Substrate binding to chloramphenicol acetyltransferase: evidence for negative cooperativity from equilibrium and kinetic constants for binary and ternary complexes

Chloramphenicol acetyltransferase (CAT) catalyzes the acetyl-CoA-dependent acetylation of chloramphenicol by a ternary complex mechanism with a rapid equilibrium and essentially random order of addition of substrates. Such a kinetic mechanism for a two-substrate reaction provides an opportunity to compare the affinity of enzyme for each substrate in the binary complexes (1/Kd) with corresponding values (1/Km) for affinities in the ternary complex where any effect of the other substrate should be manifest. The pursuit of such information for CAT involved the use of four independent methods to determine the dissociation constant (Kd) for chloramphenicol in the binary complex, techniques which included stopped-flow measurements of on and off rates, and a novel fluorometric titration method. The binary complex dissociation constant (Kd) for acetyl-CoA was measured by fluorescence enhancement and steady-state kinetic analysis. The ternary complex dissociation constant (Km) for each substrate (in the presence of the other) was determined by kinetic and fluorometric methods, using CoA or ethyl-CoA to form nonproductive ternary complexes. The results demonstrate an unequivocal decrease in affinity of CAT for each of its substrates on progression from the binary to the ternary complex, a phenomenon most economically described as negative cooperativity. The binary complex dissociation constants (Kd) for chloramphenicol and acetyl-CoA are 4 microM and 30 microM whereas the corresponding dissociation constants in the ternary complex (Km) are 12 microM and 90 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenine nucleotides affect the binding of 3-phosphoglycerate to pig muscle 3-phosphoglycerate kinase

Pig muscle 3-phosphoglycerate kinase was complexed with 1-anilino-8-naphthalenesulfonate (ANS) in order to monitor the binding of substrates to the enzyme. The enzyme-dye interaction did not influence the enzymic activity under the experimental conditions used. By measuring the substrate-dependent change in the fluorescence emission of ANS molecules tightly bound to the enzyme (Kd less than or equal to 0.05 mM), fluorimetric titrations were carried out in 0.1 M Tris/HCl buffer pH 7.5, containing 5 mM mercaptoethanol, at 20 degrees C. The dissociation constants obtained for the separate bindings of 3-phosphoglycerate, MgATP, 1,3-bisphosphoglycerate and MgADP were 0.03 +/- 0.01 mM, 0.15 +/- 0.10 mM, 0.00005 +/- 0.00001 mM and 0.15 +/- 0.10 mM respectively. binding of 3-phosphoglycerate is weakened when MgATP is also bound to the enzyme: the dissociation constant of 3-phosphoglycerate in this ternary complex (0.25 +/- 0.08 mM) is comparable to its Km value (0.38 +/- 0.10 mM). The same weakening can be observed in the non-productive ternary complexes where MgATP is replaced by MgADP (Kd = 0.20 +/- 0.10 mM) or AMP (Kd = 0.12 +/- 0.05 mM), whereas adenosine has no such effect. This indicates the importance of the negatively charged phosphate(s) of nucleotides in influencing the binding of 3-phosphoglycerate. In contrast to 3-phosphoglycerate, the binding of the substrate analogue, glycerol 3-phosphate is practically not affected by the presence of MgATP: the dissociation constant to the free enzyme (0.40 +/- 0.10 mM) is comparable to its inhibitory constant (0.70 +/- 0.20 mM). This finding and the similarity of the dissociation constant of glycerol 3-phosphate binding (0.40 +/- 0.10 mM) and the Km value of 3-phosphoglycerate (0.38 +/- 0.10 mM) suggest that, during the enzymic reaction, binding of 3-phosphoglycerate occurs probably without involvement of the carboxyl group.     

The halide complexes of myeloperoxidase and the mechanism of the halogenation reactions

The spectral changes caused by the addition of halides to myeloperoxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) have been investigated and the dissociation constants of the enzyme-halide complexes have been determined. The pH dependence of the dissociation constants suggests that halide binding is associated with a protonation step in myeloperoxidase. Myeloperoxidase catalyzes the peroxidative chlorination and bromination of monochlorodimedone. It is shown that at low pH, chloride acts as a competitive inhibitor with respect to H2O2, whereas at higher pH, H2O2 inhibits the chlorination reaction. The dissociation constant (Kd) of the spectroscopically detectable complex and the Km for chloride are considerably smaller than the inhibition constant (Ki) for chloride. These halogenation reactions are strongly pH dependent, the logarithm of the Km for chloride varies linearly with pH. The position of the pH optimum of the chlorination and bromination reaction is a linear function of the logarithm of the [halide]/[H2O2] ratio. A mechanism of the chlorination and bromination reaction is suggested with substrate inhibition for both hydrogen peroxide and the halide.     

Two-dimensional electrophoretic analysis of human erythrocyte cylindrin

In the absence of a peptidylproline substrate, the oxidative decarboxylation of 2-oxoglutarate by prolyl 4-hydroxylase (prolyl-glycyl-peptide,2-oxoglutarate:oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2) is stoicheiometrically coupled to the oxidation of ascorbate. The Km and Kd for O2 in this partial reaction are 1.5 mM, this value being one order of magnitude higher than the Km and Kd for O2 in the complete reaction in the presence of (Pro-Pro-Gly)5, indicating that in this case O2 can become enzyme-bound predominantly after the interaction of the peptide substrate with the enzyme. The Km values for 2-oxoglutarate in the partial and the complete reactions are the same. In the absence of both a peptide substrate and ascorbate 2 mol CO2 per mol enzyme are produced in the first 1-1.5 min, during which the enzyme becomes inactivated and, as shown earlier (De Jong , L., Albracht , S.P.J. and Kemp, A. (1982) Biochim. Biophys. Acta 704, 326-332) enzyme-bound Fe2+ becomes oxidized to Fe3+. The results are consistent with a mechanism in which a Fe2+O complex is the O-transferring intermediate involved in peptidylproline hydroxylation.     

Kinetic isotope effect and the presteady-state kinetics of the reaction catalyzed by the bacterial formate dehydrogenase

The primary kinetic isotope effect of the reaction catalyzed by NAD+-dependent formate dehydrogenase (EC 1.2.1.2.) from the methylotrophic bacterium Pseudomonas sp. 101 has been studied. Analysis of the ratios HVm/DVm and H(Vm/KM)/D(Vm/KM) in the pH range 6.1-7.9 showed that the transfer of hydride ion in ternary enzyme-substrate complex is a limiting step of the reaction, and the formate binding to the binary complex (formate dehydrogenase + NAD+) reached equilibrium when the pH of the medium was increased. An approach has been developed to determine the elementary constants of substrate association (kon) and dissociation (koff) at the stages of the binary--ternary enzyme-substrate complexes for the random equilibrium 2-substrate kinetic mechanism. The kon and koff values obtained for the bacterial formate dehydrogenase by using the proposed approach for NAD+ were (4.8 +/- 0.8)*10(5)M-1s-1 and (90 +/- 10) s-1, and for formate (2.0 +/- 1.0)*10(4) M-1s-1 and (60 +/- 20) s-1, respectively.     

Substrate specificities and structure-activity relationships for the nucleotidylation of antibiotics catalyzed by aminoglycoside nucleotidyltransferase 2'-I

Aminoglycoside nucleotidyltransferase 2'-I (formerly gentamicin adenylyltransferase) conveys antibiotic resistance to Gram-negative bacteria by transfer of AMP to the 2'-hydroxyl group of 4,6-substituted deoxystreptamine-containing aminoglycosides. The kinetics constants of thirteen aminoglycoside antibiotics and the magnesium chelates of eight nucleotide triphosphates were determined with purified enzyme. Eleven of the antibiotics exhibit substrate inhibition attributed to secondary binding of the aminoglycoside to an enzyme-AMP-aminoglycoside complex. Maximal velocities vary by only 4-fold, versus variation of values of Vmax/Km for the aminoglycosides of nearly 4000-fold, consistent with a Theorell-Chance kinetic mechanism as proposed for this enzyme [Gates, C. A., & Northrop, D. B. (1988) Biochemistry (second of three papers in this issue)] with the added specification that the binding of aminoglycosides is in rapid equilibrium. Under these conditions, Vmax/Km becomes kcat/Kd, where kcat is the net rate constant for catalysis (but not turnover) and Kd is the dissociation constant of aminoglycosides from a complex with enzyme and nucleotide. Values of kcat fall closely together into three distinct sets, with the 3',4'-dideoxygentamicins greater than gentamicins greater than kanamycins. These sets reflect unusual structure-activity correlations which are specific for catalysis but have nothing to do with the maximal velocity of this enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

The binding of Ca2+ ions to pig heart NAD+-isocitrate dehydrogenase and the 2-oxoglutarate dehydrogenase complex.

1. The binding of Ca2+ ions to purified pig heart NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase, freed of contaminating Ca2+ by parvalbumin/polyacrylamide chromatography, has been studied by flow dialysis and by the use of fura-2. 2. For the 2-oxoglutarate dehydrogenase complex, 3.5 mol of Ca2+-binding sites/mol of complex were apparent, with an apparent dissociation constant (Kd value) for Ca2+ of 2.0 microM. These values were little affected by Mg2+ ions, ADP or 2-oxoglutarate. 3. By contrast, binding of Ca2+ to NAD+-isocitrate dehydrogenase (Kd = 14 microM) required ADP, isocitrate and Mg2+ ions. The number of Ca2+-binding sites associated with NAD+-isocitrate dehydrogenase was then 0.9 mol/mol of tetrameric enzyme. 4. The 2-oxoglutarate dehydrogenase complex bound ADP (as ADP3-) to a group of tight-binding sites (Kd = 3.1 microM) with a stoichiometry, 3.3 mol/mol of complex, similar to that for the binding of Ca2+; a variable number of much weaker sites (Kd = 100 microM) for ADP3- was also apparent.     

Purification and characterization of the 2-oxoglutarate dehydrogenase complex from Dictyostelium discoideum

The 2-oxoglutarate dehydrogenase complex was isolated from the cellular slime mould, Dictyostelium discoideum, and purified 113-fold. The enzyme exhibited Michaelis-Menten kinetics and the Km values for 2-oxoglutarate, CoA, and NAD were 1.0 mM, 0.002 mM, and 0.07 mM, respectively. The Ki value for succinyl-CoA was determined to be 0.004 mM and the Ki for NADH was 0.018 mM. AMP had positive effects whereas ATP had negative effects on the enzyme activity. The kinetic constants determined in this study and the reaction mechanism suggested can now be incorporated into a transition model of the tricarboxylic acid cycle during differentiation of D. discoideum.     

Characterization of steady state, single-turnover, and binding kinetics of the TaqI restriction endonuclease.

The TaqI restriction endonuclease recognizes and cleaves the duplex DNA sequence T decreases CGA. Steady state kinetic analysis with a small oligodeoxyribonucleotide substrate showed that the enzyme obeyed Michaelis-Menten kinetics (Km = 53 nM, kcat = 1.3 min-1 at 50 degrees C and Km = 0.5 nM, kcat = 2.9 min-1 at 60 degrees C). At 0 degree C, the enzyme was completely inactive, while at 15 degrees C, turnover produced nicked substrate as the major product in excess of enzyme indicating dissociation between nicking events. Above 37 degrees C, both strands in the duplex were cleaved prior to dissociation. In contrast to the tight, temperature-dependent binding of substrate, binding of the Mg2+ cofactor was weak (Kd = 2.5 mM) and the same at either 50 degrees C or 60 degrees C. Single-turnover experiments using oligonucleotide substrate showed that hydrolysis of duplex DNA occurred via two independent nicking events, each with a first order rate constant (kst) of 5.8 min-1 at 60 degrees C and 3.5 min-1 at 50 degrees C. The pH dependence of Km (pKa = 9) and kst (pKa = 7) suggests Lys/Arg and His, respectively, as possible amino acids influencing these constants. Moreover, although kst increased significantly with pH, kcat did not, indicating that at least two steps can be rate-controlling in the reaction pathway. Binding of protein to canonical DNA in the presence of Mg2+ at 0 degree C or in the absence of Mg2+ at 50 degrees C was weak (Kd = 2.5 microM or 5,000-fold weaker than the optimal measured Km) and equal to the binding of noncanonical DNA as judged by retention on nitrocellulose. Similar results were seen in gel retardation assays. These results suggest that both Mg2+ and high temperature are required to attain the correct protein conformation to form the tight complex seen in the steady state analysis. In the accompanying paper (Zebala, J. A., Choi, J., Trainor, G. L., and Barany, F. (1992) J. Biol. Chem. 267, 8106-8116), we report how these kinetic constants are altered using substrate analogues and propose a model of functional groups involved in TaqI endonuclease recognition.     

Catalytic and regulatory site composition of yeast AMP deaminase by comparative binding and rate studies. Resolution of the cooperative mechanism

Yeast AMP deaminase is allosterically activated by ATP and MgATP and inhibited by GTP and PO4. The tetrameric enzyme binds 2 mol each of ATP, GTP, and PO4/subunit with Kd values of 8.4 +/- 4.0, 4.1 +/- 0.6, and 169 +/- 12 microM, respectively. At 0.7 M KCl, ATP binds to the enzyme, but no longer activates. Titration with coformycin 5'-monophosphate, a slow, tight-binding inhibitor, indicates a single catalytic site/subunit. ATP and GTP bind at regulatory sites distinct from the catalytic site and their binding is mutually exclusive. Inorganic phosphate competes poorly with ATP for the ATP sites (Kd = 20.1 +/- 4.1 mM). However, near-saturating ATP reduces the moles of phosphate bound per subunit to 1 PO4, which binds with a Kd = 275 +/- 22 microM. In the presence of ATP, PO4 cannot effectively compete with ATP for the nucleotide triphosphate sites. The PO4 which binds in the presence of ATP is competitive with AMP at the catalytic site since the Kd equals the kinetic inhibition constant for PO4. Initial reaction rate curves are a cooperative function of AMP concentration and activation by ATP is also cooperative. However, no cooperativity is observed in the binding of any of the regulator ligands and ATP binding and kinetic activation by ATP is independent of substrate analog concentration. Cooperativity in initial rate curves results, therefore, from altered rate constants for product formation from each (enzyme.substrate)n species and not from cooperative substrate binding. The traditional cooperative binding models of allosteric regulation do not apply to yeast AMP deaminase, which regulates catalytic activity by kinetic control of product formation. The data are used to estimate the rates of AMP hydrolysis under reported metabolite concentrations in yeast.     

The AdhS alleloenzyme of alcohol dehydrogenase from Drosophila melanogaster. Variation of kinetic parameters with pH.

The variation with pH of the kinetic parameters for the alcohol and acetaldehyde reactions were studied for the alleloenzyme AdhS from Drosophila melanogaster. The variation of Ki (KEO,I) with pH for two ethanol-competitive inhibitors, pyrazole and 2,2,2-trifluoroethanol, was also studied. Both alcohol oxidation and acetaldehyde reduction follow a compulsory ordered pathway, with coenzyme binding first. The rate-limiting step for ethanol oxidation is complex and involves at least hydride transfer and dissociation of the enzyme-NADH complex (ER). In contrast with this, the rate-limiting step for the back reaction, i.e. the reduction of acetaldehyde, is dissociation of the enzyme-NAD+ complex (EO). A rate-limiting ER dissociation appears in the oxidation of the secondary alcohol propan-2-ol, whereas for the back reaction, i.e. acetone reduction, hydride transfer in the ternary complexes is rate-limiting. There is one group in the free enzyme, with a pK of approx. 8.0, that regulates the kon velocity for NADH, whereas for NAD+ several groups seem to be involved. A group in the enzyme is drastically perturbed by the formation of the binary EO complex. Protonation of this group with a pK of 7.6 in the EO complex resulted in weakened alcohol and inhibitor binding, in addition to an increased dissociation rate of NAD+ from the binary EO complex. Neither the binding of acetaldehyde nor the dissociation rate of NADH from the binary ER complex varied within the pH region studied.     

Dissecting the domain structure of the regulatory subunit of cAMP-dependent protein kinase I and elucidating the role of MgATP

A truncated regulatory subunit of cAMP-dependent protein kinase I was constructed which contained deletions at both the carboxyl terminus and at the amino terminus. The entire carboxyl-terminal cAMP-binding domain was deleted as well as the first 92 residues up to the hinge region. This monomeric truncated protein still forms a complex with the catalytic subunit, and activation of this complex is mediated by cAMP. The affinity of this mutant holoenzyme for cAMP and its activation by cAMP are nearly identical to holoenzyme formed with a regulatory subunit having only the carboxyl-terminal deletion and very similar to native holoenzyme. The off rate for cAMP from both mutant regulatory subunits, however, is monophasic and very fast relative to the biphasic off rate seen for the native regulatory subunit. The effects of NaCl, urea, and pH on cAMP binding are also very similar for the mutant and native holoenzymes. Like the native type I holoenzyme, both mutant holoenzymes bind ATP with a high affinity. The positive cooperativity seen for MgATP binding to the native holoenzyme, however, is abolished in the double deletion mutant. The Hill coefficient for ATP binding to this mutant holoenzyme is 1.0 in contrast to 1.6 for the native holoenzyme. The Kd (cAMP) is increased by approximately 1 order of magnitude for both mutant forms of the holoenzyme in the presence of MgATP. A similar shift is seen for the native holoenzyme. Further characterization of the MgATP-binding properties of the wild-type holoenzyme indicates that a binary complex containing catalytic subunit and MgATP is required, in particular, for reassociation with the cAMP-bound regulatory subunit. This binary complex is required for rapid dissociation of the bound cAMP and is probably responsible for the observed reduction in cAMP-binding affinity for the type I holoenzyme in the presence of MgATP.     

Some kinetic and other properties of the isoenzymes of aspartate aminotransferase isolated from sheep liver.

A method for the purification of mitochondrial isoenzyme of sheep liver aspartate aminotransferase (EC 2.6.1.1) is described. The final preparation is homogeneous by ultracentrifuge analyses and polyacrylamide-gel electrophoresis and has a high specific activity (182 units/mg). The molecular weight determined by sedimentation equilibrium is 87,100 +/- 680. The amino acid composition is presented; it is similar to that of other mitochondrial isoenzymes, but with a higher content of tyrosine and threonine. Subforms have been detected. On isoelectric focusing a broad band was obtained, with pI 9.14. The properties of the mitochondrial aspartate aminotransferase are compared with those of the cytoplasmic isoenzyme. The Km for L-aspartate and 2-oxoglutarate for the cytoplasmic enzyme were 2.96 +/- 0.20 mM and 0.093 +/- 0.010 mM respectively; the corresponding values for the mitochondrial form were 0.40 +/- 0.12 mM and 0.98 +/- 0.14 mM. Cytoplasmic aspartate aminotransferase showed substrate inhibition by concentrations of 2-oxoglutarate above 0.25 mM in the presence of aspartate up to 2mM. The mitochondrial isoenzyme was not inhibited in this way. Pi at pH 7.4 inhibited cytoplasmic holoenzyme activity by up to about 60% and mitochondrial holoenzyme activity up to 40%. The apparent dissociation constants for pyridoxal 5'-phosphate were 0.23 micrometer (cytoplasmic) and 0.062 micrometer (mitochondrial) and for pyridoxamine 5'-phosphate they were 70 micrometer (cytoplasmic) and 40 micrometer (mitochondrial). Pi competitively inhibited coenzyme binding to the apoenzymes; the inhibition constants at 37 degree C were 32 micrometer for the cytoplasmic isoenzyme and 19.5 micrometer for the mitochondrial form.     

Evidence that rabbit muscle protein kinase has two kinetically distinct binding sites for adenosine 3' ; 5'-cyclic monophosphate.

Two distinct populations of binding sites for cyclic AMP are associated with the regulatory moity of cyclic AMP dependent protein kinase (E.C. 2.7.1.37), as judged from the kinetics of the interaction between the nucleotide and the binding protein. The two types of sites were present at the proportion 1:1. The rate of dissociation of bound cyclic AMP was more rapid for one type of site than for the other type. High ionic strength accentuated the difference in the rate of dissociation of cyclic AMP from the two sites.The two binding sites and protein kinase activity copurified during the entire procedure for preparation of protein kinase holoenzyme. The kinetic properties of each of the two sites and the proportion between them was the same in a highly purified preparation of the regulatory moiety of protein kinase and in binding protein freshly prepared in the presence of protease-inhibitor.     

Chloride affects the interaction between tyrosyl-tRNA synthetase and tRNA

The physiological concentration of free magnesium in Escherichia coli cells is about 1 mM, and there is almost no chloride in the cell. When the aminoacylation of tRNA by tyrosyl-tRNA synthetase was assayed at 1 mM free Mg2+, chloride (and sulphate) ions inhibited the reaction but acetate at the same concentration (< 200 mM) was not inhibitory. When the magnesium concentration was increased to 10 mM there was almost no chloride inhibition any more. Chloride strengthened the PPi inhibition, the Ki(app)(PPi) values at 1 mM free Mg2+ were 140, 120, and 56 microM at 0, 50 and 150 mM KCl, respectively. Chloride weakened the AMP inhibition, the corresponding values for Ki(app)(AMP) were 0.35, 0.5, and 0.9 mM. The value of Km(app)(tRNA(Tyr)) was clearly increased by chloride, being 22, 37, 93, and 240 nM at 0, 50, 100, and 150 mM KCl, respectively. Best-fit analyses of the PPi inhibition, AMP inhibition and Km(app)(tRNA) assays were accomplished using total rate equations. The analysis showed that the only kinetic events which are obligatory to explain the chloride effects are a weakened binding of Mg2+ to the tRNA before the transfer reaction and a weakened binding of Mg2+ to the Tyr-tRNA-enzyme complex after the transfer reaction. The dissociation constants for the former were 0.11, 0.3, and 2.8 mM and for the latter 0.6, 2.5, and 13 mM at 0, 50 and 150 mM KCl, respectively. Mg2+ is required for the reactive conformation of tRNA in the transfer reaction but chloride weakens its formation. After the transfer reaction the dissociation of Mg2+ from the aa-tRNA-enzyme complex enhances the dissociation of the aa-tRNA from the enzyme. The kinetics and the chloride effect were similar in the tyrosyl-tRNA synthetases from both Bacillus stearothermophilus and E. coli.     

Chemical cross-linking of cyclic AMP-dependent protein kinase and its dissimilar subunits

Previous kinetic studies have demonstrated that the activation of cyclic AMP-dependent protein kinase by cyclic AMP involves the formation of a ternary complex of cyclic AMP, the regulatory subunit (R) and the catalytic subunit (C). It is suggested that only this ternary complex breaks down to liberate the enzymically active catalytic subunit. We have performed cross-linking experiments with the holoenzyme and its dissimilar subunits in the presence of MgATP and various concentrations of cyclic AMP. Results from these cross-linking studies indicate that regulatory subunits exist as dimers in the native form. Moreover, dissociation of the holoenzyme or the reconstituted enzyme is promoted by cyclic AMP, and the effect of MgATP is to stabilize the enzyme in the tetrameric form. The success in cross-linking the regulatory and the catalytic subunits of protein kinase with the lysine-specific bifunctional cross-linking reagent dimethyl suberimidate may be attributed to the presence of a large number of lysine residues in the enzyme.     

Interaction of rat muscle AMP deaminase with myosin. II. Modification of the kinetic and regulatory properties of rat muscle AMP deaminase by myosin.

The problems of whether the kinetic and regulatory properties of AMP deaminase were modified by formation of a deaminase-myosin complex were investigated with an enzyme preparation from rat skeletal muscle. Results showed that AMP deaminase was activated by binding to myosin. Myosin-bound AMP deaminase showed a sigmoidal activity curve with respect to AMP concentration in the absence of ATP and ADP, but a hyperbolic curve in their presence. Addition of ATP and ADP doubled the V value, but did not affect the Km value. Myosin-bound AMP deaminase also gave a sigmoidal curve in the presence of alkali metal ions, whereas free AMP deaminase gave a hyperbolic curve. GTP abolished the activating effects of both myosin and ATP.     

Purification of the 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase complexes of Neurospora crassa mitochondria

A simple purification procedure for the 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase complexes of Neurospora crassa mitochondria is described. After fractionated precipitations with polyethylene glycol, elimination of thiol proteins, and gel-filtration chromatography, the resulting preparations contained both activities. Covalent chromatography on thiol-activated Sepharose CL-4B allowed the specific binding of the 2-oxoglutarate dehydrogenase complex activity in the presence of 2-oxoglutarate, whereas the pyruvate dehydrogenase complex activity was retained in the presence of pyruvate. The purified 2-oxoglutarate dehydrogenase complex showed 4 protein bands by electrophoresis under dissociating conditions with apparent molecular weights of 160,000, 56,200, 55,600, 52,600 and a Km value of 3.8 X 10(-4) M for 2-oxoglutarate. The purified pyruvate dehydrogenase complex showed 5 protein bands with apparent molecular weights of 160,000, 57,600, 55,600, 52,500 and 37,100 and a Km value of 3.2 X 10(-4) M for pyruvate.     

12 alpha-hydroxysteroid dehydrogenase from Clostridium group P, strain C 48-50. Production, purification and characterization

NADP(H)-dependent 12 alpha-hydroxysteroid dehydrogenase (HSDH) from Clostridium group P, strain C 48-50, is still expressed at unusual high level (approximately 1% of total protein) under cultivation conditions where the usual expensive brain/heart infusion complex medium is replaced by inexpensive technical grade yeast autolysate. An inexpensive anaerobic bioprocess for the production of HSDH was developed provisionally up to 900-1 scale (9000 U/l, 7 g HSDH, specific activity 1.0 U/mg crude protein, 55 U/g wet cells). By a simple two-step affinity chromatography procedure, easily adaptable to a large-scale operation, using columns of small dimensions of Sephacryl-S-400-Procion-orange-P-2R (5 cm x 28 cm) and Sephacryl-S-400-Procion-red-HE-7B (2.6 cm x 14 cm) approximately 140 mg (1.8 x 10(4) U), HSDH was purified to apparent homogeneity and concentrated directly from a crude cell extract (overall yield 53%, specific activity 128 U/mg). As confirmed by fast native and SDS/PAGE, isoelectric focussing and electron microscopy, HSDH has a molecular mass of approximately 105 kDa and consists of four flattened tetrahedrically arranged identical subunits (26 kDa). The enzyme exhibits a rather low isoelectric point of 4.6, a pH optimum of 8.5-9.5 and a temperature optimum of approximately 55 C for the oxidation of cholic acid. Inhibition by SH reagents and pyridoxal 5'-phosphate has been observed. Chelating agents have no inhibitory effect. The presence of NADP increases considerably the thermostability (t 1/2 4-10 d, 25 C; 2-5 d, 37 C). Steady-state kinetic analysis for both reaction directions indicated that the reaction proceeds through an ordered bi bi mechanism with NADP(H) binding first to the free enzyme. Km, Vmax [forward (Vf) and reverse reactions (Vr)] and the dissociation constants Kd for the binary complexes with NADP and NADPH were as follows. NADP, Km = 35 microns, Kd = 35 microns; cholic acid, Km = 72 microns, deoxycholic acid, Km = 45 microns, Vf = 160 U mg; NAPDH, Kd = 16 microns; 12-oxochenodeoxylic acid, Km = 12 microns, 66 U/mg (conditions, 0.1 M potassium phosphate, pH 8.0, 25 degrees C). N6-functionalized NADP derivatives, e.g. N6-(2-aminoethyl)NADP (Km = 4.5 mM) are poorly accepted as coenzyme by HSDH.     

Cyclic AMP-dependent pig brain protein kinase: subunit structure, mechanism of autophosphorylation and holoenzyme dissociation under cyclic AMP action

Some properties of cyclic AMP-dependent pig brain protein kinase were studied. The holoenzyme was shown to exist in solution in the form of a tetramer complex R2C2 with mol. weight of 180 000. The limited proteolysis of the regulatory subunit caused the formation of a fragment with mol. weight of 35 000, capable of independent binding of 3H-cyclic AMP and containing a site, which can be phosphorylated in the autophosphorylation reaction. Autophosphorylation of the holoenzyme led to an increase in the degree of dissociation of the former into individual subunits under the effect of cyclic AMP. The ability of the phosphoform of the catalytic subunit was demonstrated. The autophosphorylation process and the phosphotransferase reaction involve the same active site of the catalytic subunit.