[Regulation of cooperative properties of alpha-ketoglutarate dehydrogenase by means of thiol-disulfide metabolism]. / Reguliatsiia kooperativnykh svoistv alpha-ketoglutaratdegidrogenazy petem tiol-disul'fidnogo obmena.

The redox state of two SH-groups per enzyme subunit has been shown to control the cooperative properties of alpha-ketoglutarate dehydrogenase. These thiols oxidized, alpha-ketoglutarate dehydrogenase does not exhibit any cooperative properties. The enzyme reduction leads to subunit interactions. It has been found that the most effective agent reducing the alpha-ketoglutarate dehydrogenase thiols essential for the cooperativity is dihydrolipoate, one of the intermediates of the overall alpha-ketoglutarate dehydrogenase reaction. The possibility of changing the properties of alpha-ketoglutarate dehydrogenase in the multienzyme complex under the conditions when the lipoic acid integrated into the complex is reduced, has been investigated. Thus, incubation of the alpha-ketoglutarate dehydrogenase complex with NADH has been found to induce the conversion from the non-cooperative form to the cooperative one, presumably through the reduction of lipoic acid bound to the complex in the reaction catalyzed by lipoyl dehydrogenase, the third component of the complex.

Effect of alpha-ketoglutarate and its structural analogues on hysteretic properties of alpha-ketoglutarate dehydrogenase.

The burst of product accumulation during the KGD reaction was investigated. It has been shown not to be the obligatory feature of catalysis, but appears when increasing the enzyme saturation by KG. Structural analogues of KG and the SH-group modification suppress the initial burst without preventing catalysis. The results obtained are in favour of the existence of the regulatory site for binding KG and its structural analogues essential for hysteretic properties of KGD.

Inactivation of alpha-ketoglutarate dehydrogenase during enzyme-catalyzed reaction.

Alpha-Ketoglutarate dehydrogenase is inactivated during the enzymatic reaction. This inactivation is revealed both in a model system with an artificial electron acceptor and in the overall reaction catalyzed by the alpha-ketoglutarate dehydrogenase complex. Neither the substrate depletion and the product accumulation nor the changing of the alpha-ketoglutarate dehydrogenase oligomeric structure induces the inactivation. There are two independent mechanisms of alpha-ketoglutarate dehydrogenase inactivation in the course of the enzymatic reaction which are consistent with the two stages of the process. The first one corresponds to an essential decrease in activity during several minutes from the beginning of the reaction. This process can be reversed, occurs only during catalysis and manifests itself in the same degree both in the model system and during the functioning of the alpha-ketoglutarate dehydrogenase complex. The second stage is slow, irreversible and more apparent in the model system; it coincides with the inactivation due to the alpha-ketoglutarate dehydrogenase preincubation with hexacyanoferrate alone. The data obtained provide evidence that irreversible inactivation of alpha-ketoglutarate dehydrogenase during the enzymatic reaction is caused by the oxidant.

Regulation of alpha-ketoglutarate dehydrogenase cooperative properties in substrate binding by thiol-disulfide exchange.

The influence of reducing the KGD non-cooperative form by DTT on the KG binding by the enzyme was investigated. The chemical modification of KGD by DEP has revealed that reduction of KGD cysteine residues results in the appearance of the interaction of the dimer active sites upon the enzyme-substrate complex formation. The reduction of 2 SH-groups per KGD subunit: the most reactive one and a buried one--was established to be sufficient for the appearance of KGD cooperative properties in substrate binding as well as for the change in the enzyme activity plots versus substrate concentration. It is suggested that KGD can be regulated by thiol-disulfide exchange in the cell.

Change in alpha-ketoglutarate dehydrogenase cooperative properties due to dihydrolipoate and NADH.

Reducing 2 SH-groups of KGD by dihydrolipoate leads to cooperativity in substrate binding. Cooperative properties of KGD in the KGD complex are modulated by NADH. Physiological significance of these observations is discussed.

Structural and functional peculiarities of alpha-ketoglutarate dehydrogenase with non-interacting active sites.

The properties of alpha-ketoglutarate dehydrogenase with non-interacting active sites were investigated. The substrate and coenzyme saturation curves are found to be hyperbolic, which is consistent with the absence of cooperativity between the active sites of the enzyme. The peculiarities of KGD of this form, determining its functional properties, were revealed. Thus, 6 cysteine residues of the enzyme possess different properties in comparison with the form of the enzyme with interacting active sites. 3 Sulfhydryl groups of the "non-cooperative" enzyme form were rapidly oxidized in the process of the enzyme isolation and storage; thereafter they could not be reduced by dithiols. Three other cysteine residues are probably involved in the formation of disulfide bonds. Two of them are supposed to form intramolecular disulfide, whereas the third one is thought to be modified by some low molecular weight disulfide. The reduction of these sulfhydryl groups by dithiols is shown to be accompanied by the appearance of the kinetic cooperativity with respect to the substrate. It is suggested that the thiol/disulfide exchange in vivo can regulate a reversible conversion of the "non-cooperative" KGD form into one with interacting sites.

[Functional role of histidine residues of alpha-ketoglutarate dehydrogenase]. / O funktsional'noi roli gistidinovykh ostatkov alpha-ketoglutaratdegidrogenazy.

The protective effect of alpha-ketoglutarate dehydrogenase substrate and its analogs on the enzyme inactivation by diethylpyrocarbonate was studied. The values of true rate constants for diethylpyrocarbonate-induced inactivation and the Kd values for the enzyme complexes with ligands were determined. A comparison of Kd values for a number of ligands suggests that the histidine residue of the enzyme active center interacts with the alpha-keto group of the substrate. A mechanism of this histidine residue involvement in the catalytic act is proposed. According to this mechanism, the imidazole ring of histidine which is responsible for the substrate activation causes a simultaneous formation of a catalytically active form of the coenzyme--thiamine pyrophosphate ilide. It is assumed that the lower (as compared with the enzyme-substrate complexes) values of rate constants of inactivation by diethylpyrocarbonate for alpha-ketoglutarate dehydrogenase complexes with succinate, glutarate, and oxaloacetate are due to additional protonation of the histidine residue, eventually resulting in the blocking of the analogs interaction with the coenzyme.

[The form of alpha-ketoglutarate dehydrogenase showing no cooperative properties during substrate binding]. / Forma alpha-ketoglutaratdegidrogenazy, ne proiavliaiushchaia kooperativnykh svoistv pri sviazyvanii substrata.

A form of alpha-ketoglutarate dehydrogenase was detected, which is characterized by the non-equivalency of active centers for substrate binding normally revealed by chemical modification techniques and typical for other enzyme forms. The properties of various forms of alpha-ketoglutarate dehydrogenase (both soluble and immobilized on Sepharose) were compared. It was shown that despite its dimeric structure the newly detected enzyme form binds alpha-ketoglutarate in a way similar to the monomer; in this case no substrate-induced non-equivalency of the subunits due to intersubunit interactions is observed. It was found that the independent functioning of the active centers of the enzyme is due to the loosening of intersubunit contacts.

[Dehydrogenases of alpha-keto acids from pigeon breast muscle]. / Degidrogenazy alpha-ketokislot grudnoi myshtsy golubia.

The structure, function and regulation of pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase complexes from pigeon breast muscle are reviewed. The nature of essential groups involved in the formation of active centers of the first components of the complexes, i.e., pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase, is described. The catalytic mechanism of the pyruvate dehydrogenase reaction and the peculiarities of cooperative interactions of the active centers of the above enzymes are discussed.

[Non-equivalency of active centers of alpha-ketoglutarate dehydrogenase detected by modification of histidine residues]. / Neékvivalentnost' aktivnykh tsentrov alpha-ketoglutaratdegidrogenazy,vyiavliaemaia pri modifikatsii gistidinovykh ostatkov.

Using selective chemical modification of histidine residues of the alpha-ketoglutarate dehydrogenase component within the alpha-ketoglutarate dehydrogenase complex, the existence of interconvertible forms of the enzyme was demonstrated. These forms are distinguished by kinetics of inactivation caused by diethylpyrocarbonate. The interconversion of the enzyme forms involves alpha-ketoglutarate. Studies on substrate effects on the inactivation kinetics of individual enzyme forms revealed the non-equivalency of the enzyme active centers within the dimeric molecule of the alpha-ketoglutarate dehydrogenase component. The accessibility of an essential histidine residue in the active center of a neighbouring substrate-free monomer to the modifier increases as a result of interaction of the enzyme active centers during alpha-ketoglutarate binding by one of the subunits. The non-equivalency of the active centers manifests itself in different rates of interaction and in the unequal stability of binding of alpha-ketoglutarate to the alternate sites of the dimer. It is assumed that the biphasic kinetics of inactivation of pigeon breast muscle alpha-ketoglutarate dehydrogenase is due to tight binding of alpha-ketoglutarate in one of active centers of the enzyme dimeric molecule.

Evidence for alpha-ketoglutarate dehydrogenase monomer activity with the aid of enzyme immobilization.

Immobilization of pigeon breast muscle alpha-ketoglutarate dehydrogenase on CNBr-activated Sepharose 4B was carried out. Conditions for dissociation of the dimeric enzyme bound to the carrier via a single subunit were determined. Immobilized enzyme monomers with a specific activity higher than that of the dimer were obtained. The immobilized subunits are capable of reassociating with the soluble ones; this is accompanied by the restoration of the initial amount of the matrix-bound protein and the reconstitution of the activity typical of the immobilized enzyme original preparations.

[Amino acid composition of the alpha-ketoglutarate dehydrogenase from the breast muscle of the pigeon]. / Aminokislotnyi sostav al'fa-ketoglutaratdegidrogenazy iz grudnoi myshtsy golubia.

The amino acid composition of alpha-ketoglutarate dehydrogenase--a component of alpha-ketoglutarate dehydrogenase complex--from the pigeon breast muscle has been determined. A significant similarity of the enzyme amino acid composition isolated from systematically remote species has been revealed by comparison of the data obtained with the literary one concerning the alpha-ketoglutarate dehydrogenase from Escherichia coli and pig heart.

Essential histidine residues of alpha-ketoglutarate dehydrogenase from pigeon breast muscle.

Modification of histidine residues of alpha-ketoglutarate dehydrogenase results in complete inactivation of the enzyme. The kinetic curves for histidine residues modification and for alpha-ketoglutarate dehydrogenase inactivation are biphasic. The rate constants for both processes show a good coincidence at two phases. The complete inactivation of the enzyme is due to modification of one histidine at each step of the reaction. At saturating concentrations of the substrate, alpha-ketoglutarate, the inactivating effect of the modifier is lowered; the cofactors, thiamine pyrophosphate and Mg2+, exert no protective effect.

Essential arginine residues for catalytic and regulatory functions of alpha-ketoglutarate dehydrogenase from pigeon breast muscle.

The number of arginine residues of pigeon breast muscle alpha-ketoglutarate dehydrogenase modified by 2,3-butanedione and 2,4-pentanedione was determined. It was shown that two of the 40 arginine residues in the enzyme monomer (Mr = 86,000) are accessible to the action of dicarbonyl compounds and are functionally significant. The protective effect of alpha-ketoglutarate and ADP against enzyme modification by 2,3-butanedione suggests the participation of alpha-ketoglutarate dehydrogenase arginine residues in the binding of substrate and allosteric activator, ADP.

[Sulfhydryl groups of alpha-ketoglutarate dehydrogenase from pigeon breast muscle]. / Sul'fgidril'nye gruppy al'fa-ketoglutaratdegidrogenazy iz grudno myshtsy golubia.

Using various thiol reagents (DTNB, pCMB, NBD-chloride), the number and reactivity of SH-groups of the alpha-ketoglutarate dehydrogenase component (KGD) of the alpha-ketoglutarate dehydrogenase complex were established. The total number of SH-groups as determined in the presence of a detergent is 12 per monomer of KGD. In the native enzyme DTNB titrates 3 or 4 SH-groups which, according to their reactivity, can be divided into 3 types. pCMB and NBD-chloride modify 5 to 6 SH-groups of KGD. In the presence of alpha-ketoglutarate and its active analog, alpha-ketoadipate, two SH-groups of the enzymes, the most and the least reactive ones, become inaccessible to the action of DTNB but the titrated by a 30-100-fold molar excess of pCMB. Binding of alpha-ketoacids which are competitive inhibitors of the enzyme causes no masking of SH-groups of KGD. When 3-4 SH-groups of KGD are blocked, the enzyme activity is reduced by 30% due to modification of the same two SH-groups which are inaccessible to DTNB in the presence of substrate. The fluorescence spectra of S-NBD derivatives of KGD suggest that these two SH-groups have hydrophobic environment. In terms of the number and reactivity of the thiol groups the apoenzyme does not differ from the holoenzyme; however, an addition of alpha-ketoglutarate to the apoenzyme does not decrease the number of SH-groups interacting with DTNB or the rate of their modification. The masking of two SH-groups of the holoenzyme results from conformational changes accompanying the formation of a productive ternary enzyme -- coenzyme -- substrate complex. Possible localization of these groups in the proximity of the active site is discussed.

[Regulation of alpha-ketoglutarate dehydrogenase complex from pigeon breast muscle]. / Reguliatsiia al'fa-ketoglutaratdegidrogenaznogo kompleksa iz grudnoi myshtsy golubia.

The activity of alpha-ketoglutarate dehydrogenase complex from pigeon breast muscle is controlled by ADP and the reaction products, i. e. succinyl-CoA and NADH. ADP activates the alpha-ketoglutarate dehydrogenase component of the complex, whereas NADH inhibits alpha-ketoglutarate dehydrogenase and lipoyl dehydrogenase. In the presence of NADH the kinetic curve of the complex with respect to alpha-ketoglutarate and NAD and the dependence of upsilon versus [NAD] and upsilon versus [Lip (SH)2] in the lipoyl dehydrogenase reaction are S-shaped. In the absence of inhibitor ADP had no activating effect on lipoyl dehydrogenase; however, in the presence of NADH ADP decreases the cooperativity for NAD. The cooperative kinetics of the constituent enzymes of the complex are indicative of its allosteric properties. Isolation of the alpha-ketoglutarate dehydrogenase complex and its lipoyl dehydrogenase and alpha-ketoglutarate dehydrogenase components in a desensitized state confirms their allosteric nature. It is assumed that NADH effects of isolated alpha-ketoglutarate dehydrogenase is due to a shift in the equilibrium between different oligomeric forms of the enzyme.

[Kinetic properties of the alpha-ketoglutarate dehydrogenase complex from pigeon breast muscle]. / Kineticheskie svoistva al'fa-ketoglutaratderidorgenaznogo kompleksa nz grudnoi myshtsy golubia.

A complex kinetic behaviour of the alpha-ketoglutarate dehydrogenase component isolated from the pigeon breast muscle alpha-ketoglutarate dehydrogenase complex was established. The dependence curve of the reaction rate versus substrate concentration has an intermediate plateau and a maximum. The shape of the kinetic curve and specific activity depend on the enzyme concentration both in the reaction mixture and in the original solution of enzyme. The kinetic pattern of isolated alpha-ketoglutarate dehydrogenase suggests that the enzyme is a multi-step dissociating system characterized by different rates of oligomeric forms interconversions at various steps of dissociation. It is assumed that the process of alpha-ketoglutarate dehydrogenase oligomerization is a cooperative one. The kinetic behaviour of the complex and its constituent alpha-ketoglutarate dehydrogenase component is characterized by the same type of the v + [S] dependence curves as is the isolated enzyme. The correlation between the kinetic properties of the enzyme complex and its alpha-ketoglutarate dehydrogenase component and the kinetic behaviour of free alpha-ketoglutarate dehydrogenase suggest that the association-dissociation processes which are probably typical for the component within the complex determine the kinetic behaviour of the complex to a large extent. It is assumed that the anomalous kinetic behaviour of the alpha-ketoglutarate dehydrogenase component is due to the cooperative effects during substrate binding, which are mediated by a shift in the equilibrium between the oligomeric forms of enzyme.