Inversion of sucrose with β- -fructofuranosidase (invertase) immobilized on bead DEAHP-cellulose: Batch process

The inversion of sucrose with β- -fructofuranosidase (EC 3.2.1.26) immobilized by an ionic bond on bead cellulose containing weak basic N,N-diethylamino-2-hydroxypropyl groups has been investigated. The immobilized enzyme is strongly bound at an ionic strength up to 0.1 M in the pH range 3–6. The amount adsorbed is proportional to porosity and to the exchange capacity of the ion exchange cellulose, reaching values up to 200 mg/g dry carrier, with an activity in 10% sucrose solution at 30°C, pH 5, >8000 μmol min⁻¹ g⁻¹. The inversion of sucrose with immobilized β- -fructofuranosidase was carried out in a stirred reactor. The dependence of activity on pH (3–7), temperature (0–70°C) and concentration of the substrate (2–64 wt%) were determined, and the inversion was compared with that obtained using non-immobilized enzyme under similar conditions. The rate of inversion at low substrate concentration (2–19 wt%) was described by Michaelis-Menten kinetics.     

Generalized rate equation for single-substrate enzyme catalyzed reactions

The most widely used rate expression for single-substrate enzyme catalyzed reactions, namely the Michaelis-Menten kinetics is based upon the assumption that enzyme concentration is in excess of the substrate in the medium and the rate is mainly limited by the substrate concentration according to saturation kinetics. However, this is only a special case and the actual rate expression varies depending on the initial enzyme/substrate ratio (E₀/S₀). When the substrate concentration exceeds the enzyme concentration the limitation is due to low enzyme concentration and the rate increases with the enzyme concentration according to saturation kinetics. The maximum rate is obtained when the initial concentrations of the enzyme and the substrate are equal. A generalized rate equation was developed in this study and special cases were discussed for enzyme catalyzed reactions.     

Inversion of sucrose with β-d-fructofuranosidase (invertase) immobilized on bead DEAHP-cellulose: Batch process

The inversion of sucrose with β-d-fructofuranosidase (EC 3.2.1.26) immobilized by an ionic bond on bead cellulose containing weak basic N,N-diethylamino-2-hydroxypropyl groups has been investigated. The immobilized enzyme is strongly bound at an ionic strength up to 0.1 M in the pH range 3–6. The amount adsorbed is proportional to porosity and to the exchange capacity of the ion exchange cellulose, reaching values up to 200 mg/g dry carrier, with an activity in 10% sucrose solution at 30°C, pH 5, >8000 μmol min^?1 g^?1. The inversion of sucrose with immobilized β-d-fructofuranosidase was carried out in a stirred reactor. The dependence of activity on pH (3–7), temperature (0–70°C) and concentration of the substrate (2–64 wt%) were determined, and the inversion was compared with that obtained using non-immobilized enzyme under similar conditions. The rate of inversion at low substrate concentration (2–19 wt%) was described by Michaelis-Menten kinetics.     

Improvement of the indolo-{alpha}-pyrone fluorescence method for quantitative determination of endogenous indole-3-acetic acid in lettuce seedlings

The indolo--pyrone fluorescence method was modified to measurethe endogenous amount of indole-3-acetic acid (IAA) in lettuceseedlings, because impurities in lettuce extracts made the indolo--pyronesolution turbid, if the reaction of the extracts with aceticanhydride to give indolo--pyrone was disrupted with 3 ml distilledwater. This turbid solution caused light scattering, resultingin overestimating the IAA amount in lettuce seedlings. We could,however, obtain a clear indolo--pyrone solution by disruptingthe reaction with 0.2 ml distilled water and 2.8 ml acetic acidin place of 3 ml distilled water. We examined the reliabilityof the new method and found that the use of acetic acid substantiallyslowed down the rate of indolo--pyrone breakdown and did notdecrease the sensitivity of the method. The effect of gibberellicacid on the endogenous amount of IAA in lettuce seedlings wasalso examined. ¹ One of the authors (S. K.) received for this work a grantfrom Osaka City University to study abroad. (Received January 11, 1977; )     

Strategy for the simulation of batch reactors when the enzyme-catalyzed reaction is accompanied by enzyme deactivation

The balance equations pertaining to the modelling of batch reactors performing an enzyme-catalyzed reaction in the presence of enzyme deactivation are developed. The functional form of the solution for the general situation where both the rate of the enzyme-catalyzed reaction and the rate of enzyme deactivation are dependent on the substrate concentration is obtained, as well as the condition that applies if a maximum conversion of substrate is sought. Finally, two examples of practical interest are explored to emphasize the usefulness of the analysis presented.List of Symbols C _E mol/m³ concentration of active enzyme - C _E,O mol/m³ initial concentration of active enzyme - C _S mol/m³ concentration of substrate - C _S,O mol/m³ initial concentration of substrate - C _S,min mol/m³ minimum value for the concentration of substrate - k 1/s first order rate constant associated with conversion of enzyme/substrate complex into product - k ₁ 1/s first order deactivation constant of enzyme (or free enzyme) - k ₂ 1/s first order deactivation constant of enzyme in enzyme/substrate complex form - K _m mol/m³ Michaelis-Menten constant - p mol/(m³s) time derivative of C _S - q mol/m³ auxiliary variable - t s time elapsed after reactor startup Greek Symbols 1/s univariate function expressing the dependence of the rate of enzyme deactivation on C _S - mol/m³ dummy variable of integration - mol/m³ dummy variable of integration - 1/s univariate function expressing the dependence of the rate of substrate depletion on C _S - m³/(mol s) derivative of with respect to C _S     

Osmoregulation inDunaliella. Catalysis of the glycerol-3-phosphate dehydrogenase reaction in a chloroplast-enriched fraction ofDunaliella tertiolecta

The glycerol-3-phosphate dehydrogenase (NAD-dependent) reaction was studied in a chloroplast-enriched fraction fromDunaliella tertiolecta. The reaction has widely separated pH optima for each direction. Reduction of dihydroxyacetone phosphate proceeded with Michaelis-Menten kinetics but sigmoidal double reciprocal plots were obtained with glycerol phosphate as variable substrate. NADP served as an alternative substrate but it was somewhat less effective than NAD. The reaction was inhibited by inorganic orthophosphate and by adenine nucleotides in a manner indicative of anion inhibition. Inhibition by inorganic phosphate was competitive with DHAP and possibly also with NADH. The enzyme was activated by Na⁺ at concentrations below 200 m and inhibited at higher concentrations, the region of maximum activation being affected by substrate concentration. Inhibition by Na⁺, present as a counterion of the substrate, was evidently responsible for apparent substrate inhibition by glycerol phosphate. Several important differences were apparent between the reaction in the unfractionated chloroplast-enriched fraction and the properties of a partly purified enzyme described by Haus and Wegmann (1984a, b).In toto, the results suggest that the regulatory potential of the reaction is probably more relevant to homeostatic control of glycerol content under steady state conditions than to controlling response to water stress.Abbreviations DHAP Dihydroxyacetone phosphate - CHES 2-(N-cyclohexylamino)ethanesulphonic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid     

Effect of alpha2 macroglobulin on some kinetic parameters of trypsin.

1. Complex formation of trypsin with alpha2 macroglobulin results in marked changes of the Michaelis-Menten constant, pH optimum and sensitivity to ionic strength in a system using N-carbobenzoxy-glycylglycyl-L-arginine-2-naphthylamide as substrate. 2. In contrasts to the inhibition (50%) observed when alpha2 macroglobulin-bound trypsin is assayed under conditions optimal for the free enzyme, there is minimal reduction of activity when determinations are performed at a substrate concentration and pH optimal for the bound enzyme. 3. The changes in substrate concentration and ionic environment required for maximum activity of alpha2 macroglobulin-bound trypsin are similar to those observed with enzymes embedded in polyelectrolyte matrices and may reflect alterations in the microenvironment of the enzyme resulting from conformational changes of the macromolecule during interaction with trypsin. 4. Enzymatic activity of trypsin towards casein is greatly reduced by alpha2 macroglobulin, even under assay conditions optimal for the bound enzyme, confirming previous findings that access to the active center for high-molecular weight substrates is sterically hindered by alpha2 macroglobulin.     

Cost minimization in the predesign of an enzymatic CSTR: an overall approach

The balance equations pertaining to the modelling of a CSTR performing an enzyme-catalyzed reaction in the presence of enzyme deactivation are developed. Combination of heuristic correlations for the size-dependent cost of equipment and the purification-dependent cost of recovery of product with the mass balances was used as a basis for the development of expressions relating a (suitably defined) dimensionless economic parameter with the optimal outlet substrate concentration under the assumption that overall production costs per unit mass of product were to be minimized. The situation of Michaelis-Menten kinetics for the substrate depletion and first order kinetics for the deactivation of enzyme (considering that the free enzyme and the enzyme in the enzyme/substrate complex deactivate at different rates) was explored, and plots for several values of the parameters germane to the analysis are included.List of Symbols C _E mol m^–3 concentration of active enzyme - C _E,0 mol m^–3 initial concentration of active enzyme - C _p mol m^–3 concentration of product of interest - C _s mol m^–3 concentration of substrate - C _s,0 mol m^–3 initial concentration of substrate - I $ capital cost of equipment - k _d s^–1 deactivation constant of free enzyme - k _d s^–1 deactivation constant of enzyme in enzyme/substrate complex - K _m mol m^–3 Michaelis-Menten constant - K _m dimensionless counterpart of K _m - k _r s^–1 rate constant associated with conversion of enzyme/substrate complex into product - M _w kg mol^–1 molecular weight of product of interest - P $ kg^–1 cost of recovery of product of interest in pure form - Q m³s^–1 volumetric flow rate - V m³ volume of reactor - X $ kg^–1 global manufacture cost of product of interest in pure form - X dimensionless counterpart of X Greek Symbols ₁ $ m^–1.8 constant - ₂ $ m^–3 constant - t s useful life of CSTR - ₀ ratio of initial concentrations of enzyme and substrate - ratio of deactivation constant of free enzyme to rate constant of depletion of substrate - ratio of deactivation constants - univariate function expressing the dependence of the rate of enzyme deactivation on C _S - univariate function expressing the dependence of the rate of substrate depletion on C _S - dimensionless economic parameter     

Studies on a testosterone glucuronyltransferase from the cytosol fraction of human liver

An enzyme that conjugates the 16alpha-hydroxyl group of oestriol with glucuronic acid was found in the cytosol fraction of human liver. The enzymic activity could not be sedimented when the cytosol fraction was centrifuged at 158000g(av.) for 120min. The oestriol 16alpha-glucuronyltransferase was purified 100-fold by 0-30% saturation of the cytosol fraction with ammonium sulphate followed by filtration of the precipitate through Sephadex G-200. The activity was eluted at the void volume. The product of the reaction, oestriol 16alpha-monoglucuronide, was identified by paper chromatography and by crystallization of radioactive product to constant specific radioactivity. The optimum temperature was 37 degrees C, and the activation energy was calculated to be 11.1kcal/mol. The apparent Michaelis-Menten constants for oestriol and UDP-glucuronic acid were 13.3 and 100mum respectively. Cu(2+), Zn(2+) and Hg(2+) inhibited, whereas Mg(2+), Mn(2+) and Fe(2+) stimulated the enzyme. Substrate-specificity studies indicated that the amount of oestradiol-17beta, oestradiol-17alpha and oestrone conjugated was not more than about 5% of that found for oestriol. Oestriol 16alpha-monoglucuronide, a product of the reaction, did not inhibit the 16alpha-oestriol glucuronyltransferase; in contrast, UDP, another product of the reaction, inhibited the enzyme competitively with respect to UDP-glucuronic acid as the substrate, and non-competitively with respect to oestriol as the substrate. ATP and UDP-N-acetylglucosamine did not affect the oestriol 16alpha-glucuronyltransferase. 17-Epioestriol acted as a competitive inhibitor and 16-epioestriol as a non-competitive inhibitor of the glucuronidation of oestriol. 5alpha-Pregnane-3alpha,20alpha-diol also inhibited the enzyme non-competitively. It is most likely that the oestriol 16alpha-glucuronyltransferase described here is bound to the membranes of the endoplasmic reticulum.     

Purification and characterization of {alpha}-L-fucosidase from Chinese hamster ovary cell culture supernatant

In this study, -L-fiicosidase from Chinese hamster ovary (CHO)cell culture supernatant was purified 11 200-fold to apparenthomogeneity to assess the rate of fucose hydrolysis from oligosaccharideand glycoprotein substrates. The fuco-sidase migrated as a singleband of 51 kDa on SDS-PAGE and is a glycoprotein, as determinedby retention on con-canavalin A-Sepharose, and by lectin blottingwith concana-valin A. Hydrolysis of the artificial substrate4-methyl-umbelliferyl--L-fucoside (4MU-Fuc) followed simpleMichaelis-Menten kinetics, and was competitively inhibited byfree fucose and by two known fucosidase inhibitors, fucosylamineand deoxyfuconojirimycin. Hydrolysis of fucose from oligosaccharidesincluding 2-fucosyllactose, 3-fucosyllactose, Fuca(l,6)GlcNAcand pooled gpl20 oligosaccharides with the Fuca(l,6)GlcNAc linkagealso followed simple Michaelis-Menten kinetics. However, activitytoward 4MU-Fuc was optimal near pH 7, while activities towardthe oligosaccharide substrates were optimal near pH 5. No fucosewas released from the recombinant CHO cell-produced glycoproteinsgpl20 or soluble CD4 with the Fuca(l,6)GlcNAc linkage, or fromhuman serum a,-acid glycoprotein with the Fuca(l,3)GlcNAc linkage.Enzymatic removal of sialic acid and galactose from gpl20 oligosaccharidesdid not alter the susceptibility of gpl20 to fucosidase attack.These data suggest that released CHO cell fucosidase does notcontribute to the heterogeneity of fuco-sylation that has beenobserved in CHO cell culture-produced glycoproteins. Chinese hamster ovary cells extracellular flucosidase mammalian substrate specificity     

Studies of mammalian glucoside conjugation

The mammalian glucoside-conjugation pathway was studied by using p-nitrophenol as the model substrate and mouse liver microsomal preparations as the source of enzyme. The microsomal preparations supplemented with UDP-glucose glucosylated p-nitrophenol; p-nitrophenyl glucoside was identified by chromatography in six solvent systems. The unsolubilized glucosyltransferase of fresh microsomal preparations did not follow the usual Michaelis-Menten kinetics and was easily inhibited by many steroids. All the steroids tested inhibited glucosylation of p-nitrophenol to a greater degree than glucuronidation of p-nitrophenol when assayed in the same microsomal preparations. The steroids inhibited glucosylation with the following decreasing effectiveness: pregnan-3alpha-ol-20beta-one (3alpha-hydroxypregnan-20-beta-one)>oestradiol-17beta 3-methyl ether>oestradiol-17beta>oestriol>pregnane-3alpha,20beta-diol>oestrone. Pregnan-3alpha-ol-20beta-one, pregnane-3alpha,20beta-diol and oestrone had negligible effect on glucuronidation.     

The three-dimensional structure of a glutathione S-transferase from the mu gene class. Structural analysis of the binary complex of isoenzyme 3-3 and glutathione at 2.2-A resolution.

The crystal structure of a mu class glutathione S-transferase (EC 2.5.1.18) from rat liver (isoenzyme 3-3) in complex with the physiological substrate glutathione (GSH) has been solved at 2.2-A resolution by multiple isomorphous replacement methods. The enzyme crystallized in the monoclinic space group C2 with unit cell dimensions of a = 87.98 A, b = 69.41 A, c = 81.34 A, and beta = 106.07 degrees. Oligonucleotide-directed site-specific mutagenesis played an important role in the solution of the structure in that the cysteine mutants C86S, C114S, and C173S were used to help locate the positions of mercuric ion sites in nonisomorphous derivatives with ethylmercuric phosphate and to align the sequence with the model derived from MIR phases. A complete model for the protein was not obtained until part of the solvent structure was interpreted. The dimer in the asymmetric unit refined to a crystallographic R = 0.171 for 19,298 data and I > or = 1.5 sigma (I). The final model consists of 4150 atoms, including all non-hydrogen atoms of 434 amino acid residues, two GSH molecules, and oxygen atoms of 474 water molecules. The dimeric enzyme is globular in shape with dimensions of 53 x 62 x 56 A. Crystal contacts are primarily responsible for conformational differences between the two subunits which are related by a noncrystallographic 2-fold axis. The structure of the type 3 subunit can be divided into two domains separated by a short linker, a smaller alpha/beta domain (domain I, residues 1-82), and a larger alpha domain (domain II, residues 90-217). Domain I contains four beta-strands which form a central mixed beta-sheet and three alpha-helices which are arranged in a beta alpha beta alpha beta beta alpha motif. Domain II is composed of five alpha-helices. Domain I can be considered the glutathione binding domain, while domain II seems to be primarily responsible for xenobiotic substrate binding. The active site is located in a deep (19-A) cavity which is composed of three relatively mobile structural elements: the long loop (residues 33-42) of domain I, the alpha 4/alpha 5 helix-turn-helix segment, and the C-terminal tail. GSH is bound at the active site in an extended conformation at one end of the beta-sheet of domain I with its backbone facing the cavity and the sulfur pointing toward the subunit to which it is bound.(ABSTRACT TRUNCATED AT 400 WORDS)     

Maximum likelihood estimation of the heterogeneity of substitution rate among nucleotide sites

This paper presents a maximum likelihood approach to estimating the variation of substitution rate among nucleotide sites. We assume that the rate varies among sites according to an invariant+gamma distribution, which has two parameters: the gamma parameter alpha and the proportion of invariable sites theta. Theoretical treatments on three, four, and five sequences have been conducted, and computer program have been developed. It is shown that rho = (1 + theta alpha)/(1 + alpha) is a good measure for the rate heterogeneity among sites. Extensive simulations show that (1) if the proportion of invariable sites is negligible, i.e., theta = 0, the gamma parameter alpha can be satisfactorily estimated, even with three sequences; (2) if the proportion of invariable sites is not negligible, the heterogeneity rho can still be suitably estimated with four or more sequences; and (3) the distances estimated by the proposed method are almost unbiased and are robust against violation of the assumption of the invariant + gamma distribution.      

Enzyme kinetics at high enzyme concentration

We re-visit previous analyses of the classical Michaelis-Menten substrate-enzyme reaction and, with the aid of the reverse quasi-steady-state assumption, we challenge the approximation d[C]/dt ≈ 0 for the basic enzyme reaction at high enzyme concentration. For the first time, an approximate solution for the concentrations of the reactants uniformly valid in time is reported. Numerical simulations are presented to verify this solution. We show that an analytical approximation can be found for the reactants for each initial condition using the appropriate quasi-steady-state assumption. An advantage of the present formalism is that it provides a new procedure for fitting experimental data to determine reaction constants. Finally, a new necessary criterion is found that ensures the validity of the reverse quasi-steady-state assumption. This is verified numerically.     

Activity and kinetic characteristics of glutathione reductase in vitro in reverse micellar waterpool

The enzyme activity of glutathione reductase (NAD(P)H:oxidized-glutathione oxidoreductase, EC 1.6.4.2) incorporated in CTAB/H2O/CHCl3-isooctane (1:1, v/v) reverse micelles has been investigated. Enzyme follows the Michaelis-Menten kinetics within a specified concentration range. Effects of pH, waterpool (W0), and surfactant concentration on the activity of glutathione reductase have been studied in detail. Optimum pH for the maximum enzyme activity was found to be dependent on the size of the waterpool. Further, a substrate inhibition was observed when concentration of one of the substrates was present in large excess over the other substrate. Km values for the substrate, oxidized glutathione (GSSG) and NADPH in CTAB/H2O/CHCl3-isooctane (1:1, v/v) were determined at W0 values of 14.4, 20.0, 25.5 and 29.7, at pH 8.0. These values are close to those obtained in aqueous solution, whereas the kcat values vary with W0 values of 8.8 to 32.3. Studies on the storage stability in the reverse micelle at W0 29.7 and pH 8.0 showed that glutathione reductase retained about 80% of its activity even after a month. The enzyme showed a higher stability at high waterpool. Oxidized glutathione (GSSG) provides protection to glutathione reductase against denaturation on storage in reverse micellar solution. Apparently, the enzyme is able to acquire a suitable native conformation at waterpool 29.7 and pH 8.0 and thereby exhibits an activity and stability inside the micellar cavity that are almost equivalent to that in aqueous solution.     

Lipase kinetics in organic-water solvent with amphipathic substrate for chiral reaction

Lipase from Pseudomonas cepacia was used for asymmetric hydrolysis of the substrate (+/-)1-chloro-2-acetoxy-3-(1-naphthyloxy)-propane, which is a precursor for (S)-(-)-beta-blocker synthesis. Because this substrate is insoluble in water and partially soluble in hydrophobic solvents such as hexane and octane, a mixture of hydrophilic organic solvents and aqueous buffer was used to study the initial reaction rates. Because of the amphipathic nature of the substrate, it can remain in three different forms: (1) monomeric (solution); (2) micellar; and (3) emulsion, depending on the acetone and substrate concentrations in the medium. This behavior is presented in a phase diagram. The enzyme was found to be active with micelle as well as emulsion form of the substrate, whereas it showed negligible activity with the monomeric form. Michaelis-Menten constants were determined experimentally for the emulsion and micellar part of the substrate. The initial rate of hydrolysis (v(0)) goes through a maximum with respect to the acetone content of the mixture. It is due to the combined effect of various factors occurring simultaneously with the increase in acetone content in the solvent. These phenomena are discussed based on the interfacial activation of lipase, deactivation of the enzyme at very high acetone concentration, and increase in critical micelle concentration (CMC) and critical emulsion concentration (CEC) with the increase in acetone content in the solvent. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 399-407, 1997.     

A graphical method of determining the Michaelis-Menten constant free of external mass transfer resistance for immobilized enzymes in a packed bed reactor

Summary A graphical method of determining the Michaelis-Menten constant free of the external mass transfer resistance for a packed bed immobilized enzyme system was illustrated with examples from 3 different enzyme reactions. The intercept at the ordinate obtained by the straight line extrapolation of data points in the plot of apparent K_m value vs. the reciprocal of superficial velocity in column allowed an easy calculation of K_m free of external mass transfer resistance. An asymptotic value of apparent K_m value at infinite zero superficial velocity was ascribed to the fact that the mass transfer coefficient k_L, approached a definite value at this condition.Nomenclature K_m Michaelis-Menten constant, M/L³ - K_m' K_m free of external mass transfer resistance in a given ionic strength, M/L³ - Km" apparent K_m with external mass transfer resistance, M/L³ - S substrate concentration, M/L³ - S_o initial substrate concentration, M/L³ - k₂ rate constant, t^-1 - E enzyme concentration in support, M/L³ - void volume per unit volume of reactor, dimensionless - u superficial velocity of substrate, L/t - K_L mass transfer coefficient in liquid film, L/t - a external surface area of support per unit volume of reactor, L^-1 - ratio of average channeling length to particle diameter, dimensionless - d_p diameter of support particle, L - X fractional conversion of substrate, dimensionless - H partition coefficient, dimensionless - k a constant, 3 k₂E(1-)d_p/4 - T space time, t - N molecular flux, M/L²t - r radius of immobilized enzyme particle, L     

Kinetics of Phenylalanine Ammonia-Lyase and the Effect of L-{alpha}-aminooxy-{beta}-phenylpropionic Acid on Enzyme Activity and Radicle Growth in Germinating Lettuce Seeds

The effects of different concentrations of L--aminooxy-ß-phenyIpropionicacid (AOPP), an analog of L-phenylalanine, on the activity ofphenylalanine ammonia-lyase (PAL, EC 4.3.1.5 [EC] ) and the growthof radicles in 24 h old germinating lettuce (Lactuca salivaL.) seeds were investigated. AOPP causes a significant inhibitionof PAL activity in the seeds (85% inhibition at 10⁴ M). It alsocauses a stimulation of radicle growth at that concentration.The results show that the inhibition of PAL by AOPP may be dueto an irreversible binding of the inhibitor to the enzyme leadingto its inactivation. AOPP also inhibits ethylene biosynthesisin germinating lettuce seeds which could probably explain thestimulation of radicle growth in these seeds. The enzyme shows typical Michaelis-Menten kinetics. The K_m forL-phenylalanine is 4.2 x 10⁵ M. The enzyme does not show anytyrosine ammonia-lyase activity. Various substrate analogs suchas D-phenylalanine, p-fluorophenylalanine, ß-phenyllacticacid, tryptophan and the product of the enzyme reaction, trans-cinnamicacid, inhibit the enzyme competitively. A number of intermediatesand endproducts of the phenylpropanpid pathway, except chlorogenicacid, do not show any inhibition. ¹Scientific contribution number 1423 from the New HampshireAgricultural Experiment Station. (Received May 9, 1986; Accepted September 8, 1986)     

A Model for Single-Substrate Trimolecular Enzymatic Kinetics

We developed a kinetic model for a single-substrate trimolecular enzymatic system, where a receptor binds and stretches a substrate to expose its cleavage site, allowing an enzyme to bind and cleave it into product. We demonstrated that the general kinetics of the trimolecular enzymatic system is more complex than the Michaelis-Menten kinetics. Under a limiting condition when the enzyme-substrate binding is in fast equilibrium, the enzymatic kinetics of the trimolecular system reduces to the Michaelis-Menten kinetics. In another limiting case when the receptor dissociates negligibly slowly from the substrate, the trimolecular system is simplified to a bimolecular system, which follows the Michaelis-Menten equation if and only if there is no enzyme-substrate complex initially. We applied this model to a particular trimolecular system important to hemostasis and thrombosis, consisting of von Willebrand factor (substrate), platelet glycoprotein Ibα (receptor), and ADAMTS13 (enzyme). Using parameters from independent experiments, our model successfully predicted published data from two single-molecule experiments and fitted/predicted published data from an ensemble experiment.