) pointing at a high mobility of the lipoyl moieties. Two of them are found per molecule FAD in the presence of Mg 2+
Furthermore, in the absence of pyruvate, bromopyruvate slowly inactivates the overall activity in a Mg 2+
.TPP process. During the early stages of the latter process, partial reactivation of the PDC can be observed upon incubating with DTT prior to the assay. Two processes are involved. The first enzyme (PDH) is the main target.
Aerobically, in the absence of a sulfhydryl reagent, but in the presence of pyruvate plus Mg 2+
.TPP, also a slow irreversible inactivation of the overall reaction (without affecting the partial activities) is observed. Most probably, S-S bridge formation due to oxidation of two protein-bound lip(SH)-S-acetyl species occurs. Although not responsible for the latter phenomenon it was discovered that the PDH component is also able to use oxygen instead of ferricyanide as electron acceptor in its partial reaction, catalyzing: Mg 2+.Tpppyruvate + O 2
+ H 2 O
-----------> H 2 O 2
+ CO 2
A HILL-coefficient of 1 for the dependence on the pyruvate concentration is found in this reaction.
In accordance with these studies, composition and catalytical behaviour of PDC from Azotobacter vinelandii
are schematically given in CHAPTER 6. In the Azotobacter
complex, four identical LTA units ( M
= 80,000 daltons) each containing one molecule of covalently bound lipoic acid, are distributed around one dimeric lipoamide dehydrogenase unit ( M
= 112,000 daltons) in noncovalent interaction. It is calculated that six to eight identical PDH units ( M =
90,000 daltons) and consequently four to zero PTA units ( M =
50,000 daltons) are joined to this sub-complex in a regular manner. Moreover, it is suggested that the presence of PTA in the complex actually is due to a purification-artefact and that the intact complex contains eight PDH units.
The reaction-sequence for the oxidation of pyruvate by PDC has been extended. From the pH-influence on the rate of the overall activity and of the PDH-catalyzed partial reactions, in relation to the pyruvate and AMP concentration (CHAPTER 4 and 5), and from the inactivation experiments (CHAPTER 5), participation of a protonated (unknown) group (p K
= 8.0), probably a sulfhydryl group, in catalysis is obvious. It has a function in stabilizing the carbanion intermediate (α-hydroxyethyl TPP) and the transfer of reducing equivalents to either oxygen and ferricyanide or by preparing the reaction with protein-bound oxidizing lipoic acid. It is concluded from the present studies that since the allosteric control of PDC from Azotobacter
cannot be the consequence of pyruvate-binding, it must be caused by the transfer reaction via this unknown group - which is located at each of the PDH components - to the four lipoyl moieties and by interaction of LTA-bound reduced lipoic acid with lipoamide dehydrogenase. It seems that the state of reduction of the flavin is related to the cooperativity.