High Substrate Inhibition Research Articles

Strategy for rapid recovery of simultaneous sulfide and nitrite removal under high substrate inhibition

The paper deals with the strategy for a quick recovery of reactor treating sulfide and nitrite simultaneously under inhibition caused by high substrate concentration. For influent sulfide concentration of 360 mg S/L, respective sulfide and nitrite removal percentages dropped to 74.19% and 14.33% due to inhibition caused by high sulfide and nitrite concentrations. It was found that reduction in the influent substrate concentration (300 mg S/L) could not revive the nitrite removal performance in 4 days’ operation, which still showed a declining tendency from 47.16% to 18.52%. Regulating the influent pH around 6.70 ± 0.10, it only took 4 days to recover the performance for 300 mg S/L. Furthermore, at influent sulfide concentration increased to 360 mg S/L, respective sulfide and nitrite removal percentages were 99.76 ± 0.27% and 100.00%. The strategy of regulating influent pH could recover the process performance in a short term, which would provide great convenience for subsequent process research.

Substrate Inhibition of 5β-Δ4-3-Ketosteroid Dehydrogenase in Sphingobium sp. Strain Chol11 Acts as Circuit Breaker During Growth With Toxic Bile Salts.

In contrast to many steroid hormones and cholesterol, mammalian bile salts are 5β-steroids, which leads to a bent structure of the steroid core. Bile salts are surface-active steroids excreted into the environment in large amounts, where they are subject to bacterial degradation. Bacterial steroid degradation is initiated by the oxidation of the A-ring leading to canonical Δ4-3-keto steroids with a double bond in the A-ring. For 5β-bile salts, this Δ4-double bond is introduced into 3-keto-bile salts by a 5β-Δ4-ketosteroid dehydrogenase (5β-Δ4-KSTD). With the Nov2c019 protein from bile-salt degrading Sphingobium sp. strain Chol11, a novel 5β-Δ4-KSTD for bile-salt degradation belonging to the Old Yellow Enzyme family was identified and named 5β-Δ4-KSTD1. By heterologous production in Escherichia coli, 5β-Δ4-KSTD function could be shown for 5β-Δ4-KSTD1 as well as the homolog CasH from bile-salt degrading Rhodococcus jostii RHA1. The deletion mutant of 5β-Δ4-kstd1 had a prolonged lag-phase with cholate as sole carbon source and, in accordance with the function of 5β-Δ4-KSTD1, showed delayed 3-ketocholate transformation. Purified 5β-Δ4-KSTD1 was specific for 5β-steroids in contrast to 5α-steroids and converted steroids with a variety of hydroxy groups regardless of the presence of a side chain. 5β-Δ4-KSTD1 showed a relatively low Km for 3-ketocholate, a very high specific activity and pronounced substrate inhibition. With respect to the toxicity of bile salts, these kinetic properties indicate that 5β-Δ4-KSTD1 can achieve fast detoxification of the detergent character as well as prevention of an overflow of the catabolic pathway in presence of increased bile-salt concentrations.

Optimal designs in enzymatic reactions with high-substrate inhibition

The Michaelis-Menten (MM) model is extensively used in biochemistry to describe reaction kinetics. However, deviations from MM kinetics can occur, usually due to an inhibition effect on the amount of substrate. Two different models, describing the kinetic reaction when this type of inhibition occurs, are introduced. Optimal experimental designs methods are used in order to find the best estimation of the parameters in these models. An application to the semicarbazide-sensitive amine oxidase (SSAO) activity towards benzylamine is presented.

Acetylcholinesterase of Rhipicephalus (Boophilus) microplus and Phlebotomus papatasi: Gene identification, expression, and biochemical properties of recombinant proteins

The southern cattle tick Rhipicephalus (Boophilus) microplus (Bm) is a vector of bovine babesiosis and anaplasmosis. Tick resistance to organophosphate (OP) acaricide involves acetylcholinesterase (AChE) insensitivity to OP and metabolic detoxification. Sequencing and in vitro expression of Bm genes encoding AChE allowed biochemical characterization of three BmAChEs expressed in tick synganglion. rBmAChE1, rBmAChE2 and rBmAChE3 exhibited substrate preference for acetylthiocholine, high substrate inhibition and sensitivity to AChE-specific inhibitors. OP-insensitivity mutations were demonstrated in rBmAChE1 and rBmAChE3. Gene silencing suggested functional complementation of BmAChEs in vivo. BmAChE genes were amplified in copy number and multiple transcript polymorphisms were expressed in individual tick synganglia for each of the BmAChEs, suggesting allelic diversity within individuals. Studies also identified a gene encoding AChE of the sand fly, Phlebotomus papatasi, a vector of leishmaniasis in humans and animals. Expression of recombinant P. papatasi AChE (rPpAChE) enabled biochemical characterization and identification of effective inhibitors that selectively target rPpAChE.

Cloning, expression, characterization and inhibition studies on trypanothione synthetase, a drug target enzyme, from Leishmania donovani

Trypanothione synthetase, a validated drug target, synthesizes trypanothione from glutathione and spermidine. Here we report the gene cloning, expression, characterization and inhibition studies of trypanothione synthetase from Leishmania donovani (LdTryS). The purified recombinant LdTryS enzyme obeyed Michaelis-Menten kinetics. High substrate inhibition was observed with glutathione (K(m)=33.24 μm, k(cat)=1.3 s(-1), K(i)=866 μm). The enzyme shows simple hyperbolic kinetics with fixed glutathione concentration and with other substrates limiting K(m) values for Mg. ATP and spermidine of 14.2 μm and 139.6 μm, respectively. LdTryS was also screened for inhibitors. Tomatine, conessine, uvaol and betulin were identified as inhibitors of the enzyme and were tested for leishmanicidal activity. Finally, the effect of LdTryS inhibitors on redox homeostasis of the parasite gives a broader picture of their action against leishmaniasis.

Cloning, expression, functional validation and modeling of cinnamyl alcohol dehydrogenase isolated from xylem of Leucaena leucocephala

A cDNA encoding cinnamyl alcohol dehydrogenase (CAD), catalyzing conversion of cinnamyl aldehydes to corresponding cinnamyl alcohols, was cloned from secondary xylem of Leucaena leucocephala. The cloned cDNA was expressed in Escherichia coli BL21 (DE3) pLysS cells. Temperature and Zn2+ ion played crucial role in expression and activity of enzyme, such that, at 18°C and at 2mM Zn2+ the CAD was maximally expressed as active enzyme in soluble fraction. The expressed protein was purified 14.78-folds to homogeneity on Ni–NTA agarose column with specific activity of 346nkat/mg protein. The purified enzyme exhibited lowest Km with cinnamyl alcohol (12.2μM) followed by coniferyl (18.1μM) and sinapyl alcohol (23.8μM). Enzyme exhibited high substrate inhibition with cinnamyl (beyond 20μM) and coniferyl (beyond 100μM) alcohols. The in silico analysis of CAD protein exhibited four characteristic consensus sequences, GHEXXGXXXXXGXXV; C100, C103, C106, C114; GXGXXG and C47, S49, H69, L95, C163, I300 involved in catalytic Zn2+ binding, structural Zn2+ binding, NADP+ binding and substrate binding, respectively. Tertiary structure, generated using Modeller 9v5, exhibited a trilobed structure with bulged out structural Zn2+ binding domain. The catalytic Zn2+ binding, substrate binding and NADP+ binding domains formed a pocket protected by two major lobes. The enzyme catalysis, sequence homology and 3-D model, all supported that the cloned CAD belongs to alcohol dehydrogenase family of plants.

Characterization of two soluble inorganic pyrophosphatases from Arabidopsis thaliana

Inorganic pyrophosphatases (siPPiase) catalyze the irreversible hydrolysis of pyrophosphate and their function is known to be essential to all living organisms. In this work, the cDNAs coding for two Arabidopsis thaliana siPPiase were obtained from the TAIR initiative and cloned in an expression vector as fusions with gluthathione- S-transferase. The digested pure forms were also similar to each other, both in amino acid sequence, and in kinetic and molecular properties. They showed absolute requirement for Mg 2+, and could only be replaced for Mn 2+. The only substrate found to be hydrolyzed was pyrophosphate. Their activity was retained during size exclusion chromatography only when eluted with ethylene glycol and Mg 2+, and the observed molecular size corresponded to the monomers. The fluorescence of both proteins was consistent with their low tryptophan content, but an unusual fluorescence was found with excitation at 231 nm. The CD spectrum was consistent with a protein rich in antiparallel β sheets and random coils. The pyrophosphate saturation kinetics indicated the ordered binding of free Mg 2+ and of the Mg–pyrophosphate complex, which explains an apparent high substrate inhibition found at low levels of Mg 2+. Both enzymes showed similar catalytic constants and affinities for the Mg–pyrophosphate complex, while differed in their affinity for free Mg 2+. Finally, the available microarray expression data, and the chromatography siPPiase activity profiles are consistent with a housekeeping expression of these enzymes, but the complexity of the profiles suggest a complex role in plant metabolism.

Characterization of cinnamyl alcohol dehydrogenase of Helicobacter pylori

Cinnamyl alcohol dehydrogenases (CAD; 1.1.1.195) catalyse the reversible conversion of p-hydroxycinnamaldehydes to their corresponding alcohols, leading to the biosynthesis of lignin in plants. Outside of plants their role is less defined. The gene for cinnamyl alcohol dehydrogenase from Helicobacter pylori (HpCAD) was cloned in Escherichia coli and the recombinant enzyme characterized for substrate specificity. The enzyme is a monomer of 42.5 kDa found predominantly in the cytosol of the bacterium. It is specific for NADP(H) as cofactor and has a broad substrate specificity for alcohol and aldehyde substrates. Its substrate specificity is similar to the well-characterized plant enzymes. High substrate inhibition was observed and a mechanism of competitive inhibition proposed. The enzyme was found to be capable of catalysing the dismutation of benzaldehyde to benzyl alcohol and benzoic acid. This dismutation reaction has not been shown previously for this class of alcohol dehydrogenase and provides the bacterium with a means of reducing aldehyde concentration within the cell.

Properties of trypanothione synthetase from Trypanosoma brucei.

Trypanothione [N(1),N(8)-bis(glutathionyl)spermidine] plays a central role in defence against oxidant damage, ribonucleotide metabolism and in resistance to certain drugs in trypanosomatids. In Crithidia fasciculata, synthesis of trypanothione involves sequential conjugation of two molecules of glutathione (GSH) to spermidine by two enzymes: glutathionylspermidine synthetase (GspS; EC 6.3.1.8) and trypanothione synthetase (TryS; EC 6.3.1.9), whereas in Trypanosoma cruzi both steps are catalysed by an unusual TryS with broad substrate specificity. To determine which route operates in T. brucei, we have cloned and expressed a single copy gene with similarity to C. fasciculata and T. cruzi TRYS. The purified recombinant protein catalyses formation of trypanothione from either spermidine and GSH, or glutathionylspermidine and GSH. The enzyme displays high substrate inhibition with GSH as variable substrate (apparent K(m)=56 microM, K(i)(s)=37 microM, k(cat)=2.9s(-1)). At a fixed subsaturating GSH concentration (100 microM), the enzyme obeys simple hyperbolic kinetics yielding apparent K(m) values for spermidine, glutathionylspermidine and MgATP of 38, 2.4, and 7.1 microM, respectively. Recombinant TryS can also catalyse conversion of spermine to glutathionylspermine and bis(glutathionyl)spermine, as recently reported for T. cruzi. The enzyme has amidase activity that can be inhibited by iodoacetamide. Studies using GSH and polyamine analogues identified GSH as the critical determinant for recognition by the amidase domain. Thus, the biosynthesis and degradation of trypanothione are similar in African and American trypanosomes, and different from the insect trypanosomatid, C. fasciculata.

Effect of thiols on the activity of urease from dehusked seeds of watermelon ( Citrullus vulgaris)

Urease from seeds of watermelon ( Citrullus vulgaris) was purified to apparent homogeneity, using two acetone fractionation steps, heat treatment at 48 °C and gel filtration through Sephadex G-200. The effect of various thiols like 2-mercaptoethanol (ME), dl-dithiothreitol (DTT), and l-cysteine was investigated. The enzyme was significantly activated by thiol and the order of effectiveness as activator was ME>DTT>cysteine. All the thiols exhibited two peaks of activation, one at lower concentration and another at higher concentration. Time-dependent activation of urease exhibited very rapid activation; about 5.3-fold with 8.0 mM ME and 4.9-fold with 30 μM DTT within 10 min of treatment. Moreover, the inhibition caused by high urea concentration was completely abolished by ME. Cysteine and DTT were ineffective in abolishing high substrate inhibition. Enzyme pretreated with ME also exhibited elevated activity in the urea concentration range of 10–2000 mM. The significance of these observations is discussed.

Kinetic characterisation of primer mismatches in allele-specific PCR: a quantitative assessment

A novel method of estimating the kinetic parameters of Taq DNA polymerase during rapid cycle PCR is presented. A model was constructed using a simplified sigmoid function to represent substrate accumulation during PCR in combination with the general equation describing high substrate inhibition for Michaelis–Menten enzymes. The PCR progress curve was viewed as a series of independent reactions where initial rates were accurately measured for each cycle. Kinetic parameters were obtained for allele-specific PCR (AS-PCR) amplification to examine the effect of mismatches on amplification. A high degree of correlation was obtained providing evidence of substrate inhibition as a major cause of the plateau phase that occurs in the later cycles of PCR.

A Single Enzyme Catalyses Formation of Trypanothione from Glutathione and Spermidine in Trypanosoma cruzi

Protozoa of the order Kinetoplastida differ from other organisms in their ability to conjugate glutathione (l-gamma-glutamyl-cysteinyl-glycine) and spermidine to form trypanothione [N(1),N(8)-bis(glutathionyl)spermidine], a metabolite involved in defense against chemical and oxidant stress and other biosynthetic functions. In Crithidia fasciculata, trypanothione is synthesized from GSH and spermidine via the intermediate glutathionylspermidine in two distinct ATP-dependent reactions catalyzed by glutathionylspermidine synthetase (GspS; EC ) and trypanothione synthetase (TryS; EC ), respectively. Here we have cloned a single copy gene (TcTryS) from Trypanosoma cruzi encoding a protein with 61% sequence identity with CfTryS but only 31% with CfGspS. Saccharomyces cerevisiae transformed with TcTryS were able to synthesize glutathionylspermidine and trypanothione, suggesting that this enzyme is able to catalyze both biosynthetic steps, unlike CfTryS. When cultures were supplemented with aminopropylcadaverine, yeast transformants contained glutathionylaminopropylcadaverine and homotrypanothione [N(1),N(9)-bis(glutathionyl)aminopropylcadaverine], metabolites that have been previously identified in T. cruzi, but not in C. fasciculata. Kinetic studies on recombinant TcTryS purified from Escherichia coli revealed that the enzyme displays high-substrate inhibition with glutathione (K(m) and K(i) of 0.57 and 1.2 mm, respectively, and k(cat) of 3.4 s(-1)), but obeys Michaelis-Menten kinetics with spermidine, aminopropylcadaverine, glutathionylspermidine, and MgATP as variable substrate. The recombinant enzyme possesses weak amidase activity and can hydrolyze trypanothione, homotrypanothione, or glutathionylspermidine to glutathione and the corresponding polyamine.

Time-dependent activation of the semicarbazide-sensitive amine oxidase (SSAO) from ox lung microsomes

The activity of ox lung microsomal semicarbazide-sensitive amine oxidase (EC 1.4.3.6; SSAO) towards benzylamine increased 20-fold during incubation at 37 degrees C. After an initial lag-period, activation was first-order with time and complete after approx. 20 h. No significant changes in activity towards methylamine, histamine or 2-phenylethylamine were observed, although mixed-substrate experiments were consistent with the same enzyme being involved in the oxidation of all these substrates, both before and after time-dependent activation. The enzyme-tryptophan fluorescence increased on incubation at 37 degrees C in parallel with the increase in activity towards benzylamine. Treatment of the activated-enzyme preparation with 6 M guanidinium chloride followed by dialysis, caused both the activity towards benzylamine and the fluorescence to fall to that occurring before activation. However, incubation of this preparation at 37 degrees C resulted in increases in fluorescence and activity similar to those seen with the unactivated enzyme. Benzylamine oxidation was inhibited, uncompetitively with respect to oxygen, by high substrate concentrations but no such inhibition was observed with the other amines. Activation resulted in an increase in V(max) for benzylamine oxidation, with no significant alterations in the K(m) or the K(si) for high-substrate inhibition. Kinetic studies were consistent with sequential mechanisms being followed for the oxidation of both benzylamine and methylamine but the dependence on oxygen concentration was complex. These results might indicate that benzylamine follows a different reaction pathway from the other substrates, with substrate-specific activation involving a reaction step that is rate-limiting for benzylamine oxidation but not for the others.

Modelling effects of high product and substrate inhibition on oscillatory behavior in continuous bioreactors

In this study we consider a model for continuous bioreactors which incorporates the effects of high product and substrate inhibition on the kinetics as well as biomass and product yields. We theoretically investigate the possibility of various dynamic behaviors in the bioreactor over different ranges of operating parameters to determine the delineating process conditions which may lead to oscillatory behavior. Application of the singular perturbation technique allows us to derive explicit conditions on the system parameters which specifically ascertain the existence of limit cycles composed of concatenations of catastrophic transitions occurring at different speeds. We discover further that the interactions between the limiting substrate and the growing microorganisms can give rise to high frequency oscillations which can arise during the transients toward the attractor or during the low-frequency cycle. Such a study not only can describe more fully the kinetics in a fermentor but also assist in formulating optimum fermentor operating conditions and in developing control strategy for maintaining optimum productivity.

Activation of two types of brain glutamate dehydrogenase isoproteins by gabapentin

The stimulatory effects of gabapentin on the activities of two types of glutamate dehydrogenase (GDH) isoproteins homogeneously purified from bovine brain have been studied at various conditions. When the effects of different gabapentin concentrations on GDH activities were studied in the direction of reductive amination of 2-oxoglutarate with NADPH as a coenzyme, a marked activation was observed for both isoproteins, whereas both isoproteins showed activation to a lesser extent with NADH as a coenzyme. Stimulatory effects of gabapentin on GDH activities in the direction of the oxidative deamination of glutamate were also observed, but to a much lesser extent than reductive amination. There were big differences between the two GDH isoproteins in their sensitivity to the action of gabapentin. The largest activation was observed with GDH II when NADPH was used as a coenzyme. Half-maximal stimulation was reached at around 1.5 mM. Gabapentin relieved the inhibition of GDH isoproteins by GTP and this resulted in an increase in the apparent activation by gabapentin in the presence of GTP. 2-Oxoglutarate was found to give rise to high substrate inhibition and gabapentin reduced the substrate inhibition in the presence of 0.2 mM NADH. Since there are neurodegenerative disorders in which GDH activity is decreased, the therapeutic modulation of the activity of this enzyme may be clinically useful.

Steady-state kinetic analysis of aldehyde dehydrogenase from human erythrocytes.

The steady-state kinetics of purified cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from human erythrocytes have been studied at 37 degrees C. Previous studies of the enzyme from several mammalian sources, which used a lower assay temperature, have been difficult to interpret because of the substrate activation by acetaldehyde which led to complex kinetic behaviour. At 37 degrees C the initial-rate data do not depart significantly from Michaelis-Menten kinetics. Studies of the variation of initial rates as a function of the concentrations of both substrates and studies of the inhibition by NADH were consistent with a sequential mechanism being followed. High-substrate inhibition by acetaldehyde was competitive with respect to NAD+. The enzyme was not inhibited by the product acetate and thus the results of these studies, although consistent with an ordered mechanism in which NAD+ was the first substrate to bind, were inconclusive. That such a mechanism was followed was confirmed by determination of the initial-rate behaviour in the presence of acetaldehyde and glycolaldehyde as alternative substrates. When the reciprocal of the initial rate of NADH formation was plotted against the acetaldehyde concentration at a series of fixed ratios between that substrate and glycolaldehyde, a linear 'mixed inhibition' pattern was obtained, confirming the mechanism to be ordered with NAD+ being the leading substrate and with kinetically significant ternary complex-formation.

Inhibition of ox brain glutamate dehydrogenase by perphenazine

Factors affecting the inhibition of ox brain glutamate dehydrogenase (GDH) by the anti-psychotic drug perphenazine have been studied. Inhibition was found to be of mixed type with respect to 2-oxoglutarate and competitive towards NADH. However the data indicate that perphenazine binds to a site distinct from the catalytic site to which NADH binds. Perphenazine also enhanced the high-substrate inhibition by these two substrates. Inhibition by perphenazine was not affected by the allosteric effector GTP but it was enhanced by increasing pH, in the range of 6.3 to 7.6, and diminished by increasing ionic strength. Low concentrations of perphenazine relieved the inhibition of GDH by phosphatidylserine and cardiolipin. However, at higher concentrations phosphatidylserine did not interfere with the inhibition by perphenazine whereas cardiolipin relieved it. The possible significance of these interactions in terms of the behaviour of this antipsychotic drug in vivo are discussed.

Activation of glutamate dehydrogenase by l-leucine

The activation of glutamate dehydrogenase ( l-glutamate: NAD(P) + oxidoreductase (deaminating), EC 1.4.1.3) by l-leucine has been studied. Apparently homogeneous preparations from ox liver and brain were found to respond similarly. Commercially obtained preparations of the enzyme, which had suffered limited proteolysis during the purification procedure, were shown to behave similarly to preparations which had not suffered such proteolysis when the effects of l-leucine on the oxidative deamination reaction were studied using either NAD + or NADP + as the coenzyme. There was also no significant difference in the responses when the reductive reaction was determined with NADPH or with 40 μM NADH. At higher concentrations of NADH (160 μM) the unproteolysed preparations were activated by l-leucine to a considerably greater extent than those which had suffered limited proteolysis. These results accord with the greater sensitivity of the former preparations to inhibition by high concentrations of NADH and the relief of such inhibition by l-leucine. This amino acid was also found to relieve the inhibition of the enzyme by GTP, resulting in an apparent increase in the activation observed in the presence of this nucleotide. In contrast, under the conditions used in this work, the apparent degree of activation by l-leucine was found to be decreased in the presence of the activators ATP or ADP. The presence of high concentrations of NADH (200 μM) potentiated the high substrate inhibition by 2-oxoglutarate, and l-leucine significantly reduced this effect. The effects of l-leucine on the activity of glutamate dehydrogenase thus appear to be composed of a direct effect on the activity of the enzyme together with a relief of high substrate inhibition. The effects of GTP and 2-oxoglutarate in potentiating inhibition by NADH can account for their effects in enhancing the apparent activation by l-leucine. The marked differences in the respones of proteolysed and unproteolysed preparations of the enzyme result from the effects of proteolysis in decreasing the sensitivity to high concentrations of NADH.

Purification and characterization of NADP+-dependent 3 alpha-hydroxysteroid dehydrogenase from mouse liver cytosol.

A monomeric 3 alpha-hydroxysteroid dehydrogenase with a molecular weight of 34,000 was purified to apparent homogeneity from mouse liver cytosol. The enzyme catalyzed the reversible oxidation of the 3 alpha-hydroxy group of C19-, C21-, and C24-steroids, reduced a variety of carbonyl compounds, and was inhibited by SH-reagents, synthetic estrogens, anti-inflammatory drugs, prostaglandins, and delta 4-3-ketosteroids. Although these properties are similar to those of the enzyme from rat liver cytosol, the mouse enzyme exhibited low dehydrogenase activity toward benzene dihydrodiol and some alicyclic alcohols, it showed a strict cofactor specificity for NADP(H), and high substrate inhibition was observed in the reverse reaction. In addition, dexamethasone, deoxycorticosterone, and medroxyprogesterone acetate inhibited the mouse enzyme competitively at low concentrations and noncompetitively at high concentrations, whereas hexestrol, indomethacin, and prostaglandin A1 were competitive inhibitors. Steady-state kinetic measurements in both directions indicated that the reaction proceeds through an ordered bi bi mechanism with the cofactors binding to the free enzyme. The 3-ketosteroid substrates inhibited the enzyme uncompetitively at elevated concentrations, suggesting that the substrates bind to the enzyme.NADPH complex and to the enzyme NADP+ complex.

In situ behaviour of D(-)-lactate dehydrogenase from Escherichia coli.

Some kinetic properties of the D(-)-lactate dehydrogenase (EC 1.1.1.28) of Escherichia coli have been investigated. There were marked differences between the kinetic properties of the enzyme studied in situ compared with the in vitro D(-)-lactate dehydrogenase. D(-)-Lactate dehydrogenase in situ showed high substrate inhibition with pyruvate over the pH range 6.0-7.0, whereas the enzyme in vitro did not. The pH optimum for pyruvate reduction by the in situ D(-)-lactate dehydrogenase ranged between pH 7.5 and 7.8, whereas the in vitro enzyme showed its pH optimum between pH 6.8 and 7.0. The pK values of the prototropic groups that controlled the enzymatic activity shift to the acidic region for the in vitro enzyme with respect to the in situ enzyme. In vitro D(-)-lactate dehydrogenase exhibits homotropic interactions with its substrate, pyruvate and its coenzyme, NADH, at pH values ranging between pH 6.0 and 8.5, but the in situ enzyme showed homotropic interactions neither with pyruvate nor with NADH at all pH values studied.