Bisubstrate Reaction Research Articles

Single stage bi-substrate transglycosylation reaction for the synthesis of ascorbic acid 2 glucoside using immobilized α-glucosidase

Alpha glucosidases are multifunctional glycoside hydrolases with hydrolysis and transglycosylation ability. It can be utilized for the glycosidic bond synthesis or glycosylation of Ascorbic acid/Vitamin C to its stable analogue, Ascorbic acid 2 glucoside (AA2G), a compound with wide applications in cosmetics and pharma. The application of α-glucosidases for industrial scale transglycosylation is limited due to the low transglycosylation yield of free enzymes. Enzyme immobilization techniques could enable the development of efficient, reusable catalysts. Only a few glycoside hydrolases have been studied in immobilized form for transglycosylation reactions, and α-glucosidases are probably the least explored in this form. Transglycosylation activity of immobilized α-glucosidase from Aspergillus carbonarius BTCF 5 was studied for AA2G synthesis, where different immobilization techniques like calcium alginate encapsulation, adsorption on chitosan beads, covalent cross-linking on magnetic nanoparticles, and cross-linked enzyme aggregates (CLEA) were employed for the immobilization. The immobilization yield of calcium alginate encapsulated enzyme, enzyme immobilized on Fe-MNP support, enzyme immobilized on chitosan beads and as CLEA were 107%, 99%, 46% and 486%, respectively. CLEA was identified as the best immobilization technique for this bi-substrate reaction due to the high immobilization yield and activity retention (30% activity retained after 5 consecutive cycles). Enzyme immobilization increased the transglycosylation activity by 38%, yielding 118 mM AA2G against 72 mM by the free enzyme. This indicates the potential of immobilized α-glucosidase as a catalyst for synthesizing AA2G at an industrial scale.

How is the activity of shikimate dehydrogenase from the root of Petroselinum crispum (parsley) regulated and which side reactions are catalyzed?

Inhibitors of the shikimate pathway are widely used as herbicides, antibiotics, and anti-infectious drugs. However, the regulation of the shikimic pathway is complex, and little is known about the feedback regulation of the shikimate dehydrogenase (SDH, EC 1.1.1.25) in plants. Thus, the aim of this study was to elucidate the kinetic mechanism of SDH purified from the root of Petroselinum crispum (parsley), to determine all possible reaction products and to identify phenylpropanoid compounds that affect its activity. Our results showed that the bisubstrate reaction catalyzed by P. crispum SDH follows a sequential ordered mechanism, except for three dead-end complexes. The main and lateral reactions of SDH were monitored by mass spectrometry, thereby detecting protocatechuic acid as a byproduct. Gallic acid was formed non-enzymatically, whereas quinate was not detected. Several polyphenolic compounds inhibited SDH activity, especially tannic, caffeic and chlorogenic acids, with IC50 0.014 mM, 0.15 mM, and 0.19 mM, respectively. The number of hydroxyl groups influenced their inhibition effect on SDH, and p-coumaric, t-ferulic, sinapic, syringic and salicylic acids were less effective SDH inhibitors. Nevertheless, one branch of the phenylpropanoid pathway may affect SDH activity through feedback regulation.

Enzyme Kinetics of Uridine Diphosphate Glucuronosyltransferases (UGTs).

Glucuronidation, catalyzed by uridine diphosphate glucuronosyltransferases (UGTs), is an important process for the metabolism and clearance of many lipophilic chemicals, including drugs, environmental chemicals, and endogenous compounds. Glucuronidation is a bisubstrate reaction that requires the aglycone and the cofactor, UDP-GlcUA. Accumulating evidence suggests that the bisubstrate reaction follows a compulsory-order ternary mechanism. To simplify the kinetic modeling of glucuronidation reactions in vitro, UDP-GlcUA is usually added to incubations in large excess. Many factors have been shown to influence UGT activity and kinetics in vitro, and these must be accounted for during experimental design and data interpretation. While the assessment of drug-drug interactions resulting from UGT inhibition has been challenging in the past, the increasing availability of UGT enzyme-selective substrate and inhibitor "probes" provides the prospect for more reliable reaction phenotyping and assessment of drug-drug interaction potential. Although extrapolation of the in vitro intrinsic clearance of a glucuronidated drug often underpredicts in vivo clearance, careful selection of in vitro experimental conditions and inclusion of extrahepatic glucuronidation may improve the predictivity of in vitro-in vivo extrapolation. Physiologically based pharmacokinetic (PBPK) modeling has also shown to be of value for predicting PK of drugs eliminated by glucuronidation.

Mathematical study of a memory induced biochemical system

In this work, to study the effect of memory on a bi-substrate enzyme kinetic reaction, we have introduced an approach to fractionalize the system, considering it as a threecompartmental model. Solutions of the fractionalized system are compared with the corresponding integer-order model. The equilibrium points of the fractionalized system are derived analytically. Their stability properties are discussed from numerical aspect. We determine the changes of the substances due to the changes of &#x201C memory effect &#x201D. The effect is discussed critically from the perspective of product formation. We have also analyzed the memory induced system with a control measure in view of optimizing the product. Our numerical result reveals that the solutions of the fractionalized system, when it is free from memory, are in good agreement with the integer-order system. It is noticed that the effect of memory influences the reaction in the forward direction and assists in yielding the product more quickly. However, an extensive use of memory makes the system slower, but introduction of a control input makes the reaction faster. It is possible to overcome the slowness of the reaction due to the undue effect of memory by appropriate use of a control measure.

New insights and tools for the elucidation of lipase catalyzed esterification reaction mechanism in n-hexane: The synthesis of ethyl butyrate

The enzyme catalyzed esterification of butyric acid by ethanol under anhydrous conditions in n-hexane, through continuous removal of the formed water, follows a ping pong bi-bi reaction mechanism; immobilized lipase-B from Candida antarctica on acrylic resin (Novozyme 435) was used as biocatalyst. For first time, the kinetic data of an enzymatic bi-substrate reaction generating simultaneous double dead-end substrate inhibitions were processed by surface fitting through multiparametric non-linear equations. Both anhydrous CH3CH2OH and/or CH3CH2OD were employed in an attempt to apply the technique of kinetic isotope effects and validate the selection of the best system among two (ping pong bi-bi and ordered bi-bi), which could describe the aforementioned reaction.

Mevalonate 5-diphosphate mediates ATP binding to the mevalonate diphosphate decarboxylase from the bacterial pathogen Enterococcus faecalis

The mevalonate pathway produces isopentenyl diphosphate (IPP), a building block for polyisoprenoid synthesis, and is a crucial pathway for growth of the human bacterial pathogen Enterococcus faecalis The final enzyme in this pathway, mevalonate diphosphate decarboxylase (MDD), acts on mevalonate diphosphate (MVAPP) to produce IPP while consuming ATP. This essential enzyme has been suggested as a therapeutic target for the treatment of drug-resistant bacterial infections. Here, we report functional and structural studies on the mevalonate diphosphate decarboxylase from E. faecalis (MDDEF). The MDDEF crystal structure in complex with ATP (MDDEF-ATP) revealed that the phosphate-binding loop (amino acids 97-105) is not involved in ATP binding and that the phosphate tail of ATP in this structure is in an outward-facing position pointing away from the active site. This suggested that binding of MDDEF to MVAPP is necessary to guide ATP into a catalytically favorable position. Enzymology experiments show that the MDDEF performs a sequential ordered bi-substrate reaction with MVAPP as the first substrate, consistent with the isothermal titration calorimetry (ITC) experiments. On the basis of ITC results, we propose that this initial prerequisite binding of MVAPP enhances ATP binding. In summary, our findings reveal a substrate-induced substrate-binding event that occurs during the MDDEF-catalyzed reaction. The disengagement of the phosphate-binding loop concomitant with the alternative ATP-binding configuration may provide the structural basis for antimicrobial design against these pathogenic enterococci.

Kinetic, oxygen mass transfer and hydrodynamic studies in a three-phase stirred tank bioreactor for the bioconversion of (+)-valencene on Yarrowia lipolytica 2.2ab

The oxidation of (+)-valencene on Yarrowia lipolytica 2.2ab in a three-phase partitioning bioreactor using orange essential oil is a promising technology to market natural (+)-nootkatone overcoming both substrate and product inhibitions. The adequate determination and selection of thermodynamic, kinetic, deactivation, hydraulic and mass transport parameters are essential to perform a suitable strategy of scaling-up. This study is aimed at determining these parameters to identify through a regime analysis the mechanisms limiting the bioconversion process. The volumetric oxygen transfer coefficient (kLa) and the Sauter mean drop diameter (d32) values ranged from 10 to 116h−1 and 8 to 18μm, respectively. The substrate (kS) and product (kP) global interfacial mass transfer coefficients were determined from a modified Lewis cell. The kS and kP values ranged from 0.6 to 3.0×10−5 and 2 to 3×10−5ms−1, respectively. Finally, a kinetic model, considering a bi-substrate reaction and accounting for cell deactivation, was developed. The affinity constants for oxygen and (+)-valencene were KO2=7.11×10−2mgO2L−1 and KS=7.865×10−3mgSL−1, respectively, while catalytic constant related to (+)-nootkatone formation was kcat=5.025×103 mgP mgE−1h−1. Thus, the regime analysis in terms of the characteristic times suggested that kinetics, namely the consumption rate of (+)-valencene was the main mechanism limiting the bioconversion process.

Ultrasonic Treatment Effect on Enzymolysis Kinetics and Activities of ACE-Inhibitory Peptides from Oat-Isolated Protein

Pretreatment of oat protein with ultrasound showed excellent prospects in increasing the efficiencies of enzymatic hydrolysis and inhibitory activities of peptides against angiotensin converting enzyme (ACE). The mechanism of the ultrasound pretreatment on enhancement of protein hydrolysis rate and the characterization of the enzymolysis process was investigated. The Michaelis-Menten model was used to study the effects of ultrasound on the hydrolysis rate. An ordered sequential bi-substrate reaction mechanism model was applied to describe the characteristics of enzymolysis of the protein with ultrasonic pretreatment. The protein concentration and the enzyme loading were factors in this model. The kinetic parameters of the models were estimated by experimental results and evaluated. The results showed that ultrasonic power, time and enzymolysis time greatly influenced the degree of hydrolysis and ACE inhibitory activities of the peptides. The best conditions for hydrolysis and ACE inhibitory activities were 750 W, 20 min of sonication followed by 60 min of enzymolysis. After the treatment, the hydrolysis rate and the ACE inhibitory activities of peptides were significantly (P < 0.001) increased by 32.1 and 53.8 %, respectively compared to the samples without ultrasonic pretreatment. The Michaelis constant K m indicated the ultrasonic treatment enhanced the affinities between the alcalase and oat protein. The enzymolysis kinetic model fitted well the enzyme catalyzed hydrolysis trend for the ultrasonic pretreated oat protein. The model provided a theoretical basis for describing the complex enzymatic process and preparing the ACE inhibitory peptides efficiently.

Production of lactobionic acid and sorbitol from lactose/fructose substrate using GFOR/ GL enzymes from Zymomonas mobilis cells: A kinetic study

In this work, we have investigated the kinetics of the biotechnological production of lactobionic acid ( LBA) and sorbitol by the catalytic action of glucose–fructose oxidoreductase ( GFOR) and glucono-δ-lactonase ( GL) enzymes. The cells of bacterium Zymomonas mobilis ATCC 29191 containing this enzymatic complex were submitted to permeabilization and reticulation procedures. The effect of the concentration of substrates on the rate of product formation using a mobilized cell system was investigated. The application of higher fructose concentration seems to not affect the initial rate of formation of the bionic acid. Under conditions of low initial concentration of lactose, the experimental kinetic data of the bi-substrate reaction were modelled by assuming a rate equation of the classical ping-pong mechanism. The found kinetic parameters displayed a low affinity of the GFOR enzyme for both substrates. The enzymatic system did not exhibit normal Michaelis–Menten kinetics in response to a change of concentration of lactose, when fructose was held constant, presenting a sigmoid relationship between initial velocity and substrate concentration. A rate equation based on Hill kinetics was used to describe the kinetic behaviour of this enzyme-substituted reaction at higher lactose concentrations. The results from batch experiments using immobilized cells within Ca-alginate beads revealed that there is no pronounced occurrence of mass transfer limitations on LBA production for beads with 1.2 mm in average diameter. This discussion aids for defining the best operating conditions to maximize the productivity for LBA and sorbitol in this bioconversion and also for reducing the complexity of downstream separation processes.

Kinetic properties of lactate dehydrogenase from trout muscle

The kinetics of pyruvate reduction by LDH isozymes in the presence of NADH is said to be an ordered bisubstrate reaction characterized by Michaelian concentration dependence (Borgmann et al., 1975). If the reaction is ordered, no cooperativity can occur between the two substrates (NADH and pyruvate) (Neet, 1995). Experiments with fish LDH showed, however, that this enzyme might display two types of saturation profile, hyperbolic and sigmoidal, depending on the pH in the reaction medium. At pH 9, for example, the initial velocity of pyruvate reduction by epaxial muscle LDH from goldfish showed a sigmoidal dependence upon substrate concentration, whereas at pH 8 the kinetics were of the hyperbolic type (Hochachka, 1965). Our experiments showed, in addition, that LDH from trout muscle (Onchorhynchus mykiss) could display both types of saturation profile at one and the same pH, but in the presence of different concentrations of the LDH preparation. Rainbow trout (Onchorhyncus mykiss) were obtained in October from the Goc Fishpond of the School of Forestry, University of Belgrade, Serbia. White skeletal muscle was immediately excised and kept in liquid nitrogen until use. After thawing, muscle samples were deprived of skin and spine, cut into small pieces, and homogenized in 5 volumes (w/v) of Tris-EDTA buffer. The homogenate was centrifuged for 10 min at 10 000× g. Partial purification with ammonium sulfate was performed according to Place and Powers, 1984. After centrifugation, solid ammonium sulfate was added to the supernatant to give 30% saturation at 0oC. The suspension was stirred for 1 hour and centrifuged for 30 min at 10 000× g. The pellet was discarded, and the supernatant brought to 70% saturation. The suspension was then stirred again for 1 h at 0oC, and centrifuged as already described. This time, the supernatant was discarded and the pellet redissolved in Tris-EDTA buffer and dialyzed overnight at 4oC against four 1liter replacements of the same buffer. The concentration of protein, determined by the method of Lowry (Lowry et al., 1951), amounted to 5.3 mg/ml of the final enzyme suspension.

A Kinetic Study of Human Protein Arginine N-Methyltransferase 6 Reveals a Distributive Mechanism

Human protein arginine N-methyltransferase 6 (PRMT6) transfers methyl groups from the co-substrate S-adenosyl-L-methionine to arginine residues within proteins, forming S-adenosyl-L-homocysteine as well as omega-N(G)-monomethylarginine (MMA) and asymmetric dimethylarginine (aDMA) residues in the process. We have characterized the kinetic mechanism of recombinant His-tagged PRMT6 using a mass spectrometry method for monitoring the methylation of a series of peptides bearing a single arginine, MMA, or aDMA residue. We find that PRMT6 follows an ordered sequential mechanism in which S-adenosyl-L-methionine binds to the enzyme first and the methylated product is the first to dissociate. Furthermore, we find that the enzyme displays a preference for the monomethylated peptide substrate, exhibiting both lower K(m) and higher V(max) values than what are observed for the unmethylated peptide. This difference in substrate K(m) and V(max), as well as the lack of detectable aDMA-containing product from the unmethylated substrate, suggest a distributive rather than processive mechanism for multiple methylations of a single arginine residue. In addition, we speculate that the increased catalytic efficiency of PRMT6 for methylated substrates combined with lower K(m) values for native protein methyl acceptors may obscure this distributive mechanism to produce an apparently processive mechanism.

Purification and characterization of a novel glucosyltransferase specific to 27β-hydroxy steroidal lactones from Withania somnifera and its role in stress responses

Sterol glycosyltransferases catalyze the synthesis of diverse glycosterols in plants. Withania somnifera is a medically important plant, known for a variety of pharmacologically important withanolides and their glycosides. In this study, a novel 27β-hydroxy glucosyltransferase was purified to near homogeneity from cytosolic fraction of W. somnifera leaves and studied for its biochemical and kinetic properties. The purified enzyme showed activity with UDP-glucose but not with UDP-galactose as sugar donor. It exhibited broad sterol specificity by glucosylating a variety of sterols/withanolides with β-OH group at C-17, C-21 and C-27 positions. It transferred glucose to the alkanol at C-25 position of the lactone ring, provided an α-OH was present at C-17 in the sterol skeleton. A comparable enzyme has not been reported earlier from plants. The enzyme is distinct from the previously purified W. somnifera 3β-hydroxy specific sterol glucosyltransferase and does not glucosylate the sterols at C-3 position; though it also follows an ordered sequential bisubstrate reaction mechanism, in which UDP-glucose and sterol are the first and second binding substrates. The enzyme activity with withanolides suggests its role in secondary metabolism in W. somnifera. Results on peptide mass fingerprinting showed its resemblance with glycuronosyltransferase like protein. The enzyme activity in the leaves of W. somnifera was enhanced following the application of salicylic acid. In contrast, it decreased rapidly on exposure of the plants to heat shock, suggesting functional role of the enzyme in biotic and abiotic stresses.

Purification and properties of a new S-adenosyl-L-methionine:flavonoid 4'-O-methyltransferase from carnation (Dianthus caryophyllus L.).

A new enzyme, S-adenosyl-l-methionine:flavonoid 4'-O-methyltransferase (EC 2.1.1.-) (F 4'-OMT), has been purified 1 399-fold from the tissues of carnation (Dianthus caryophyllus L). The enzyme, with a molecular mass of 43-45 kDa and a pI of 4.15, specifically methylates the hydroxy substituent in 4'-position of the flavones, flavanones and isoflavones in the presence of S-adenosyl-l-methionine. A high affinity for the flavone kaempferol was observed (Km = 1.7 micro m; Vmax = 95.2 micro mol.min-1.mg-1), while other 4'-hydroxylated flavonoids proved likewise to be suitable substrates. Enzyme activity had no apparent Mg++ requirement but was inhibited by SH-group reagents. The optimum pH value for F 4'-OMT activity was found to be around neutrality. Kinetic analysis of the enzyme bi-substrate reaction indicates a Ping-Pong mechanism and excludes the formation of a ternary complex. The F 4'-OMT activity was increased, in both in vitro and in vivo carnation tissues, by the inoculation with Fusarium oxysporum f. sp. dianthi. The enzyme did not display activity towards hydroxycinnamic acid derivatives, some of which are involved, as methylated monolignols, in lignin biosynthesis; the role of this enzyme could be therefore mainly defensive, rather than structural, although its precise function still needs to be ascertained.

2-Cys peroxiredoxin PfTrx-Px1 is involved in the antioxidant defence of Plasmodium falciparum.

Peroxiredoxins (Trx-Px) are ubiquitous antioxidant enzymes that catalyse the thioredoxin-dependent reduction of hydroperoxides. The number of characteristic active site (VCP/T) motifs defines these proteins as 1-Cys and 2-Cys Trx-Px. Steady-state kinetic parameters of Plasmodium falciparum 2-Cys Trx-Px (PfTrx-Px1) were determined using stopped flow rapid kinetics. The bi-substrate reaction displays ping-pong kinetics and the K(m) values for H2O2 and thioredoxin were determined to be 0.78+/-0.14 microM and 18.94+/-3.01 microM, respectively. The Vmax(app) and kcat(app) for H2O2 were found to be 4+/-0.6 U mg(-1) and 1.67+/-0.25 s(-1), respectively and those for thioredoxin are 23.0+/-0.2 U mg(-1) and 9.65+/-0.1 s(-1), emphasising the specificity of the enzyme for the substrate H2O2. After subjection to exogenous and endogenous oxidative stress, P. falciparum blood stage forms showed a marked elevation of PfTrx-Px1 mRNA and protein levels consistent with the hypothesis that it is an important component of the parasite's antioxidant machinery. Gel filtration, cross-linking and electron microscopy (EM) revealed that the protein forms decamers consisting of pentamers of homodimers that have a doughnut-like shape consistent with the structures of related proteins. No dimeric forms of the protein were detectable after gel filtration suggesting that PfTrx-Px1 predominantly exists as an oligomer.

Catalytic and immunochemical properties of NADPH-cytochrome P450 reductase from fungus Rhizopus nigricans

Flavoprotein NADPH-cytochrome P450 reductase (CPR, EC 1.6.2.4) from filamentous fungus Rhizopus nigricans is a membrane bound enzyme which is involved in the reduction of cytochrome P450 during the hydroxylation of progesterone at 11α position. After purification of the enzyme from induced mycelia three forms of fungal CPR were detected on SDS-PAGE: a predominant form with an apparent molecular mass of 78 kDa and two truncated forms. N-terminal sequences of all three forms were determined as well as some internal sequences of 78 kDa form. Dose-dependent immunoinhibition of NADPH-cytochrome c reductase and progesterone 11α-hydroxylase activities was observed with mouse anti-CPR antisera. No cross-reactions were obtained on Western blots between mouse anti-CPR antisera and protein preparations from noninduced mycelia and microsomal fraction from fungus Pleurotus osteatus, plant Ginkgo biloba or chicken liver. The kinetic mechanism of CPR was proposed on the basis of model reaction with cytochrome c 3+. Results obtained at high ionic strength suggest a nonclassical two-site ping pong mechanism and at low ionic strength a sequential mechanism of bisubstrate reaction.

Species-specific Inhibition of Porphobilinogen Synthase by 4-Oxosebacic Acid

Porphobilinogen synthase (PBGS) catalyzes the condensation of two molecules of 5-aminolevulinic acid (ALA), an essential step in tetrapyrrole biosynthesis. 4-Oxosebacic acid (4-OSA) and 4,7-dioxosebacic acid (4,7-DOSA) are bisubstrate reaction intermediate analogs for PBGS. We show that 4-OSA is an active site-directed irreversible inhibitor for Escherichia coli PBGS, whereas human, pea, Pseudomonas aeruginosa, and Bradyrhizobium japonicum PBGS are insensitive to inhibition by 4-OSA. Some variants of human PBGS (engineered to resemble E. coli PBGS) have increased sensitivity to inactivation by 4-OSA, suggesting a structural basis for the specificity. The specificity of 4-OSA as a PBGS inhibitor is significantly narrower than that of 4,7-DOSA. Comparison of the crystal structures for E. coli PBGS inactivated by 4-OSA versus 4,7-DOSA shows significant variation in the half of the inhibitor that mimics the second substrate molecule (A-side ALA). Compensatory changes occur in the structure of the active site lid, which suggests that similar changes normally occur to accommodate numerous hybridization changes that must occur at C3 of A-side ALA during the PBGS-catalyzed reaction. A comparison of these with other PBGS structures identifies highly conserved active site water molecules, which are isolated from bulk solvent and implicated as proton acceptors in the PBGS-catalyzed reaction.

Substrate Specificity and Kinetic Mechanism of the Insect Sulfotransferase, Retinol Dehydratase

Spodoptera frugiperda retinol dehydratase catalyzes the conversion of retinol to the retro-retinoid anhydroretinol. It shares sequence homology with the family of mammalian cytosolic sulfotransferases and provides the first link between sulfotransferases and retinol metabolism. In this study the enzymatic properties of retinol dehydratase were examined using bacterially expressed protein. We show that retinol dehydratase can catalyze the transfer of the sulfonate moiety to small phenolic compounds and exhibits many functional similarities to the mammalian cytosolic sulfotransferases. The bisubstrate reaction that it catalyzes between retinol and the universal sulfonate donor 3'-phosphoadenosine 5'-phosphosulfate seems to involve ternary complex formation and to proceed via a Random Bi Bi mechanism. In addition to the low nanomolar Km value for free retinol, retinol dehydratase is strongly inhibited by retinol metabolites, suggesting a preference for retinoids. Conversely, a number of tested mammalian cytosolic sulfotransferases do not utilize retinol, indicating that retinol is not a general substrate for sulfotransferases.

Species-related inhibition of human and rat dihydroorotate dehydrogenase by immunosuppressive isoxazol and cinchoninic acid derivatives

The isoxazol leflunomide (N-(4-trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide) and its active metabolite A77-1726 (N-(4-trifluoromethyl)-phenyl-2-cyano-3-hydroxy-crotonic acidamide) are promising disease-modifying antirheumatic drugs now in clinical trials. The malononitrilamides MNA279 (2-cyano-3-cyclopropyl-3-oxo-(4-cyanophenyl)propionamide) MNA715(N-(4-trifluoromethyl)-phenyl-2-cyano-3-hydroxy-hept-2-en-6-in-carboxylic acidamide) and HR325 (1(3-methyl-4-trifluoromethylphenyl-carbamoyl)-2-cyclopropyl-2-oxo-propionitrile) were shown to block rejection after allograft and xenograft transplantation in animals. Brequinar and other cinchoninic acid derivatives have also been evaluated as immuno-suppressive agents. A77-1726, HR325 and brequinar have been shown to have strong inhibitory effects on mitochondrial dihydroorotate dehydrogenase [EC 1.3.99.11], the fourth enzyme of pyrimidine de novo synthesis, with concomitant reduction of pyrimidine nucleotide pools. Pyrimidine nucleotides are essential for normal immune cell functions. Because most investigations had been carried out with cells, cell homogenates or mitochondrial fractions, it was the rationale of the present study to differentiate, under standardized conditions, the effect of leflunomide, A77-1726, MNA279, MNA715, HR 325 and brequinar on the recombinant rat and human enzymes, which were purified in our laboratory. Whereas leflunomide was a relatively weak inhibitor of the rat ( ic 50 = 6.3 μM) and human ( ic 50 = 98 μM) dihydroorotate dehydrogenase, the influence of A77-1726, MNA 279, MNA715 and HR325 was of comparable efficacy for either the rat (range of ic 50, 19–53 nM) or the human enzyme (range of ic 50, 0.5–2.3 μM). From the ic 50 values, it was deduced that brequinar was a more potent inhibitor of the human dihydroorotate dehydrogenase activity ( ic 50 = 10 nM) than of the rat enzyme ( ic 50 = 367 nM). The rat enzyme was influenced by all isoxazol derivatives to a greater extent ( ic 50 = 19 nM A77-1726) than the human enzyme ( ic 50 = 1.1 μM A77-1726). These results may provide a plausible explanation for the findings of other laboratories with cultured cell lines and lymphocytes: in comparison to cells derived from human tissues, rat and other rodent cells were more susceptible to the isoxazol derivatives and less susceptible to brequinar. Our detailed kinetic investigations of the bisubstrate reaction catalyzed by rat dihydroorotate dehydrogenase revealed a noncompetitive type of inhibition by A77-1726 with respect to the substrate dihydroorotate and the cosubstrates ubiquinone or decylubiquinone. For brequinar, the inhibition was noncompetitive with respect to the substrate dihydroorotate, whereas with the quinone it was found to follow the “mixed typed” inhibition. In addition, brequinar acted as a “slow-binding” inhibitor of the human dihydroorotate dehydrogenase, a feature that might be of consequence for the reversibility of the reaction with the target.

Sequential ordered mechanism for the sodium-glutamate transport in intestinal brush border membrane vesicles

The glutamate/aspartate carrier localized in the brush-border membrane vesicles from enterocytes is known as a transport system catalyzing a sodium-substrate cotransport driven by the sodium gradient across the membrane. The kinetics of this transport system is studied by analogy with an enzymatic bi-substrate reaction. The results of this approach can be summarized as follows: (1) The dependence of the l-glutamate transport rate on the sodium concentration is sigmoidal, and the stoichiometry of the transport is 2 Na +/1 glutamate/1 carrier molecule. (2) The mechanism is sequential ordered, with l-glutamate binding after both the sodium cations. In addition, there is a very high degree of cooperativity between the two sodium binding sites.

Contribution of a salt bridge to binding affinity and dUMP orientation to catalytic rate: mutation of a substrate-binding arginine in thymidylate synthase.

Invariant arginine 179, one of four arginines that are conserved in all thymidylate synthases (TS) and that bind the phosphate moiety of the substrate 2'-deoxyuridine-5'-monophosphate (dUMP), can be altered even to a negatively charged glutamic acid with little effect on kcat. In the mutant structures, ordered water or the other phosphate-binding arginines compensate for the hydrogen bonds made by Arg179 in the wild-type enzyme and there is almost no change in the conformation or binding site of dUMP. Correlation of dUMP Kds for TS R179A and TS R179K with the structures of their binary complexes shows, that the positive charge on Arg179 contributes significantly to dUMP binding affinity. kcat/K(m) for dUMP measures the rate of dUMP binding to TS during the ordered bi-substrate reaction, and in the ternary complex dUMP provides a binding surface for the cofactor. kcat/K(m) reflects the ability of the enzyme to accept a properly oriented dUMP for catalysis and is less sensitive than is Kd to the changes in electrostatics at the phosphate binding site.