An analysis of the reaction kinetics of the hexahaem nitrite reductase of the anaerobic rumen bacterium Wolinella succinogenes.

The reduction kinetics of both the resting and redox-cycled forms of the nitrite reductase from the anaerobic rumen bacterium Wolinella succinogenes were studied by stopped-flow reaction techniques. Single-turnover reduction of the enzyme by dithionite occurs in two kinetic phases for both forms of the enzyme. When the resting form of the enzyme is subjected to a single-turnover reduction by dithionite, the slower of the two kinetic phases exhibits a hyperbolic dependence of the rate constant on the square root of the reductant concentration, the limiting value of which (approximately 4 s-1) is assigned to a slow internal electron-transfer process. In contrast, when the redox-cycled form of the enzyme is reduced by dithionite in a single-turnover experiment, both kinetic phases exhibit linear dependences of the rate on the square root of dithionite concentration, with associated rate constants of 150 M-1/2.s-1 and 6 M-1/2.s-1. Computer simulations of both the reduction processes shows that no unique set of rate constants can account for the kinetics of both forms, although the kinetics of the redox-cycled species is consistent with a much enhanced rate of internal electron transfer. Under turnover conditions the time course for reduction of the enzyme, in the presence of millimolar levels of nitrite and 100 mM-dithionite, is extremely complex. A working model for the mechanism of the turnover activity of the enzyme is proposed which very closely describes the reaction kinetics over a wide range of substrate concentrations, as shown by computer simulation. The similarity in the action of the nitrite reductase enzyme and mammalian cytochrome c oxidase is commented upon.     

An immobilized alpha-galactosidase continuous flow reactor

An α-galactosidase which will hydrolyze the oligosaccharides melibiose, raffinose, and stachyose has been immobilized on nylon microfibrils suitable for use in large flow-through reactors. This catalyst system is stable for many months, both under use and storage conditions. The immobilized enzyme behaves similarly to the enzyme in solution, characteristically exhibiting both product and substrate inhibition. The catalyst is prepared in situ and a large, 8-liter reactor has been made. The catalyst has been used to reduce the raffinose concentration in beet sugar molasses.     

Role of phospholipid in the low affinity reactions between cytochrome c and cytochrome oxidase

The steady-state oxidation of ferrocytochrome c by cytochrome oxidase monitored spectrophotometrically showed that: (1) the kinetics were strictly biphasic with purified enzyme, while mitochondrial membrane-bound enzyme exhibited multiphasic kinetics with extended low affinity phases; (2) the TNmax for the highest affinity phase was as slow as 5-10 electron X s-1 for both preparations, while for the low affinity phases it was about 45 electron X s-1 for the purified enzyme and 150 electron X s-1 for the mitochondrial membrane-bound enzyme; (3) reconstitution of purified enzyme into acidic phospholipid vesicles partially repleted the extended low affinity phases, while reconstitution into uncharged vesicles had no effect.     

Bordetella pertussis adenylate cyclase. Identification of multiple forms of the enzyme by antibodies

Two forms of Bordetella pertussis adenylate cyclase of 200 and 47 kDa have been purified from dialyzed urea extract of the bacteria to specific activities of 466 and 1685 mumol.min-1.mg-1, respectively. Both forms are activated 50-200-fold by calmodulin. The half-maximum concentration required for the activation of the 200-kDa catalyst is 5.4.10(-9) M, whereas the one required for activation of the 47-kDa catalyst is 1.8.10(-10) M. Polyclonal antibodies raised against the 47-kDa catalyst specifically recognize both forms of the enzyme in purified state as well as in bacterial extracts on immunoblots. The antibody inhibits at similar titer adenylate cyclase activity of the purified forms as well as the activity present in dialyzed urea extract of the bacteria. It also prevents the penetration of the invasive B. pertussis adenylate cyclase into human lymphocytes. The inhibition induced by the antisera is specific to B. pertussis enzyme, since both calmodulin-dependent brain and sperm adenylate cyclase are not affected by the antibody.     

The simulation of thymidine kinase enzyme kinetics in cultured cells using the computer language cellsim

Thymidine kinase is an enzyme that occurs in cells actively synthesizing DNA. In studies of synchronized cell populations, it has been shown that the enzyme activity disappears during the G1 phase of the cell cycle and reappears during the S and G2 phases. Its reappearance is consistent with the synthesis of the mRNA for this enzyme during the S and G2 phases and its immediate translation into active enzyme by the protein synthesis machinery within the cell. The disappearance of the enzyme is consistent with the cessation of mRNA synthesis by mitotic cells. We have now tested this concept by computer simulation of a growing cell population in which a specific mRNA is generated while cells are in the S and G2 phases of the cell cycle. The computer simulation was done using the simulation language Cellsim designed for modeling populations of cells. The Cellsim program which we developed allowed each cell to make about 1 mRNA molecule per min during the S and G2 phases. Every 3 min each mRNA molecule generated a protein enzyme molecule. The mRNA had a half-life of about 9 min, and the enzyme had a half-life of about 150 min. When these molecular parameters were coupled to the cell cycle parameters for Chinese hamster fibroblasts, the resulting curve of enzyme production with time closely matched the observed kinetics of enzyme activity seen in synchronized cells. The only part of the curve that did not fit was the rapid drop in enzyme activity which was seen as the population of mitotic cells was permitted to enter G1. This drop in activity was not seen in mitotic cells blocked with Colcemid where mRNA synthesis must be lacking. Earlier studies have shown that the Gl cells do not contain any inhibitor of enzyme activity. It therefore appears that the enzyme molecule is more unstable during the G1 phase than in any of the other phases of the cell cycle.     

Immobilization of glucoamylase and glucose oxidase in activated carbon: effects of particle size and immobilization conditions on enzyme activity and effectiveness

Glucoamylase and glucose oxidase have been immobilized on carbodiimide-treated activated carbon particles of various sizes. Loading data indicate nonuniform distribution of immobilized enzyme within the porous support particles. Catalysts with different enzyme loading and overall activities have been prepared by varying enzyme concentration in the immobilizing solution. Analysis of these results by a new method based entirely upon experimentally observable catalyst properties indicates that intrinsic catalytic activity is reduced by immobilization of both enzymes. Immobilized glucoamylase intrinsic activity decreases with increasing enzyme loading, and similar behavior is suggested by immobilized glucose oxidase data analysis. The overall activity data interpretation method should prove useful in other immobilized enzyme characterization research, especially in situations where the intraparticle distribution of immobilized enzyme is nonuniform and unknown.     

Decarboxylation of oxalacetate by pyruvate carboxylase

The decarboxylation of oxalacetate by pyruvate carboxylase in the absence of ADP and Pi is stimulated 400-fold by the presence of oxamate, which is an inhibitory analogue of pyruvate. The observation of substrate inhibition when either oxamate or oxalacetate is varied at a fixed concentration of the other indicates that both molecules bind at the same site on the enzyme. The pH profiles for this reaction show no evidence of the involvement of an enzymic acid-base catalyst, suggesting that the proton and CO2 units may be exchanged directly between the reactants (although CO2 sequestered in the active site may be an intermediate in the process). The pH profiles of the full reverse reaction of pyruvate carboxylase in which oxalacetate decarboxylation is coupled to ATP formation and where Pi is the variable substrate do, however, indicate that such an acid-base catalyst is involved in the other partial reaction of the enzyme in proton transfer to and from biotin. The enzyme also displays two oxamate-independent oxalacetate decarboxylating activities, one of which is biotin-dependent and the other is independent of biotin.     

Formation of factors IXa and Xa by the extrinsic pathway: differential regulation by tissue factor pathway inhibitor and antithrombin III

The activation of factor X by VIIa/TF and the Xa-dependent inhibition of the enzyme complex by tissue factor pathway inhibitor (TFPI) are considered primary steps in the initiation of coagulation. IX activation by VIIa/TF is considered to contribute catalyst necessary for further Xa production in the ensuing amplification phase. We have investigated Xa and IXabeta production by VIIa-TF in a system reconstituted with both X and IX and the principal physiologic inhibitors of this pathway TFPI and antithrombin III (AT). Kinetic studies without inhibitors established that IX and X functioned as competitive alternate substrates for VIIa/TF with similar kinetic constants. When both IX and X were present, TFPI significantly inhibited the extent of formation of either IXabeta or Xa. In contrast, AT rapidly depleted active Xa with a small effect on IXabeta formation. When both AT and TFPI were present, active IXabeta formation significantly exceeded the formation of active Xa regardless of the VIIa/TF concentration. These findings could be quantitatively accounted for by a model encompassing the kinetics of the individual activation and inhibition steps. Active Xa formation by this pathway is regulated in a principal way by its rapid inactivation by AT. In contrast, the Xa-dependent inhibitory reactions of TFPI play a primary role in limiting zymogen consumption and the formation of active IXabeta. These regulatory phenomena yield active IXabeta as a major rather than secondary product of VIIa/TF. Our findings raise the possibility that IXabeta produced by the extrinsic pathway, and its ability to function within the intrinsic Xase complex to activate X may play a significant role in producing Xa necessary for both the initiation and sustained phases of the procoagulant response following vascular damage.     

Batch reactor performance for the enzymatic synthesis of cephalexin: influence of catalyst enzyme loading and particle size

A mathematical model is presented for the kinetically controlled synthesis of cephalexin that describes the heterogeneous reaction-diffusion process involved in a batch reactor with glyoxyl-agarose immobilized penicillin acylase. The model is based on equations considering reaction and diffusion components. Reaction kinetics was considered according to the mechanism proposed by Schro?n, while diffusion of the reacting species was described according to Fick's law. Intrinsic kinetic and diffusion parameters were experimentally determined in independent experiments. It was found that from the four kinetic constants, the one corresponding to the acyl-enzyme complex hydrolysis step had the greatest value, as previously reported by other authors. The effective diffusion coefficients of all substances were about 5×10(-10)m(2)/s, being 10% lower than free diffusion coefficients and therefore agreed with the highly porous structure of glyoxyl-agarose particles. Simulations made from the reaction-diffusion model equations were used to evaluate and analyze the impact of internal diffusional restrictions in function of catalyst enzyme loading and particle size. Increasing internal diffusional restrictions decreases the Cex synthesis/hydrolysis ratio, the conversion yield and the specific productivity. A nonlinear relationship between catalyst enzyme loading and specific productivity of Cex was obtained with the implication that an increase in catalyst enzyme loading will not increase the volumetric productivity by the same magnitude as it occurs with the free enzyme. Optimization of catalyst and reactor design should be done considering catalyst enzyme loading and particle size as the most important variables. The approach presented can be extended to other processes catalyzed by immobilized enzymes.     

The reaction of trimethylamine dehydrogenase with trimethylamine

The reductive half-reaction of trimethylamine dehydrogenase with its physiological substrate trimethylamine has been examined by stopped-flow spectroscopy over the pH range 6.0-11.0, with attention focusing on the fastest of the three kinetic phases of the reaction, the flavin reduction/substrate oxidation process. As in previous work with the slow substrate diethylmethylamine, the reaction is found to consist of three well resolved kinetic phases. The observed rate constant for the fast phase exhibits hyperbolic dependence on the substrate concentration with an extrapolated limiting rate constant (klim) greater than 1000 s-1 at pH above 8.5, 10 degrees C. The kinetic parameter klim/Kd for the fast phase exhibits a bell-shaped pH dependence, with two pKa values of 9.3 +/- 0.1 and 10. 0 +/- 0.1 attributed to a basic residue in the enzyme active site and the ionization of the free substrate, respectively. The sigmoidal pH profile for klim gives a single pKa value of 7.1 +/- 0. 2. The observed rate constants for both the intermediate and slow phases are found to decrease as the substrate concentration is increased. The steady-state kinetic behavior of trimethylamine dehydrogenase with trimethylamine has also been examined, and is found to be adequately described without invoking a second, inhibitory substrate-binding site. The present results demonstrate that: (a) substrate must be protonated in order to bind to the enzyme; (b) an ionization group on the enzyme is involved in substrate binding; (c) an active site general base is involved, but not strictly required, in the oxidation of substrate; (d) the fast phase of the reaction with native enzyme is considerably faster than observed with enzyme isolated from Methylophilus methylotrophus that has been grown up on dimethylamine; and (e) a discrete inhibitory substrate-binding site is not required to account for excess substrate inhibition, the kinetic behavior of trimethylamine dehydrogenase can be readily explained in the context of the known properties of the enzyme.     

Lipase-catalyzed kinetic resolution of (R,S)-1-phenylethyl propionate in an enzyme membrane reactor

Enzymatic stereoselective hydrolysis of (R,S)-1-phenylethyl propionate was performed in a stirred tank and in a biphasic enzyme membrane reactor. Lipase from Pseudomonas sp. was proved to be a good enantioselective catalyst for this reaction. The enzyme was covalently immobilized in a porous polyamide membrane (flat sheet as well as hollow-fibres) via glutaraldehyde. An influence of membrane hydrophobicity on reactor performance was observed. Initial lipase activity and productivity in the processes were equal to 1.05 × 10^?4, 1.3 × 10^?5 and 1.0 × 10^?5 mole/(h × mg of enzyme) in the case of native lipase, in the aromatic polyamide hydrophobic membrane reactor and in the hydrophilic polyamide-6 membrane reactor, respectively. The influence of some factors such as temperature, pH, buffer concentration, initial substrate concentration and addition of β-cyclodextrin derivatives on reaction rate and enantioselectivity was investigated and discussed. In the enzyme membrane reactor both organic and aqueous phases circulated countercurrently on both sides of the membrane. At a conversion degree of under 55–60%, pure enantiomer of the remaining ester (i.e. > 98%) was obtained.     

Epoxidation of fatty acids,fatty methyl esters,and alkenes by immobilized oat seed peroxygenase

Fatty epoxides are used as plasticizers and plastic stabilizers and are intermediates for the production of other chemical substances. The currently used industrial procedure for fatty epoxide synthesis requires a strong acid catalyst which can cause oxirane ring opening and side product formation. To find a replacement for the acid catalyst, we have been conducting research on a peroxygenase enzyme from oat (Avena sativa) seeds and have devised a method for immobilization of this enzyme using a hydrophobic membrane support. In this study, fatty acids and fatty methyl esters commonly encountered in commercial vegetable oils were tested as substrates for immobilized peroxygenase, and the epoxide products were characterized. The epoxidation time course of linoleic acid showed two distinct phases with nearly complete conversion to monoepoxide before diepoxide was produced. The diepoxide formed from linolenic acid was found to be 9,10-15,16-diepoxy-12-octadecenoic acid, and only a trace of triepoxide was obtained. Additionally it was discovered that acyclic alkenes with internal double bonds, a cyclic alkene, and an alkene with an aromatic substituent were substrates of peroxygenase. However, alkenes with terminal unsaturation were unreactive. With every substrate examined, oat seed peroxygenase exhibited specificity for epoxidation, producing no other products, and oxirane ring opening did not occur.     

Characterization of benzaldehyde lyase from Pseudomonas fluorescens: A versatile enzyme for asymmetric C-C bond formation

The thiamin-diphosphate-dependent enzyme benzaldehyde lyase is a very import catalyst for chemoenzymatic synthesis catalyzing the formation and cleavage of (R)-hydroxy ketones. We have studied the stability of the recombinant enzyme and some enzyme variants with respect to pH, temperature, buffer salt, cofactors and organic cosolvents. Stability of BAL in chemoenzymatic synthesis requires the addition of cofactors to the buffer. Reaction temperature should not exceed 37 degrees C. The enzyme is stable between pH 6 and 8, with pH 8 being the pH-optimum of both the lyase and the ligase reaction. Potassium phosphate and Tris were identified as optimal reaction buffers and the addition of 20 vol% DMSO is useful to enhance both the solubility of aromatic substrates and products and the stability of BAL. The initial broad product range of BAL-catalyzed reactions has been enlarged to include highly substituted hydroxybutyrophenones and aliphatic acyloins.     

Gluconeogenesis and P-enolpyruvate carboxykinase in liver and kidney of long-term fasted quails

The activity of cytoplasmic and mitochondrial phosphoenolpyruvate carboxykinase (PEPCK) in kidney and liver, and in vivo gluconeogenic activity, were determined during different phases of prolonged fasting in quails. The fasting-induced changes in the activity of kidney cytoplasmic PEPCK were positively correlated with the changes in gluconeogenesis. Both activities increased at the initial phase (I) of fasting to levels 65% to 100% higher than fed values, and decreased during the protein-sparing period (phase II), although remaining higher than in fed birds. At the catabolic final phase (III) both kidney cytoplasmic PEPCK activity and gluconeogenesis increased markedly, attaining levels 115% to 150% higher than fed values. The activity of liver cytoplasmic PEPCK, present in appreciable amounts in quails, did not change during phases I and II of fasting, but increased to levels 60% higher than fed values at the final phase (III). Plasma glucose levels at phase III did not differ significantly from those at phases I and II. In both kidney and liver the activity of the mitochondrial PEPCK was not significantly affected by fasting. The data suggest that the kidney cytoplasmic PEPCK is the main enzyme responsible for gluconeogenesis adjustments during food deprivation in quails, and that this function is complemented at the final phase by enzyme present in liver cytosol. Accepted: 14 April 2000     

Hydrophobic interactions of brush border alkaline phosphatases: the role of phosphatidyl inositol

Tissue-specific (intestinal) and tissue-nonspecific (kidney) rat alkaline phosphatases are released from their respective brush border membranes by different enzymes. To elucidate the mechanism underlying their membrane attachment, we tested the ability of these enzymes to partition into lipid or aqueous phases both before and after treatment with phospholipases and proteases. Interaction with Triton X-114 micelles was eliminated or decreased by treatment of intestinal enzyme with phospholipase A2 or papain, while only phosphatidylinositol (PI)-specific phospholipase C (PIPLC) and subtilisin were effective with the kidney enzyme. Binding to octyl Sepharose for the intestinal enzyme was decreased by phospholipase A2 more than by PIPLC, whereas the reverse was true for the kidney enzyme. Treatment with phospholipases decreased the apparent mass of the phosphatases by 50-80 kDa, presumably due to loss of bound lipid and detergent. PIPLC treatment of the kidney, but not the intestinal enzyme, prevented binding of the phosphatase to phospholipid vesicles. These results show that both enzymes are bound to respective membranes by hydrophobic anchor peptides to which phospholipids are bound. However, their sensitivity to phospholipases is different. The data are consistent with the hypothesis that, in the kidney enzyme, the PI is bound covalently, while with the intestinal enzyme, binding of PI appears to be tight but not covalent.     

Immobilized α-chymotrypsin: Pore diffusion control owing to pH gradients in the catalyst particles

The fast enzymatic hydrolysis of D ,L -phenylalanine methylester (DLE) to L -phenylalanine (LA) and D -phenylalanine methylester (DE) with immobilized α-chymotrypsin was chosen as a model reaction. Under the experimental conditions applied in the present investigations the pore diffusion is the rate-limiting step of this reaction owing to the pH gradient in the particles. The effectiveness of the catalyst is experimentally determined as a function of the substrate concentration based on measurements of the enzyme protein content of native and immobilized enzyme. The proteolytic reaction is theoretically treated by also using a pore diffusion model which takes into account the concentration gradients of substrate and product, pH- and enzyme activity profiles, as well as the change of buffer capacity of the solute in the catalyst particles. The model parameters were experimentally determined for the investigated system. It can be shown that conditions are possible for which the effectiveness of the catalyst exceeds unity.     

Changes in activity and mRNA levels of poly(ADP-ribose) polymerase during rat liver regeneration

ADP-ribosylation of nuclear proteins, catalysed by the enzyme poly(ADP-ribose) polymerase, is involved in the regulation of different cellular processes of DNA metabolism. To further clarify the role of the enzyme during proliferating activity of mammalian cells, we have studied the control of gene expression in regenerating rat liver. The changes in activity and mRNA levels were analysed during the early and late phases of the compensatory model. When enzyme activity was measured in isolated liver nuclei obtained at different times after hepatectomy, two different phases were observed: an early wave occurring before the onset of DNA synthesis, and a second one, starting several hours after the onset of DNA synthesis and returning to control values at later times. The evaluation of the enzymatic level in nuclear extracts and by activity gel analysis showed a more gradual increase starting 1 day after hepatectomy, in concomitance with the peak of DNA synthesis. By using a specific murine cDNA probe, a significant enhancement of mRNA levels for poly(ADP-ribose) polymerase was observed during liver regeneration, slightly preceding the onset of DNA synthesis. The results obtained show that changes in poly(ADP-ribose) polymerase activity, during liver regeneration, are associated both to early events preceding the increase in DNA synthesis and to later phases of the cell proliferation process.     

Conformational Change in the Active Site of Streptococcal Unsaturated Glucuronyl Hydrolase Through Site-Directed Mutagenesis at Asp-115

Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst.     

2,4,6-trinitrotoluene reduction by an Fe-only hydrogenase in Clostridium acetobutylicum

The role of hydrogenase on the reduction of 2,4,6-trinitrotoluene (TNT) in Clostridium acetobutylicum was evaluated. An Fe-only hydrogenase was isolated and identified by using TNT reduction activity as the selection basis. The formation of hydroxylamino intermediates by the purified enzyme corresponded to expected products for this reaction, and saturation kinetics were determined with a K(m) of 152 micro M. Comparisons between the wild type and a mutant strain lacking the region encoding an alternative Fe-Ni hydrogenase determined that Fe-Ni hydrogenase activity did not significantly contribute to TNT reduction. Hydrogenase expression levels were altered in various strains, allowing study of the role of the enzyme in TNT reduction rates. The level of hydrogenase activity in a cell system correlated (R(2) = 0.89) with the organism's ability to reduce TNT. A strain that overexpressed the hydrogenase activity resulted in maintained TNT reduction during late growth phases, which it is not typically observed in wild type strains. Strains exhibiting underexpression of hydrogenase produced slower TNT rates of reduction correlating with the determined level of expression. The isolated Fe-only hydrogenase is the primary catalyst for reducing TNT nitro substituents to the corresponding hydroxylamines in C. acetobutylicum in whole-cell systems. A mechanism for the reaction is proposed. Due to the prevalence of hydrogenase in soil microbes, this research may enhance the understanding of nitroaromatic compound transformation by common microbial communities.