Two-substrate Enzyme Research Articles

Two-substrate enzyme engineering using small libraries that combine the substrate preferences from two different variant lineages

Improving the range of substrates accepted by enzymes with high catalytic activity remains an important goal for the industrialisation of biocatalysis. Many enzymes catalyse two-substrate reactions which increases the complexity in engineering them for the synthesis of alternative products. Often mutations are found independently that can improve the acceptance of alternatives to each of the two substrates. Ideally, we would be able to combine mutations identified for each of the two alternative substrates, and so reprogramme new enzyme variants that synthesise specific products from their respective two-substrate combinations. However, as we have previously observed for E. coli transketolase, the mutations that improved activity towards aromatic acceptor aldehydes, did not successfully recombine with mutations that switched the donor substrate to pyruvate. This likely results from several active site residues having multiple roles that can affect both of the substrates, as well as structural interactions between the mutations themselves. Here, we have designed small libraries, including both natural and non-natural amino acids, based on the previous mutational sites that impact on acceptance of the two substrates, to achieve up to 630× increases in kcat for the reaction with 3-formylbenzoic acid (3-FBA) and pyruvate. Computational docking was able to determine how the mutations shaped the active site to improve the proximity of the 3-FBA substrate relative to the enamine-TPP intermediate, formed after the initial reaction with pyruvate. This work opens the way for small libraries to rapidly reprogramme enzyme active sites in a plug and play approach to catalyse new combinations of two-substrate reactions.

Modulation of enzyme activity at nano-bio interface: A case study with acetylcholinesterase and citrate synthase adsorbed on colloidal metal nanoparticles

The effects of colloidal gold and silver nanoparticles (Au and Ag NP, respectively) were studied on the activity of acetylcholinesterase (AChE), the key enzyme in the pathology of Alzheimer's Disease (AD) and citrate synthase (CS), the mitochondrial enzyme responsible for oxidative metabolism. Being established as a primary basis of neurodegeneration, mitochondrial dysfunction connects both these two enzymes with AD. The in-vitro enzymatic activities are modulated strongly in presence of NPs. The maximum AChE catalytic rate (Vmax) increases by 30% in presence of NP systems. The Michalis-Menten constant (Km) remains unaltered in presence of AuNP; while, ~61.23% increase is noted in presence of AgNP. For the two-substrate enzyme CS, NPs were found to affect only the pathway involving oxaloacetate (OAA) as substrate. In this case, Vmax and Km increased by 21.80% and 46.60%, respectively for AuNP; while the corresponding increase were 18.20% and 33.33%, respectively for AgNP. The binding of AChE on AgNP surface occurred through random contact within very short time scale, followed by slower interaction and contact optimization within tens of nanoseconds. A series of cationic surfactants exhibited optimistic results in restoring the intrinsic activity for both the enzymes. The results highlight neurotoxicity of metal NPs in certain cases like AD and emphasize their use in pharmacological milieu only with suitable accompanying delivery medium like surfactants.

Full-time dynamics of batch-wise enzymatic cycling system composed of two kinds of dehydrogenase mediated by NAD(P)H for mass production of chiral hydroxyl compounds

Enzymatic cycling system (coupled dehydrogenase-catalyzed biosystem being composed of two elementary enzymatic reactions mediated by NAD(P)H+ NAD(P)+) is industrially attractive for reducing prochiral carbonyl compounds to the corresponding chiral hydroxyl compounds. The reaction rate equation of the batch-wise biosystem was generally derived by ordered Bi Bi mechanism of two-substrate enzyme reaction on several reasonable assumptions. The rate equations of the batch-wise biosystem was generalized by transforming them into the dimensionless forms. The dimensionless forms were solved numerically. It was revealed that the batch-wise biosystem was generally made up of unique 3 phases, i.e., phases I, II and III. Phase I was very short transient so that the biosystem entered rapidly phase II. In phase II the consumption rate dynamically balanced with its formation rate so that the concentration of NAD(P)H was invariable with time (and hence NAD(P)+ concentration was, too). Phase III was substrate-exhausting phase, and the coenzyme concentration became finally only [NAD(P)+] or only [NAD(P)H] depending on the initial molar ratio of the prochiral carbonyl compound to the substrate of the coenzyme regeneration reaction ( [Formula: see text] ) > or <1.0. In phases I and II the numerically calculated values of state variables were very close to the analytical but approximate ones. Preferable initial conditions of the batch-wise enzymatic cycling system, i.e., the initial coenzyme species= NAD(P)+ and [Formula: see text] , were proposed. As the main assumption irreversibility of the two elemental enzymatic reactions was discussed. Validity of the proposed rate equations was mentioned.

Molecular system identification for enzyme directed evolution and design.

The rational design of chemical catalysts requires methods for the measurement of free energy differences in the catalytic mechanism for any given catalyst Hamiltonian. The scope of experimental learning algorithms that can be applied to catalyst design would also be expanded by the availability of such methods. Methods for catalyst characterization typically either estimate apparent kinetic parameters that do not necessarily correspond to free energy differences in the catalytic mechanism or measure individual free energy differences that are not sufficient for establishing the relationship between the potential energy surface and catalytic activity. Moreover, in order to enhance the duty cycle of catalyst design, statistically efficient methods for the estimation of the complete set of free energy differences relevant to the catalytic activity based on high-throughput measurements are preferred. In this paper, we present a theoretical and algorithmic system identification framework for the optimal estimation of free energy differences in solution phase catalysts, with a focus on one- and two-substrate enzymes. This framework, which can be automated using programmable logic, prescribes a choice of feasible experimental measurements and manipulated input variables that identify the complete set of free energy differences relevant to the catalytic activity and minimize the uncertainty in these free energy estimates for each successive Hamiltonian design. The framework also employs decision-theoretic logic to determine when model reduction can be applied to improve the duty cycle of high-throughput catalyst design. Automation of the algorithm using fluidic control systems is proposed, and applications of the framework to the problem of enzyme design are discussed.

In‐capillary reactant mixing for monitoring glycerol kinase kinetics by CE

CE was used for the first time to study the two-substrate enzyme glycerol kinase. The capillary was used as a nanoreactor in which the enzyme and its two substrates glycerol and adenosine-5'-triphosphate were in-capillary mixed to realize the enzymatic assay. For kinetic parameters determination, reactants were injected (50 mbar × 5 s) as follows: (i) incubation buffer; (ii) adenosine-5'-triphosphate; (iii) enzyme, and (iv) glycerol. Enzymatic reaction was then initiated by mixing the reactants using electrophoretically mediated microanalysis (+20 kV for 6 s) followed by a zero-potential amplification step of 3 min. Finally, electrophoretic separation was performed; the product adenosine-5'-diphosphate was detected at 254 nm and quantified. For enzyme inhibition, an allosteric inhibitor fructose-1,6-bisphosphate plug was injected before the first substrate plug and +20 kV for 8 s was applied for reactant mixing. A simple, economic, and robust CE method was developed for monitoring glycerol kinase activity and inhibition. Only a few tens of nanoliters of reactants were used. The results compared well with those reported in literature. This study indicates, for the first time, that at least four reactant plugs can be in-capillary mixed using an electrophoretically mediated microanalysis approach.

Probing the Donor and Acceptor Substrate Specificity of the γ-Glutamyl Transpeptidase

γ-Glutamyl transpeptidase (GGT) is a two-substrate enzyme that plays a central role in glutathione metabolism and is a potential target for drug design. GGT catalyzes the cleavage of γ-glutamyl donor substrates and the transfer of the γ-glutamyl moiety to an amine of an acceptor substrate or water. Although structures of bacterial GGT have revealed details of the protein-ligand interactions at the donor site, the acceptor substrate site is relatively undefined. The recent identification of a species-specific acceptor site inhibitor, OU749, suggests that these inhibitors may be less toxic than glutamine analogues. Here we investigated the donor and acceptor substrate preferences of Bacillus anthracis GGT (CapD) and applied computational approaches in combination with kinetics to probe the structural basis of the enzyme's substrate and inhibitor binding specificities and compare them with human GGT. Site-directed mutagenesis studies showed that the R432A and R520S variants exhibited 6- and 95-fold decreases in hydrolase activity, respectively, and that their activity was not stimulated by the addition of the l-Cys acceptor substrate, suggesting an additional role in acceptor binding and/or catalysis of transpeptidation. Rat GGT (and presumably HuGGT) has strict stereospecificity for L-amino acid acceptor substrates, while CapD can utilize both L- and D-acceptor substrates comparably. Modeling and kinetic analysis suggest that R520 and R432 allow two alternate acceptor substrate binding modes for L- and D-acceptors. R432 is conserved in Francisella tularensis, Yersinia pestis, Burkholderia mallei, Helicobacter pylori and Escherichia coli, but not in human GGT. Docking and MD simulations point toward key residues that contribute to inhibitor and acceptor substrate binding, providing a guide to designing novel and specific GGT inhibitors.

Paraoxonase 1 (PON1) status and substrate hydrolysis

Paraoxonase 1 (PON1) hydrolyzes a number of organophosphorus (OP) compounds including insecticides and nerve agents. The in vivo efficacy of PON1 to protect against a specific OP exposure depends on the catalytic efficiency of hydrolysis. The Q192R polymorphism affects the catalytic efficiency of hydrolysis of some substrates and not others. While PON1 R192 hydrolyzes paraoxon approximately 9-times as efficiently as PON1 Q192, the efficiency is insufficient to provide in vivo protection against paraoxon/parathion exposure. The two PON1 192 alloforms have nearly equivalent but higher catalytic efficiencies for hydrolyzing diazoxon (DZO) and provide equivalent in vivo protection against DZO exposures. On the other hand, PON1 R192 is significantly more efficient in hydrolyzing chlorpyrifos oxon (CPO) than PON1 Q192 and provides better protection against CPO exposure. Thus, for some exposures it is only the level of plasma PON1 that is important, whereas for others it is both plasma level and the PON1 192 alloform(s) present in plasma that are important. In no case is the plasma level of PON1 unimportant, provided that the catalytic efficiency is sufficient to protect against the exposure. Two-substrate enzyme assay/analysis protocols that reveal both PON1 plasma levels and PON1 192 phenotype (QQ; QR; RR) are designed to optimize the separation of PON1 192 phenotypes; however, they have not been optimized for evaluating in vivo rates of OP detoxication. This study describes the adaptation of a non-OP, two-substrate determination of PON1 status to the conversion of the PON1 status data to physiologically relevant rates of DZO and CPO detoxication. Conversion factors were generated for rates of hydrolysis of different substrates.

Substrate Specificity, Localization, and Essential Role of the Glutathione Peroxidase-type Tryparedoxin Peroxidases in Trypanosoma brucei

Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three nearly identical cysteine homologues of the classical selenocysteine-containing glutathione peroxidases. Although one of the sequences, peroxidase III, carries both putative mitochondrial and glycosomal targeting signals, the proteins are detectable only in the cytosol and mitochondrion of mammalian bloodstream and insect procyclic T. brucei. The enzyme is a trypanothione/tryparedoxin peroxidase as are the 2 Cys-peroxiredoxins of the parasite. Hydrogen peroxide, thymine hydroperoxide, and linoleic acid hydroperoxide are reduced with second order rate constants of 8.7 x 10(4), 7.6 x 10(4), and 4 x 10(4) m(-1) s(-1), respectively, and represent probable physiological substrates. Phosphatidylcholine hydroperoxide is a very weak substrate and, in the absence of Triton X-100, even an inhibitor of the enzyme. The substrate preference clearly contrasts with that of the closely related T. cruzi enzyme, which reduces phosphatidylcholine hydroperoxides but not H(2)O(2). RNA interference causes severe growth defects in bloodstream and procyclic cells in accordance with the peroxidases being essential in both developmental stages. Thus, the cellular functions of the glutathione peroxidase-type enzymes cannot be taken over by the 2 Cys-peroxiredoxins that also occur in the cytosol and mitochondrion of the parasite.

Kinetic equivalence of the subunits of liver alcohol dehydrogenase.

1 The kinetics of two-substrate enzyme reactions proceeding by an ordered ternary-complex mechanism have been analyzed theoretically with respect to the precise magnitude of amplitudes of the two slowest transients governing such reactions under single-turnover conditions. Relationships derived have been applied to the liver alcohol dehydrogenase system for detailed interpretation of amplitudes of the biphasic absorbance changes associated with the enzymic reduction of aromatic aldehydes in catalytic-suicide experiments. 2 Amplitudes of the slow phase 328–330-nm absorbance changes occurring during the enzymic reduction of various para-substituted benzaldehydes by NADH or NAD2H correspond to 6–26% of the total absorbance change under conditions of substrate and coenzyme saturation. Saturation values of amplitudes determined in the present and previous investigations agree within 2 % of the total absorbance change with those calculated theoretically for an ordered mechanism of enzyme action at equivalent sites. In particular, amplitudes observed exhibit the expected sensitivity to electronic substituent and deuterium isotope effects. 3 Enzyme recycling, aldehyde binding, and alcohol desorption provide negligible contributions to the 328–330-nm absorbance changes observed during aldehyde reduction in the presence of 20 mM pyrazole. The maximum slow phase amplitude contribution provided by pyrazole binding corresponds to 5–6% of the total absorbance change. Additional slow phase absorbance changes persisting at saturating concentrations of coenzyme and substrate reflect incomplete accumulation of the enzymic intermediate formed through hydride transfer in the rapid reaction phase. This intermediate contains no detectable amounts (less than 2 %,) of NADH, but exhibits spectral properties indistinguishable from those of enzyme. NAD+. alcohol complexes examined. 4 It has been concluded in recent publications that an ordered ternary-complex mechanism involving equivalent sites is insufficient to account for the transient-state kinetics of liver alcohol dehydrogenase catalysis. These conclusions are shown to be based on an inadequate interpretation of the observations made. Results reported up to date provide no evidence whatsoever for the kinetic significance of subunit interactions leading to a non-equivalence of the enzymic sites. Subunit interactions resulting in a “half-site7rdquo; reactivity of the enzyme are definitely not at hand.

A kinetic model of the branch-point between the methionine and threonine biosynthesis pathways in Arabidopsis thaliana.

This work proposes a model of the metabolic branch-point between the methionine and threonine biosynthesis pathways in Arabidopsis thaliana which involves kinetic competition for phosphohomoserine between the allosteric enzyme threonine synthase and the two-substrate enzyme cystathionine gamma-synthase. Threonine synthase is activated by S-adenosylmethionine and inhibited by AMP. Cystathionine gamma-synthase condenses phosphohomoserine to cysteine via a ping-pong mechanism. Reactions are irreversible and inhibited by inorganic phosphate. The modelling procedure included an examination of the kinetic links, the determination of the operating conditions in chloroplasts and the establishment of a computer model using the enzyme rate equations. To test the model, the branch-point was reconstituted with purified enzymes. The computer model showed a partial agreement with the in vitro results. The model was subsequently improved and was then found consistent with flux partition in vitro and in vivo. Under near physiological conditions, S-adenosylmethionine, but not AMP, modulates the partition of a steady-state flux of phosphohomoserine. The computer model indicates a high sensitivity of cystathionine flux to enzyme and S-adenosylmethionine concentrations. Cystathionine flux is sensitive to modulation of threonine flux whereas the reverse is not true. The cystathionine gamma-synthase kinetic mechanism favours a low sensitivity of the fluxes to cysteine. Though sensitivity to inorganic phosphate is low, its concentration conditions the dynamics of the system. Threonine synthase and cystathionine gamma-synthase display similar kinetic efficiencies in the metabolic context considered and are first-order for the phosphohomoserine substrate. Under these conditions outflows are coordinated.

A strategy for the determination of enzyme kinetics using electrospray ionization with an ion trap mass spectrometer.

A simple and rapid means of enzyme kinetic analysis was achieved using electrospray ionization mass spectrometry and a one-point normalization factor. The model system used, glutathione S-transferase from porcine liver, is a two-substrate enzyme catalyzing the conjugation of glutathione with a variety of compounds containing an electrophilic center. An internal standard that is structurally similar to the product was added to the reaction quench solution, and a single-point normalization factor was used to determine the product concentration without the need of a calibration curve. Kinetic parameters, such as Km, Vmax and Ki (for thyroxine), obtained by electrospray mass spectrometry agreed with those obtained from traditional UV-vis spectroscopy, and competitive vs noncompetitive inhibition reactions could be delineated via mass spectrometry. These results suggest that our method can be applied to enzymatic processes in which spectrophotometric or spectrofluorometric assays are not feasible or when the relevant substrates do not incorporate chromophores or fluorophores. This new method is competitive with traditional UV assays in that it is facile and it involves very little analysis time.

Donor substrate specificity of recombinant human blood group A, B and hybrid A/B glycosyltransferases expressed in Escherichia coli.

The human blood group A and B glycosyltransferases catalyze the transfer of GalNAc and Gal, to the (O)H-precursor structure Fuc alpha (1-2)Gal beta-OR to form the blood group A and B antigens, respectively. Changing four amino acids (176, 235, 266 and 268) alters the specificity from an A to a B glycosyltransferase. A series of hybrid blood group A/B glycosyltransferases were produced by interchanging these four amino acids in synthetic genes coding for soluble forms of the enzymes and expressed in Escherichia coli. The purified hybrid glycosyltransferases were characterized by two-substrate enzyme kinetic analysis using both UDP-GalNAc and UDP-Gal donor substrates. The A and B glycosyltransferases were screened with other donor substrates and found to also utilize the unnatural donors UDP-GlcNAc and UDP-Glc, respectively. The kinetic data demonstrate the importance of a single amino acid (266) in determining the A vs. B donor specificity.

Calibration of glucose biosensors by using pre-steady state kinetic data

A new method for biosensor calibration and data processing, allowing the prediction of steady state parameters from the analysis of transient response curves ( Rinken et al., 1996. Analytical Letters 29, 859), has been evaluated in the case of an oxygen sensor based two-substrate enzyme electrode for glucose determination. The electrochemical glucose biosensor was prepared by covering the surface of oxygen sensor with glucose oxidase (EC 1.1.3.4) immobilized in nylon mesh. This decreased the oxygen flow to the sensor in the presence of glucose and resulted in time-dependent decrease of the biosensor signal. Except the lag period of the response in the beginning of the assay, the oxygen consumption by the immobilized enzyme was described by an exponential function: [ O 2] c bulk O 2 =A exp(−Bt)+C . The parameter C, which corresponded to the steady state output of the biosensor, was found to be the most suitable for glucose determination. The non-linear fitting for data of over 1000 independent experiments to the equation above always revealed correlation coefficients greater than 0.97. The calculation of the steady state parameter from the transient phase data makes the analysis fast and precise, especially for sensors with thick membranes, being convenient to use in the case of enzyme electrodes. The theoretical essence of the parameter C also gives valuable information for the optimal design of biosensors.

A Model of Oximeter - Based Enzyme Electrode

A model of an oximeter-based two-substrate enzyme electrode is presented. The simplifications of the general model lead to a solution to differential equations describing the influence of the rate of the enzyme reaction in combination with the diffusion phenomena on measurable oxygen flux. Both transient phase and steady state conditions for experimental data can be considered within the frames of the model and provide possibilities for the calibration of the biosensor. The optimal parameters for the biosensor calibration can be used in practical design of enzyme electrodes.

On the Mechanism of Interaction of Steroids with Human Glucose 6-Phosphate Dehydrogenase

Previous studies have demonstrated that certain 17- and 20-ketosteroids are potent inhibitors of the NADP-linked oxidation of glucose 6-phosphate by mammalian glucose 6-phosphate dehydrogenases, This inhibition is uncompetitive with respect to both NADP+ and glucose 6-phosphate, In order to elucidate the detailed mechanism of this rare type of inhibition, we examined the effects of steroids on human glucose 6-phosphate dehydrogenase catalyzing the reverse reaction, i,e,, the reduction of the gluconolactone by NADPH. To circumvent problems associated with the known instability of 6-phospho-δ-gluconolactone, the natural product of glucose 6-phosphate oxidation, the more stable 6-phospho-γ-gluconolactone was used. Dehydroepiandrosterone, epiandrosterone, 16α-bromoepiandrosterone, and dehydroepiandrosterone sulfate all inhibited the reverse reaction uncompetitively with respect to both NADPH and the γ-lactone. The Ki values for each of these steroids, determined by varying either coenzyme or substrate in both the forward and the reverse reactions, are very similar, These results demonstrate that steroids inhibit glucose 6-phosphate dehydrogenase by binding to the ternary enzyme-coenzyme-substrate ternary complex(es). This is the first direct demonstration that uncompetitive inhibition of a two-substrate enzyme, by compounds other than its substrates or products, can occur by binding of the inhibitor to a ternary enzyme complex.

Inhibition of Spinach D-Glyceraldehyde 3-Phosphate: NADP + Oxidoreductase (Nonphosphorylating) by Adenylate Compounds: The Effect of Dead-End Inhibitors on a Steady State Random Reaction Mechanism

D-Glyceraldehyde 3-phosphate: NADP + oxidoreductase, nouphosphorylating (GNR; EC 1.2.1.9) purified from spinach leaves was investigated by initial velocity analysis. The hyperbolic saturation curves became non-hyperbolic when NADP + was varied at elevated D-glyceraldehyde 3-phosphate (G3P) concentrations (sigmoidicity) or when G3P was varied at low NADP + concentrations (pseudo-substrate inhibition), suggesting a random bi bi mechanism (Scagliarini et al. Plant Physiol. 94, 1337-1344, 1990). Free ATP was a linear competitive inhibitor of both NADP + with K I 0.5 ± 0.2 mM (SD) and G3P with K I 3.2 ± 0.2 mM as determined by data in the hyperbolic range of responses when the non-varied substrate was saturating. Similarly ADP inhibited competitively with K I 1.9 ± 0.4 mM (NADP +) and 3.5 ± 0.5 mM (G3P). Inhibition was mixed-type when the nonvaried substrate was below saturation. ATP, but not ADP, tended to enhance the nonhyperbolic behavior of GNR, resulting in potentiated inhibition at high [G3P]/[NADP +] ratios. The Mg-chelated form of ATP was less effective. The rate equation of a steady state random bi bi reaction mechanism in the presence of a dead-end inhibitor was derived. Suitable values of the rate constants were chosen to fit the kinetic data for the uninhibited enzyme. These values and the measured inhibition constants inserted in the rate equation can satisfactorily account for the nonhyperbolic inhibition patterns of ATP and ADP. The generalized model represents a possible alternative to allosteric models in interpreting nonlinear kinetics and dead-end inhibition of two-substrate enzymes.

Specificity constants for two-substrate enzymes

Specificity constants for two-substrate enzymes

Experimental Strategy to Study the pH Dependence of the Kinetic Behavior of Enzymes: Practical Application to Chicken Liver Xanthine Dehydrogenase

An experimental strategy has been developed to analyze the pH dependence of kinetic parameters in two-substrate enzyme kinetics based upon model discrimination, parameter refinement, and optimal discrete design. The experimental protocol proposed includes obtaining reproducible experimental results in different sessions, replicates of each experimental determination, and rejection of outliers. This strategy leads to an empirical kinetic equation that fits the experimental results. As a practical application of this strategy we studied the pH dependence of kinetic parameters of chicken liver xanthine dehydrogenase. We obtained an empirical rate law from the determination of the initial rates at different substrate concentrations and pH. Finally, we have proposed a plausible kinetic mechanism that may explain the experimental findings. This model is not the only one leading to the empirical equation, but it is based on previous experimental findings (Bruguera, P., López-Cabrera, A., and Canela, E. I., 1988, Biochem. J. 249, 171-178).

Digital simulation of homogeneous enzyme kinetics for amperometric redox-enzyme electrodes

An explicit point method is described for digital simulation of redox-enzyme catalysis and diffusion at soluble-enzyme electrodes. Two experimental conditions (chronopotentiometry and open-circuit potential decay) were simulated for a two-substrate enzyme (glucose oxidase) catalyzing the anaerobic oxidation of glucose, mediated by a soluble redox couple. Transient and steady-state responses are compared to experimental data and to predictions of the analytical solutions for selected cases. Simulated steady-state concentration profiles near the electrode surface are analyzed for different values of K s/ C s and K O/ C O. The validity of linear Lineweaver—Burk plots for the amperometric response of enzyme electrodes is discussed.

Stachyose synthesis in mature leaves of Cucumis melo. Purification and characterization of stachyose synthase (EC 2.4.1.67)

Galactinol: raffinose-6-galactosyltransferase (EC 2.4.1.67), a stachyose synthase, was extracted from mature leaves of Cucumis melo cv. Ranjadew and was purified to homogeneity by (NH4)2SO4 precipitation, ion-exchange chromatography, gel-filtration and non-denaturing polyacrylamide gel electrophoresis. A specific activity of 516 μkat · mg(-1) and a 160-fold purification was achieved. The pH optimum of the enzyme reaction was found to be 6.8 in sodium-phosphate buffer, and the temperature optimum 32° C. The purified enzyme was very sensitive towards SH-poisons but its reaction was hardly affected by changes in the ion composition of the assay medium. The two-substrate enzyme was specific for galactinol and raffmose; uridine-diphosphate galactose and p-nitrophenyl-α-D-galactoside as well as melibiose were not accepted by the purified enzyme. Stachyose synthesis was competitively inhibited by concentrations >4 mM raffinose as well as 2.5 mM galactinol. The K m values determined under non-saturating conditions were 3.3 mM for raffinose and 7.7 mM for galactinol. Myoinositol was a strong competitive inhibitor with a K i of 1.8mM. Galactinol was hydrolyzed in the absence of raffinose with a K m of 0.8 mM. The pure enzyme is a protein with a molecular weight of at least 95 kDa and an isoelectric point of 5.1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of two subunits of 45 and 50 kDa. Polyclonal antibodies from rabbit were obtained which were specific for the native enzyme but cross-reacted with other proteins separated under denaturing conditions.