Pig heart lactate dehydrogenase. Binding of pyruvate and the interconversion of pyruvate-containing ternary complexes.

1. Lactate oxidation catalysed by pig heart lactate dehydrogenase was studied in the presence of inhibitory concentrations of pyruvate. Experimental results show the presence of an intermediate which occurs immediately after the hydride transfer step, but before the dissociation of pyruvate and the H+ produced by the reaction. The rate constant for pyruvate dissociation and the dissociation constant for pyruvate from the ternary complex differ from those obtained in pyruvate reduction experiments. 2.In single-turnover pyruvate reduction by pig heart lactate dehydrogenase at pH8.0 pyruvate can bind to the enzyme before a H+ is taken up, and the subsequent uptake of a H+ is governed by a step that is also rate-limiting for single-turnover and steady-state NADH oxidation. 3. Observation of various intermediates in the single-turnover pyruvate reduction experiments has made it possible to determine separately the dissociation constant and Km value for pyruvate at pH8.0, and also the catalytic turnover rate and Km for pyruvate under first-order conditions at different pH values. 4. Further studies on single-turnover pyruvate reduction carried out in 2H2O, or in water at low temperature, show another step which, under these conditions, is slower than that controlling H+ uptake and rate-limiting for NADH oxidation. A scheme is presented which explains these results.     

Anion binding to yeast phosphoglycerate kinase.

The single thiol of yeast phosphoglycerate kinase was labelled with the chromophoric sulfhydryl reagent, 2-chloromercuri-4-nitrophenol. Sequential additions of individual anions to this modified enzyme brought about a decrease in absorbance at 410 nm that reflected the degree of saturation of the enzyme with anion. The binding curves were analyzed to determine the dissociation constants of a number of anions with charges varying from--1 to--4.1. A linear relationship was found between the charge of the anion and the negative logarithm of the dissociation constant for the labelled enzyme-anion complex. The highly charged anions, such as ATP, bound more tightly than did anions with less charge, such as Cl-. The average number of binding sites for those anions for which accurate results could be obtained was 1.06 mol per 47000 g of enzyme. Several lines of evidence suggested that titration of the active center was not being monitored. Anions bound to phosphoglycerate kinase decreased the rate of reaction between the enzyme thiol and 5,5'-dithiobis(2-nitrobenzoic acid). The relationship between the degree of saturation of the anion binding site and the reaction rate constant was used to calculate the dissociation constant between anion and enzyme. Dissociation constants determined in this manner were in good agreement with those determined by titration of the enzyme-mercurial complex.     

Transient kinetic studies of substrate inhibition in the horse liver alcohol dehydrogenase reaction

The rate-limiting step of ethanol oxidation by alcohol dehydrogenase (E) at substrate inhibitory conditions (greater than 500 mM ethanol) is shown to be the dissociation rate of NADH from the abortive E-ethanol-NADH complex. The dissociation rate constant of NADH decreased hyperbolically from 5.2 to 1.4 s-1 in the presence of ethanol causing a decrease in the Kd of NADH binding from 0.3 microM for the binary complex to 0.1 microM for the abortive complex. Correspondingly, ethanol binding to E-NADH (Kd = 37 mM) was tighter than to enzyme (Kd = 109 mM). The binding rate of NAD+ (7 X 10(5) M-1s-1) to enzyme was not affected by the presence of ethanol, further substantiating that substrate inhibition is totally due to a decrease in the dissociation rate constant of NADH from the abortive complex. Substrate inhibition was also observed with the coenzyme analog, APAD+, but a single transient was not found to be rate limiting. Nevertheless, the presence of substrate inhibition with APAD+ is ascribed to a decrease in the dissociation rate of APADH from 120 to 22 s-1 for the abortive complex. Studies to discern the additional limiting transient(s) in turnover with APAD+ and NAD+ were unsuccessful but showed that any isomerization of the enzyme-reduced coenzyme-aldehyde complex is not rate limiting. Chloride increases the rate of ethanol oxidation by hyperbolically increasing the dissociation rate constant of NADH from enzyme and the abortive complex to 12 and 2.8 s-1, respectively. The chloride effect is attributed to the binding of chloride to these complexes, destabilizing the binding of NADH while not affecting the binding of ethanol.     

Salt specificity of a reduced nicotinamide adenine dinucleotide oxidase prepared from a halophilic bacterium

Extracts prepared from a halophilic bacterium contained a reduced nicotinamide adenine dinucleotide (NADH(2)) oxidase active at high solute concentrations. The cation requirement was nonspecific, since KCl, RbCl, and CsCl replaced NaCl with little or no loss of activity, and NH(4)Cl was only partially effective. Only LiCl failed to replace NaCl. No specific chloride requirement was observed although not all anions replaced chloride. Bromide, nitrate, and iodide were essentially ineffective, whereas acetate, formate, citrate, and sulfate proved suitable. The presence of sulfate affected the ability of a cation to satisfy the solute requirement. Sulfate enhanced the rate of NADH(2) oxidation when compared with the rate observed in the presence of chloride. Cations which were inactive as chlorides (LiCl and MgCl(2) at high concentrations) satisfied the cation requirement when added as sulfate salts. Although magnesium satisfied the cation requirement, a concentration effect, as well as an anion effect, was observed. In the presence of MgCl(2), little NADH(2) oxidation was observed at concentrations greater than 1 m. At lower concentrations, the rate of oxidation increased, reaching a maximal value at 0.1 m and remaining constant up to a concentration of 0.05 m MgCl(2). Magnesium acetate and MgSO(4) also replaced NaCl, and the maximal rate of oxidation occurred at 0.05 m with respect to magnesium. There was no change in the rate of oxidation at high magnesium acetate concentrations, whereas the rate of NADH(2) oxidation increased at higher concentrations of MgSO(4).     

The identification of intermediates in the reaction of pig heart lactate dehydrogenase with its substrates

Pig heart lactate dehydrogenase was studied in the direction of pyruvate and NADH formation by recording rapid changes in extinction, proton concentration, nucleotide fluorescence and protein fluorescence. Experiments measuring extinction changes show that there is a very rapid formation of NADH within the first millisecond and that the amplitude of this phase (phase 1) increases threefold over the pH range 6-8. A second transient rate (phase 2) can also be distinguished (whose rate is pH-dependent), followed by a steady-state rate (phase 3) of NADH production. The sum of the amplitudes of the first two phases corresponds to 1mol of NADH produced/mol of active sites of lactate dehydrogenase. Experiments that measured the liberation of protons by using Phenol Red as an indicator show that no proton release occurs during the initial very rapid formation of NADH (phase 1), but protons are released during subsequent phases of NADH production. Fluorescence experiments help to characterize these phases, and show that the very rapid phase 1 corresponds to the establishment of an equilibrium between E(NAD) (Lactate) right harpoon over left harpoon H(+)E(NADH) (Pyruvate). This equilibrium can be altered by changing lactate concentration or pH, and the H(+)E(NADH) (Pyruvate) species formed has very low nucleotide fluorescence and quenched protein fluorescence. Phase 2 corresponds to the dissociation of pyruvate and a proton from the complex with a rate constant of 1150s(-1). The observed rate constant is slower than this and is proportional to the position of the preceding equilibrium. The E(NADH) formed has high nucleotide fluorescence and quenched protein fluorescence. The reaction, which is rate-limiting during steady-state turnover, must then follow this step and be involved with dissociation of NADH from the enzyme or some conformational change immediately preceding dissociation. Several inhibitory complexes have also been studied including E(NAD+) (Oxamate) and E(NADH) (Oxamate') and the abortive ternary complex E(NADH) (Lactate). The rate of NADH dissociation from the enzyme was measured and found to be the same whether measured by ligand displacement or by relaxation experiments. These results are discussed in relation to the overall mechanism of lactate dehydrogenase turnover and the independence of the four binding sites in the active tetramer.     

Equilibrium and water proton relaxation rate enhancement properties of formyltetrahydrofolate synthetase-manganous ion-substrate complexes.

Binding of Mn(pi)-nucleotide complexes to the enzyme formyltertrahydrofolate synthetase (EC 6.3.4.3) from Clostridium cylindrosporum has been examined in the presence and absence of other substrates by solvent proton relaxation mearurements. MnADP and MnATP form ternary complexes with the enzyme with highly enhanced proton relaxation rates for water. The enhancement parameters, epsilont, for the MnADP and MnATP ternary complexes are 19.8 and 12.5, respectively at 24.3 MHZ and 25 degrees. Titration curves with constant total concentrations of enzyme and Mn(pi) with variable nucleotide concentration are similar to those observed in similar titrations with the endp and MnATP are 175 muM and 64 muM, respectively at 25 degrees. Addition of tetrahydrofolate to solutions of the MnADP OR MnATP ternary complexes lowers the observed relaxation enhancement markedly. An analysis of titration curves with constant total concentrations of enzyme, Mn(pi), and nucleotide with variable tetrahydrofolate concentration gives the dissociation constant for tetrahydrofolate from the respective quaternary complexes. The affinity of the enzyme for tetrahydrofolate is increased 6-fold when MnADP is present at the active site whereas a 3-fold increase is observed with MnATP present. Furthermore, there is a 20-fold increase in the enzyme's affinity for tetrahydrofolate when both MnADP and the third substrate, formate, are present. The observed relaxation rate of water for solutions of the complex, enzyme-MnADP-tetrahydrofolate-formate, is deenhanced with respect to the rate observed for the simple aquo-Mn(pi) solution. Addition of nitrate to solutions of the above complex increases the affinity of the enzyme for tetrahydrofolate and MnADP by an additional factor of 5 and lowers the relaxation rate further to a value which approaches that for solutions of the enzyme and substrates which lack the paramagnetic cation.     

NADH binding to porcine mitochondrial malate dehydrogenase.

The binding of NADH to porcine mitochondrial malate dehydrogenase in phosphate buffer at pH 7.5 has been studied by equilibrium and kinetic methods. Hyperbolic binding was obtained by fluorimetric titration of enzyme with NADH, in the presence or absence of hydroxymalonate. Identical results were obtained for titrations of NADH with enzyme in the presence or absence of hydroxymalonate, measured either by fluorescence emission intensity or by the product of intensity and anisotropy. The equilibrium constant for NADH dissociation was 3.8 +/- 0.2 micrometers, over a 23-fold range of enzyme concentration, and the value in the presence of saturating hydroxymalonate was 0.33 +/- 0.02 micrometer over a 10-fold range of enzyme concentration. The rate constant for NADH binding to the enzyme in the presence of hydroxymalonate was 3.6 X 10(7) M-1 s-1, while the value for dissociation from the ternary complex was 30 +/- 1 s-1. No limiting binding rate was obtained at pseudo-first order rate constants as high as 200 s-1, and the rate curve for dissociation was a single exponential for at least 98% of the amplitude. In addition to demonstrating that the binding sites are independent and indistinguishable, the absence of effects of enzyme concentration on the KD value indicates that NADH binds with equal affinity to monomeric and dimeric enzyme forms.     

Anion-induced increases in the rate of colchicine binding to tubulin.

The rate of binding of colchicine to tubulin to tubulin is enhanced by certain anions. Among the inorganic anions tested, only sulfate was effective. The organic anions include mostly dicarboxylic acids, among which tartrate was the most effective. This effect occurs onlt at low concentrations of colchicine (less than 0.6 X 10(-5) M). The rate increase dor sulfate and L-(+)-tartrate is ca. 2.5-fold at 1.0 mM and plateaus at a limiting value of ca. 4-fold at 100mM. The overall dissociation rate of the colchicine from the complex, which includes both the true rate of dissociation and the rate of irreversible denaturation of tubulin, is not influenced by 1.0 mM tartrate. The affinity constants for colchicine determined from the rate constants are 8.7 X 10(6) and 2.1 X 10(7) M-1 in the absence and the presence of 1.0 mM L-(+)-tartrate. The limiting value is 3.2 X 10(7) M-1. The affinity constant calculated from steady-state measurements is 3.2 X 10(6) M-1 with or without anions. The binding of other ligands like podophyllotoxin, vinblastine, and 1 -anilino-8-naphthalenesulfonate to tubulin is not affected by tartrate. No major conformational changes resulting from anion treatment could be detected by circular dichroism or intrinsic fluorescence. However, the ability of tubulin to polymerize is inhibited by L-(+)-tartrate at concentrations that increase the rate of colchicine binding. We conclude that anions must have a local effect at or near the binding site which enhances the binding rate of colchicine and which may be related to inhibition of polymerization.     

Bull semen nicotinamide adenine dinucleotide nucleosidase. VI. Fluorescence studies of the enzyme with reduced nicotinamide adenine dinucleotide

The binding of NADH to bull semen NAD nucleosidase was observed to be accompanied by a considerable enhancement of the fluorescence of NADH. The fluorescence enhancement observed in the binding of NADH to the enzyme was utilized to study the stoichiometry of binding of this compound to the enzyme. Results obtained from the fluorescence titration of the enzyme with NADH indicated the binding of one mole of NADH per mole of enzyme (36,000 g). The dissociation constant for the enzyme-NADH complex was determined to be 2.52 × 10^?6m. NADH was also found to be a very effective competitive inhibitor of the NADase-catalyzed hydrolysis of NAD, and the inhibitor dissociation constant (K_I) for the enzyme-NADH complex was determined to be 2.99 × 10^?6m which was in good agreement with the value obtained from the fluorescence titration experiments.     

Effect of the lyotropic series of anions on denaturation and renaturation of 20β-hydroxysteroid dehydrogenase

The effect of the lyotropic series of anions on the stability and renaturation of tetrameric 20β-hydroxysteroid dehydrogenase (17,20β,21-trihydroxysteroid:NAD⁺ oxidoreductase, EC 1.1.1.53) was investigated. The variations in enzymatic activity were correlated with the changes in protein fluorescence, circular dichroism, reactivity of histidine residues and molecular weight. High concentrations of salting-out anions (phosphate, citrate, sulphate) were found to stabilize the enzyme markedly and increase the renaturation yield of the urea-denaturated enzyme. Phosphate, for instance, induced the highest stabilization at about 1.2 M and the maximum reactivation (66%) at 0.5 M. At low anion concentration (0.01 M), the reactivation was only 7%. The renaturation property of salting-out anions seems to be due to their stabilizing effect on the end-product, i.e., the assembled tetramer. Salting-in anions (perchlorate, thiocyanate, iodide) inactivated the enzyme. At moderate anion concentrations (no greater than 0.25 M) the inactivation, which occurred slowly, without tetramer dissociation and with minor modifications of enzyme conformation, was fully reversed by concentrated phosphate or by saturating concentrations of NADH. In contrast, the inactivation induced by high anion concentrations (1–2 M) was rapid, irreversible and linked to considerable modifications of enzyme conformation.     

Effect of the lyotropic series of anions on denaturation and renaturation of 20 beta-hydroxysteroid dehydrogenase

The effect of the lyotropic series of anions on the stability and renaturation of tetrameric 20 beta-hydroxysteroid dehydrogenase (17,20 beta,21-trihydroxysteroid:NAD+ oxidoreductase, EC 1.1.1.53) was investigated. The variations in enzymatic activity were correlated with the changes in protein fluorescence, circular dichroism, reactivity of histidine residues and molecular weight. High concentrations of salting-out anions (phosphate, citrate, sulphate) were found to stabilize the enzyme markedly and increase the renaturation yield of the urea-denatured enzyme. Phosphate, for instance, induced the highest stabilization at about 1.2 M and the maximum reactivation (66%) at 0.5 M. At low anion concentration (0.01 M), the reactivation was only 7%. The renaturation property of salting-out anions seems to be due to their stabilizing effect on the end-product, i.e., the assembled tetramer. Salting-in anions (perchlorate, thiocyanate, iodide) inactivated the enzyme. At moderate anion concentrations (no greater than 0.25 M) the activation, which occurred slowly, without tetramer dissociation and with minor modifications of enzyme conformation, was fully reversed by concentrated phosphate or by saturating concentrations of NADH. In contrast, the inactivation induced by high anion concentrations (1-2 M) was rapid, irreversible and linked to considerable modifications of enzyme conformation.     

The use of ternary complexes to study ionizations and isomerizations during catalysis by lactate dehydrogenase

1. The binding of oxamate to pig heart and pig muscle isoenzymes of lactate dehydrogenase in the presence of NADH was studied by fluorescence titration. The dissociation constant of oxamate from the heart enzyme complex is 3mum and from the muscle isoenzyme 25mum at pH5. These values quantitatively increase with pH as predicted if oxamate can bind only to the enzyme-NADH complex if a group with pK6.9 is protonated. There are four non-interacting oxamate-binding sites per tetramer. 2. o-Nitrophenylpyruvate is a poor substrate for both isoenzymes but has a reasonable affinity to the heart isoenzyme. Initially, it forms an enzyme-NADH-substrate complex, which can be detected either by protein-fluorescence quenching or by NADH-fluorescence quenching. The pH-dependence of the dissociation constant of nitrophenylpyruvate also shows that this ternary complex can only form if a group with pK6.8 is protonated. Taken with the results of chemical-modification experiments, these results allow the pK of 6.8 to be assigned to a system probably involving the imidazole side chain of histidine-195. Formation of a ternary complex from a binary one at pH8 is predicted to result in a proton being taken up from solution. 3. Isotope-effect studies with NADH and its deuterium analogue show that the rapidly formed ternary complex with o-nitrophenylpyruvate slowly isomerizes to give an active ternary complex, which then rapidly decomposes to NAD(+). The isomerization is pH-independent, and it is suggested that histidine-195 is still protonated in the activated ternary complex, which is present before hydride transfer. 4. All four subunits of the enzyme are kinetically equivalent with respect to the oxidation of bound NADH by o-nitrophenylpyruvate. 5. A partial mechanism for the enzyme is described which emphasizes the isomerizations and ionizations involved in forming the reduced ternary complex at pH6 and 8.     

Effect of the structure of phospholipid on the kinetics of intravesicle scooting of phospholipase A2

Action of pig pancreatic phospholipase A2 on vesicles of over 50 synthetic 1,2-diacylglycerol-3-phosphate derivatives and analogs is examined in the absence of any additives. In general, shorter acyl chains and small substituents on the phosphate make a better substrate, while phospholipids with large apolar substituents are not hydrolyzed. The interfacial turnover rate constant for scooting kinetics, ki, for the various phospholipids were from less than 0.1 to 1 per min. Intervesicle exchange of the bound enzyme is faster in vesicles of phospholipids with larger polar substituents, and it is promoted in the presence of anions like chloride, sulfate and thiocyanate. These factors lower the residence time of the enzyme on the bilayer and therefore effectively decrease the rate of hydrolysis. The apparent Km for the enzyme in the interface of anionic phospholipids in the presence of salts is in the 40 to 100 microM range which is 3- to 7-times larger than the dissociation constants for the bound enzyme measured by fluorescence enhancement of Trp-3. The quantum yield of the bound enzyme in vesicles of the various lipids is found to be up to 4-fold different. It is suggested that this difference is due to the E* + S to E*S equilibrium, where E*S has higher fluorescence intensity. The role of calcium in generating the enzyme binding site at the anionic interface, the role of anion anchoring site on the enzyme, and the relationship between the catalytic efficiency and the fluorescence quantum yields are discussed.     

Studies of a halophilic NADH dehydrogenase. II. Kinetic properties of the enzyme in relation to salt activation.

1. An NADH dehydrogenase, obtained from an extremely halophilic bacterium, was activated by various salts when enzyme activity was measured as the observed velocity, whereas the maximum velocity was unaffected by either the salt concentration or the nature of the salt. 2. Two ion effects were observed; a quantitative cation effect, reflected in changes in the apparent Michaelis constant for 2,6-dichlorophenolindophenol, and a qualitative anion effect, reflected in the apparent Michaelis and dissociation constants for NADH. 3. The data suggest that cations act by neutralizing electrostatic charges surrounding the 2,6-dichlorophenolindophenol-binding site, whereas the anions affect the conformation of the enzyme by altering the accessibility of the NADH-binding site to the bulk solvent. 4. Thus, the apparent activation of this enzyme, obtained from an extremely halophilic bacterium, is a reflection of measuring enzyme activity at non-saturating substrate concentrations.     

Determination of the affinity of each component of a composite quaternary transition-state analogue complex of creatine kinase

Recombinant rabbit muscle creatine kinase (CK) was titrated with MgADP in 50 mM Bicine and 5 mM Mg(OAc)2, pH 8.3, at 30.0 degrees C by following a decrease in the protein's intrinsic fluorescence. In the presence of 50 mM NaOAc, but in the absence of added creatine or nitrate, MgADP has an apparent K(d) of 135 +/- 7 microM, and the total change in fluorescence on saturation (Delta%F) is 15.3 +/- 0.3%. Acetate was used as the anion in this experiment because it does not promote the formation of a CK.MgADP.anion.creatine transition-state analogue complex (TSAC) [Millner-White and Watts (1971) Biochem. J. 122, 727-740]. In the presence of 80 mM creatine, but no nitrate, the apparent K(d) for MgADP remains essentially unchanged at 132 +/- 10 microM, while Delta%F decreases slightly to 13.2 +/- 0.3%. In the presence of 10 mM nitrate, but no creatine, the apparent K(d) is once again essentially unchanged at 143 +/- 23 microM, but the Delta%F is markedly reduced to 4.2 +/- 0.2%. The presence of both 10 mM nitrate and 80 mM creatine during titration reduces the apparent K(d) for MgADP 10-fold to 13.7 +/- 0.7 microM, and Delta%F increases to 20.6 +/- 0.3%, strongly suggesting that the simultaneous presence of saturating levels of creatine and nitrate increases the affinity of CK for MgADP and promotes the formation of the enzyme*MgADP*nitrate*creatine TSAC. When the fluorescence of CK was titrated with MgADP in the presence of 80 mM creatine and fixed saturating concentrations of various anions, apparent K(d) values for MgADP of 132 +/- 10 microM, 25.2 +/- 1.3 microM, 18.8 +/- 0.9 microM, 13.7 +/- 0.7 microM, and 6.4 +/- 0.7 microM were observed as the anion was changed from acetate to formate to chloride to nitrate to nitrite, respectively. This is the same trend reported by Millner-White and Watts for the effectiveness of various monovalent anions in forming the CK.MgADP.anion.creatine TSAC. On titration of CK with MgADP in the presence of 80 mM creatine and various fixed concentrations of NaNO3, the apparent K(d) for MgADP decreases with increasing fixed concentrations of nitrate. A plot of the apparent K(d) for MgADP vs [NO3-] suggests a K(d) for nitrate from the TSAC of 0.39 +/- 0.07 mM. Similarly, titration with MgADP in the presence of 10 mM NaNO3 and various fixed concentrations of creatine gives a value of 0.9 +/- 0.4 mM for the dissociation of creatine from the TSAC. The data were used to calculate K(TDAC), the dissociation constant of the quaternary TSAC into its individual components, of 3 x 10(-10) M3. To our knowledge this is the first reported dissociation constant for a ternary or quaternary TSAC.     

The resolution of some steps of the reactions of lactate dehydrogenase with its substrates

1. The reaction of pig heart lactate dehydrogenase (EC 1.1.1.27) with NAD(+) and lactate to form pyruvate and NADH was followed by rapid spectrophotometric methods. The distinct spectrum of enzyme-bound NADH permits the measurement of the rate of dissociation of this compound. 2. The reduction of the first mole equivalent of NAD(+) per mole of enzyme sites can also be observed, and is much more rapid than the steady-state rate of NADH production. 3. At pH8 the dissociation of the enzyme-NADH complex is rate-determining for the steady-state oxidation of lactate. At lower pH some other step after the interconversion of the ternary complex and before the dissociation of NADH is rate-determining. Other evidence for a compulsory-order mechanism is provided.     

Pre-steady-state kinetic analysis of recombinant Arabidopsis NADH:nitrate reductase: rate-limiting processes in catalysis

Recombinant Arabidopsis NADH:nitrate reductase was expressed in Pichia pastoris using fermentation. Large enzyme quantities were purified for pre-steady-state kinetic analysis, which had not been done before with any eukaryotic nitrate reductase. Basic biochemical properties of recombinant nitrate reductase were similar to natural enzyme forms. Molybdenum content was lower than expected, which was compensated for by activity calculation on molybdenum basis. Stopped-flow rapid-scan spectrophotometry showed that the enzyme FAD and heme were rapidly reduced by NADH with and without nitrate present. NADPH reduced FAD at less than one-tenth of NADH rate. Reaction of NADH-reduced enzyme with nitrate yielded rapid initial oxidation of heme with slower oxidation of flavin. Rapid-reaction freeze-quench EPR spectra revealed molybdenum was maintained in a partially reduced state during turnover. Rapid-reaction chemical quench for quantifying nitrite production showed that the rate of nitrate reduction was initially greater than the steady-state rate, but rapidly decreased to near steady-state turnover rate. However, rates of internal electron transfer and nitrate reduction were similar in magnitude with no one step in the catalytic process appearing to be much slower than the others. This leads to the conclusion that the catalytic rate is determined by a combination of rates with no overall rate-limiting individual process.     

Characterization of specific interactions of coenzymes, regulatory nucleotides and cibacron blue with nucleotide binding domains of enzymes by analytical affinity chromatography

The dissociation constant for the complex of rhodanese and Cibacron Blue, determined by analytical affinity chromatography using rhodanese immobilized on controlled-pore glass (CPG) beads (200 nm pore diameter) and aminohexyl-Cibacron Blue, was 44 microM which agreed well with the kinetic inhibition constant, suggesting that the dye binds at or near the active site of this enzyme. Formation of a binary complex of the dye and lactate dehydrogenase (LDH) was also characterized by direct chromatography of LDH on CPG/immobilized Cibacron Blue (KD = 0.29 microM). The binary complex formed between LDH and NADH was characterized by analytical affinity chromatography using both CPG/immobilized LDH and immobilized Cibacron Blue. Since the dye competes with NADH in binding to the active site of LDH, competitive elution chromatography using the immobilized dye allows determination of the dissociation constant of the soluble LDH.NADH complex. Agreement between the dissociation constants determined by direct chromatography of NADH on immobilized LDH (KD = 1.4 microM) and that determined for the soluble complex (KD = 2.4 microM) indicates that immobilization of LDH did not affect the interaction. Formation of various binary, ternary and quaternary complexes of bovine liver glutamate dehydrogenase (GDH) with glutamate, NADPH, NADH, and ADP was also investigated using immobilized GDH. This approach allows characterization of the enzyme/ligand interactions without the complicating effect of enzyme self-association. The affinity for NADPH is considerably greater in the ternary complex (including glutamate) as compared to the binary complex (0.38 microM vs 22 microM); however, occupancy of the regulatory site by ADP greatly reduces the affinity in both complexes (6.4 microM and 43 microM, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Anion interactions with Na,K-ATPase: simultaneous binding of nitrate and eosin

Nucleotide binding affinity to Na,K-ATPase is reduced by a number of anions such as nitrate and perchlorate in comparison with affinity in the presence of chloride (all with sodium as the cation). The reduction correlates with the position of these anions in the Hofmeister series. Transient kinetic experiments using the fluorescent dye eosin—which binds to the nucleotide site of the Na,K-ATPase—show that simultaneous anion binding, exemplified with nitrate, and eosin binding is possible. The effect of nitrate on eosin binding is reflected in a decreased binding-rate constant and an increased dissociation rate constant, leading to a decreased equilibrium binding constant for eosin. Since eosin binding is analogous with nucleotide binding to Na,K-ATPase, the results suggest the simultaneous presence of nucleotide and anion binding sites.Abbreviations E₁ the protein conformation in Na⁺ - E₂ the enzyme conformation in K⁺ - Eo eosin (tetrabromofluorescein) - F fluorescence - I ionic strength - k_i rate constant - K_i equilibrium dissociation constant - K_i,0 equilibrium dissociation constant at zero ionic strength - N nitrate - z_i net charge - charge product z_i·z_j     

Reduced S-adenosylhomocysteine hydrolase. Kinetics and thermodynamics for binding of 3'-ketoadenosine, adenosine, and adenine.

S-Adenosylhomocysteine hydrolase (SAHase) was resolved into apoenzyme and NAD+ by acidic ammonium sulfate treatment. The apoenzyme was catalytically inactive, but could be reconstituted to active enzyme with NAD+. Reduced SAHase (ENADH) that was prepared by reconstitution of the apoenzyme with NADH was catalytically inactive. ENADH was oxidized by 3'-ketoadenosine to active SAHase. The recovery of activity paralleled the oxidation of enzyme-bound NADH. The association rate constant for ENADH and 3'-ketoadenosine was 6.1 x 10(2) M-1 s-1, and the dissociation rate constant was calculated to be 4 x 10(-7) s-1. This association rate constant was considerably smaller than the association rate constant for adenosine and SAHase (greater than 10(7) M-1 s-1). However, the observed pseudo first-order rate constant for reaction of 3'-ketoadenosine with ENADH (0.6 s-1 with 1 mM 3'-ketoadenosine) approached kcat for the hydrolytic reaction (1.2 s-1). Thus, bound 3'-ketoadenosine probably reacted sufficiently rapidly with ENADH to be considered a kinetically competent intermediate. The dissociation constants of SAHase for adenosine and 4',5'-dehydroadenosine, substrates for the enzyme, were 9 and 14 microM, respectively. In contrast, the dissociation constants of ENADH for 3'-ketoadenosine and 4',5'-dehydro-3'-ketoadenosine, intermediates of the catalytic reaction, were significantly lower with values of 600 and 300 pM, respectively. The equilibrium constant for reduction of enzyme-bound NAD+ in the absence of an adenosine analogue, as estimated from cyanide binding studies, was 10-fold more favorable than that for free NAD+. ENADH was highly fluorescent (emission maximum 428 nm, excitation 340 nm) with a quantum yield that was six times that of free NADH. Since SAHase reduced by adenosine was not highly fluorescent, enzyme-bound intermediates quenched the fluorescence of enzyme-bound NADH. Adenosine and adenine quenched the fluorescence of ENADH. Cyanide formed a complex with SAHase that was analogous to ENADH. Adenine stabilized this complex sufficiently that addition of 65 microM adenine and 25 mM cyanide to SAHase caused total complex formation with loss of over 95% of the catalytic activity.