The form of alpha-ketoglutarate dehydrogenase showing no cooperative properties during substrate binding

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.     

Functional role of histidine residues of alpha-ketoglutarate dehydrogenase

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.     

D-glyceraldehyde-3-phosphate dehydrogenase subunit cooperativity studied using immobilized enzyme forms

Tetrameric D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) isolated from rabbit skeletal muscle was covalently bound to CNBr-activated Sepharose 4B via a single subunit. Catalytically active immobilized dimer and monomeric forms of the enzyme were prepared after urea-induced dissociation of the tetramer. A study of the coenzyme-binding properties of matrix-bound tetrameric, dimeric and monomeric species has shown that: (1) an immobilized tetramer binds NAD+ with negative cooperativity, the dissociation constants being 0.085 microM for the first two coenzyme molecules and 1.3 microM for the third and the fourth one; (2) coenzyme binding to the dimeric enzyme form also displays negative cooperativity with Kd values of 0.032 microM and 1.1 microM for the first and second sites, respectively; (3) the binding of NAD+ to a monomer can occur with a dissociation constant of 1.6 microM which is close to the Kd value for low-affinity coenzyme binding sites of the tetrameric or dimeric enzyme forms. In the presence of NAD+ an immobilized monomer acquires a stability which is not inferior to that of a holotetramer. The catalytic properties of monomeric and tetrameric enzyme forms were compared and found to be different under certain conditions. Thus, the monomers of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase displayed a hyperbolic kinetic saturation curve for NAD+, whereas the tetramers exhibited an intermediary plateau region corresponding to half-saturating concentrations of NAD+. At coenzyme concentrations below half-saturating a monomer is more active than a tetramer. This difference disappears at saturating concentrations of NAD+. Immobilized monomeric and tetrameric forms of D-glyceraldehyde-3-phosphate dehydrogenase from baker's yeast were also used to investigate subunit interactions in catalysis. The rate constant of inactivation due to modification of essential arginine residues in the holoenzyme decreased in the presence of glyceraldehyde 3-phosphate, probably as a result of conformational changes accompanying catalysis. This effect was similar for monomeric and tetrameric enzyme forms at saturating substrate concentrations, but different for the two enzyme species under conditions in which about one-half of the active centers remained unsaturated. Taken together, the results indicate that association of D-glyceraldehyde-3-phosphate dehydrogenase monomers into a tetramer imposes some constraints on the functioning of the active centers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Scanning transmission electron microscopic study of alpha-ketoglutarate dehydrogenase complex from Escherichia coli

The alpha-ketoglutarate dehydrogenase complex was resolved into its three component enzymes: alpha-ketoglutarate dehydrogenase (E1), dihydrolipoyl transsuccinylase (E2), and dihydrolipoyl dehydrogenase. Subcomplexes were prepared in vitro by incubating the resolved E2, a 24-subunit cube-shaped molecule, with E1 (dimeric). The morphology and mass of the subcomplexes were determined by scanning transmission electron microscopy of negatively stained and of freeze-dried specimens. Images of both negative stained and freeze-dried subcomplexes were consistent with E1 binding at or near the midpoints of the edges of the E2 molecule. Mass analysis of the freeze-dried specimen showed that at least 95% of E1 remains in the dimeric state (or as two closely juxtaposed monomers) when it binds to E2.     

Modification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides with the 2',3'-dialdehyde derivative of NADP+ (oNADP+)

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is irreversibly inactivated by the 2,3'-dialdehyde of NADP+ (oNADP+) in the absence of substrate. The inactivation is first order with respect to NADP+ concentration and follows saturation kinetics, indicating that the enzyme initially forms a reversible complex with the inhibitor followed by covalent modification (KI = 1.8 mM). NADP+ and NAD+ protect the enzyme from inactivation by oNADP+. The pK of inactivation is 8.1. oNADP+ is an effective coenzyme in assays of glucose-6-phosphate dehydrogenase (Km = 200 microM). Kinetic evidence and binding studies with [14C] oNADP+ indicate that one molecule of oNADP+ binds per subunit of glucose-6-phosphate dehydrogenase when the enzyme is completely inactivated. The interaction between oNADP+ and the enzyme does not generate a Schiff's base, or a conjugated Schiff's base, but the data are consistent with the formation of a dihydroxymorpholino derivative.     

Modification of pig liver dimeric dihydrodiol dehydrogenase with diethylpyrocarbonate and by rose bengal-sensitized photooxidation: evidence for an active-site histidine residue.

Dihydrodiol dehydrogenase from pig liver was inactivated by diethylpyrocarbonate (DEP) and by rose bengal-sensitized photooxidation. The DEP inactivation was reversed by hydroxylamine and the absorption spectrum of the inactivated enzyme indicated that both histidine and tyrosine residues were carbethoxylated. The rates of inactivation by DEP and by photooxidation were dependent on pH, showing the involvement of a group with a pKa of 6.4. The kinetics of inactivation and spectrophotometric quantification of the modified residues suggested that complete inactivation was caused by modification of one histidine residue per active site. The inactivation by the two modifications was partially prevented by either NADP(H) or the combination of NADP+ and substrate, and completely prevented in the presence of both NADP+ and a competitive inhibitor which binds to the enzyme-NADP+ binary complex. The DEP-modified enzyme caused the same blue shift and enhancement of NADPH fluorescence as did the native enzyme, suggesting that the modified histidine is not in the coenzyme-binding site of the enzyme. The results suggest the presence of essential histidine residues in the catalytic region of the active site of pig liver dihydrodiol dehydrogenase.     

Identification of histidine residues at the active site of Megalobatrachus japonicus alkaline phosphatase by chemical modification

Alkaline phosphatase from Megalobatrachus japonicus was inactivated by diethyl pyrocarbonate (DEP). The inactivation followed pseudo-first-order kinetics with a second-order rate constant of 176 M(-1) x min(-1) at pH 6.2 and 25 degrees C. The loss of enzyme activity was accompanied with an increase in absorbance at 242 nm and the inactivated enzyme was re-activated by hydroxylamine, indicating the modification of histidine residues. This conclusion was also confirmed by the pH profiles of inactivation, which showed the involvement of a residue with pK(a) of 6.6. The presence of glycerol 3-phosphate, AMP and phosphate protected the enzyme against inactivation. The results revealed that the histidine residues modified by DEP were located at the active site. Spectrophotometric quantification of modified residues showed that modification of two histidine residues per active site led to complete inactivation, but kinetic stoichiometry indicated that one molecule of modifier reacted with one active site during inactivation, probably suggesting that two essential histidine residues per active site are necessary for complete activity whereas modification of a single histidine residue per active site is enough to result in inactivation.     

Regulation of alpha-ketoglutarate dehydrogenase complex from pigeon breast muscle]

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.     

Evidence for a change in the structure of glutamate dehydrogenase during its catalytic cycle under cryogenic conditions

Glutamate dehydrogenase binds alpha-ketoglutarate and NADPH to form a ternary complex whose ultraviolet difference spectrum exhibits a blue-shifted coenzyme absorption band and a distinctive aromatic amino acid perturbation. When ammonia is added to this complex at -42 degrees C in 50% methanol, initiating the enzymatic reaction, these two spectral features disappear at different rates. The kinetic independence of these two features is especially evident in the presence of excess L-glutamate. We propose that, under cryogenic conditions at least, there are two forms of the enzyme. The blue shift of the coenzyme absorption band reflects only the physical presence of an alpha-ketoglutarate molecule at the active site, while the distinctive aromatic amino acid perturbation reflects a change in enzyme structure caused by alpha-ketoglutarate binding which may persist in the absence of any bound alpha-ketoglutarate molecule. Simple red and blue shifts of model tyrosine and tryptophan compounds cannot be used to simulate the observed aromatic amino acid perturbation.     

Aspartate-beta-semialdehyde dehydrogenase from Escherichia coli. Affinity labeling with the substrate analogue L-2-amino-4-oxo-5-chloropentanoic acid: an example of half-site reactivity

The substrate binding site of aspartate-beta-semialdehyde dehydrogenase from Escherichia coli was studied by affinity labeling with L-2-amino-4-oxo-5-chloropentanoic acid. The substrate analogue irreversibly inactivates the enzyme with pseudo-first-order kinetics and with a half-of-the-sites reactivity. The substrate aspartate beta-semialdehyde protects the enzyme against the inactivation. A single group is labeled at the active site and is concluded to be the side-chain of a histidine residue. The amino acid sequence around the active site residue was established from a peptic digest of the labeled enzyme: Phe-Val-Gly-Gly-Asp-(modified residue)-Thr-Val-Ser.     

Inactivation of porcine heart cytoplasmic malate dehydrogenase by pyridoxal 5'-phosphate.

Pyridoxal 5'-phosphate (pyridoxal-5'-P) has been found to act as a bifunctional reagent during the inactivation of porcine heart cytoplasmic malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37). The biphasic kinetics and X-azolidine-like structure formed were similar to those observed for mitochondrial malate dehydrogenase (Wimmer, M.J., Mo, T., Sawyers, D.L., and Harrison, J.H. (1975) J. Biol. Chem. 250, 710-715). In the cytoplasmic enzyme, however, irreversible inactivation representing X-azolidine formation was found to be the dominant characteristic of the interaction with pyridoxal-5'-P. Spectral evidence indicated that at total inactivation 2 mol of pyridoxal-5'-P were incorporated per mol of enzyme or one pyridoxal-5'-P per enzymatic active site. The presence of NADH protected the enzyme from inactivation suggesting interaction of pyridoxal-5'-P at or near the enzymatic active centers of this enzyme. Fluorometric titrations indicated that pyridoxal-5'-P-inactivated enzyme failed to bind NADH or at least failed to bind NADH in the same fashion as native enzyme.     

The presence of essential histidine residues in manganese(III)-containing acid phosphatase from sweet potato

Chemical modification studies of manganese(III)-containing acid phosphatase [EC 3.1.3.2] were carried out to investigate the contributions of specific amino-acid side-chains to the catalytic activity. Incubation of the enzyme with N-ethylmaleimide at pH 7.0 caused a significant loss of the enzyme activity. The inactivation followed pseudo-first-order kinetics. Double log plots of pseudo-first-order rate constant vs. concentration gave a straight line with a slope of 1.02, suggesting that the reaction of one molecule of reagent per active site is associated with activity loss. The enzyme was protected from inactivation by the presence of molybdate or phosphate ions. Amino acid analyses of the N-ethylmaleimide-modified enzyme showed that the 96%-inactivated enzyme had lost about one histidine and one-half lysine residue per enzyme subunit without any significant decrease in other amino acids, and also demonstrated that loss of catalytic activity occurred in parallel with the loss of histidine residue rather than that of lysine residue. Molybdate ions also protected the enzyme against modification of the histidine residue. The enzyme was inactivated by photooxidation mediated by methylene blue according to pseudo-first-order kinetics. The pH profile of the inactivation rates of the enzyme showed that an amino acid residue having a pKa value of approximately 7.2 was involved in the inactivation. These studies indicate that at least one histidine residue per enzyme subunit participates in the catalytic function of Mn(III)-acid phosphatase.     

Supramolecular organization of enzymes of the tricarboxylic acid cycle

In virtue of analysis of data on the interaction of tricarboxylic acid cycle enzymes with the mitochondrial inner membrane and data on the enzyme-enzyme interactions, the spatial structure for the tricarboxylic acid cycle enzyme complex (tricarboxylic acid cycle metabolon) is proposed. The alpha-ketoglutarate dehydrogenase complex, adsorbed on the mitochondrial inner membrane along one of its 3-fold symmetry axes, plays the key role in the formation of metabolon. Two association sites of the alpha-ketoglutarate dehydrogenase complex located on opposite sides of the complex participate in the interaction with the membrane. The tricarboxylic acid cycle enzyme complex contains one molecule of the alpha-ketoglutarate dehydrogenase complex and six molecules of each of the other enzymes of the tricarboxylic acid cycle, as well as aspartate aminotransferase and nucleosidediphosphate kinase. Succinate dehydrogenase, the integral protein of the mitochondrial inner membrane, is a component of the anchor site responsible for the assembly of metabolon on the membrane. The molecular mass of the complex (ignoring succinate dehydrogenase) is of 8.10(6) daltons. The metabolon symmetry corresponds to the D3 point symmetry group. It is supposed, that the tricarboxylic acid cycle enzyme complex interacts with other multienzyme complexes of the matrix and the electron transfer chain.     

Effects of di-isopropyl phosphorofluoridate on rat liver microsomal and lysosomal beta-glucuronidase

N-Bromosuccinimide completely inactivated the cellulase, and titration experiments showed that oxidation of one tryptophan residue per cellulase molecule coincided with 100% inactivation. CM-cellulose protected the enzyme from inactivation by N-bromosuccinimide. The cellulase was inhibited by active benzyl halides, and reaction with 2-hydroxy-5-nitrobenzyl bromide resulted in the incorporation of 2.3 hydroxy-5-nitrobenzyl groups per enzyme molecule; one tryptophan residue was shown to be essential for activity. Diazocarbonyl compounds in the presence of Cu2+ ions inhibited the enzyme. The pH-dependence of inactivation was consistent with the reaction occurring with a protonated carboxyl group. Carbodi-imide inhibited the cellulase, and kinetic analysis indicated that there was an average of 1 mol of carbodi-imide binding to the cellulase during inactivation. Treatment of the cellulase with diethyl pyrocarbonate resulted in the modification of two out of the four histidine residues present in the cellulase. The modified enzyme retained 40% of its original activity. Inhibition of cellulase activity by the metal ions Ag+ and Hg2+ was ascribed to interaction with tryptophan residues, rather than with thiol groups.     

Regulation of mitochondrial malate dehydrogenase: kinetic modulation independent of subunit interaction

Porcine heart mitochondrial malate dehydrogenase (EC 1.1.1.37), a dimeric enzyme of Mr = 70,000, is both allosterically activated and inhibited by citrate. Using an affinity elution procedure based upon citrate binding to malate dehydrogenase, the isolation of pure heterodimer (a dimeric species with one active subunit and one iodoacetamide-inactivated subunit) has been achieved. Investigations utilizing this heterodimer in conjunction with resin-bound monomers of malate dehydrogenase have allowed the formulation of a definite conclusion concerning the role of subunit interactions in catalysis and regulation of this enzyme. The citrate kinetic effects, oxaloacetate inhibition, malate activation, and the effects of 2-thenoyl-trifluoroacetone (TTFA) are shown to be independent of interaction between catalytically active subunits. Previous kinetic data thought to support a reciprocating catalytic mechanism for this enzyme may be reinterpreted upon closer analysis in relation to an allosteric, conformationally specific binding model for malate dehydrogenase.     

Modification of glutamate dehydrogenase by pyridoxal-5'-phosphate. Study of the cooperative type of inhibition by GTP

The character of allosteric inhibition of glutamate dehydrogenase by GTP was studied. The derivative of the enzyme not capable of being polymerized was taken as a model. It was shown that: in the absence of NADH every protomer of this derivative can bind one molecule of GTP; in the presence of NADH the additional binding site for GTP was induced; the modification of the enzyme derivative by pyridoxal-5-phosphate in the presence of NADH and alpha-ketoglutarate blocked the NADH-induced GTP binding site and the disappearance of positive kinetic cooperativity induced by GTP was observed; to achieve the inhibitory action of GTP the binding of the effector to only one (NADH-induced) site was enough; the role of GTP binding to the NADH-induced site is to provide better affinity of the effector to the "inhibitory" centre; the positive kinetic cooperativity of inhibition of glutamate dehydrogenase by GTP depends probable on the cooperative character of interaction between the two molecules of GTP to each protomer of the enzyme.     

Evidence for the presence of a critical histidine residue at the active site in glyceraldehyde-3-phosphate dehydrogenase of Ehrlich ascites carcinoma cells

Ehrlich ascites carcinoma (EAC) cell glyceraldehyde-3-phosphate dehydrogenase (GA3PD) (EC. 1.2.1.12) was completely inactivated by diethyl pyrocarbonate (DEPC), a fairly specific reagent for histidine residues in the pH range of 6.0-7.5. The rate of inactivation was dependent on pH and followed pseudo-first order reaction kinetics. The difference spectrum of the inactivated and native enzymes showed an increase in the absorption maximum at 242 nm, indicating the modification of histidine residues. Statistical analysis of the residual enzyme activity and the extent of modification indicated modification of one essential histidine residue to be responsible for loss of the catalytic activity of EAC cell GA3PD. DEPC inactivation was protected by substrates, D-glyceraldehyde-3-phosphate and NAD, indicating the presence of essential histidine residue at the substrate-binding region of the active site. Double inhibition studies also provide evidence for the presence of histidine residue at the active site.     

Purification and characterization of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase from porcine kidney

A NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-OH-PGDH) from porcine kidney was purified to homogeneity by acid precipitation, blue agarose affinity chromatography, hydroxyapatite-ultrogel adsorption chromatography, DEAE-Sephadex ion-exchange chromatography and NAD(+)-agarose affinity chromatography. The specific activity of the homogeneous enzyme was 31.2 U/mg. The molecular mass of the native enzyme was estimated to be 55,000 Da, whereas that of SDS-treated enzyme was 29,000 Da indicating that the native enzyme was dimeric. Compared to human placental 15-OH-PGDH, porcine kidney enzyme gave a similar general amino acid residue distribution. Chemical modification of the enzyme with N-ethyl maleimide (3 microM), N-chlorosuccinimide (20 microM) or 2,4,6-trinitrobenzenesulfonic acid (2.5 microM) followed pseudo-first-order inactivation kinetics, and inactivation could be prevented by the presence of NAD+ (1 mM) but not of prostaglandin E1 (140 microM) indicating the involvement of cysteine, methionine and lysine residues in the coenzyme binding site. Inactivation by diethyl pyrocarbonate (1.25 mM) or phenylglyoxal (10 mM) also showed pseudo-first-order kinetics suggesting that histidine and arginine residues were catalytically or structurally important in the native enzyme. These studies provide new insights into the structure and function of 15-OH-PGDH.     

Essential histidine residues in coenzyme B12-dependent diol dehydrase: dye-sensitized photooxidation and ethoxycarbonylation

The apoenzyme of diol dehydrase was inactivated by photoirradiation in the presence of rose bengal or methylene blue, following pseudo-first-order kinetics. The inactivation rates were markedly reduced under a helium atmosphere, suggesting that the inactivation is due to photooxidation of the enzyme under air. The half-maximal rate of methylene blue-sensitized photoinactivation was observed at pH around 7.5. Amino acid analyses indicated that one to two histidine residues decreased upon the dye-sensitized photoinactivation, whereas the numbers of tyrosine, methionine, and lysine did not change. Ethoxyformic anhydride, another histidine-modifying reagent, also inactivated diol dehydrase, with pseudo-first-order kinetics and a half-maximal rate at pH 7.7. It was shown spectrophotometrically that three histidine residues per enzyme molecule were modified by this reagent with loss of enzyme activity. Two tyrosine residues per enzyme molecule were also modified rapidly, irrespective of the activity. The photooxidation or ethoxycarbonylation of the enzyme did not result in dissociation of the enzyme into subunits, but deprived the enzyme of ability to bind cyanocobalamin. The percentage loss of cobalamin-binding ability agreed well with the extent of inactivation. The enzyme-bound hydroxocobalamin showed only partial protecting effect against photoinactivation and resulting loss of the cobalamin-binding ability. These results provide evidence that diol dehydrase possesses essential histidine residues which are required for the coenzyme binding.