Ketol Isomerase Research Articles

Termofilik Anoxybacillus gonensis glukoz izomerazının DEAE-Sefaroz üzerine iyonik ve kovalent immobilizasyonu

High fructose corn syrup (HFCS), which is produced by the conversion of one sugar into another (glucose to fructose), has a marketing value. Hence, different glucose isomerases [(GI) (D-xylose ketol isomerase, EC 5.3.1.5)] isolated from different sources (macro-and microorganisms) were researched until today. In addition, the cost reduction of GI production for industrial applications has been investigated and applied with different techniques. Enzyme immobilization approaches have prominent features because they allow enzymes to be used repeatedly. In the current study, Anoxybacillus gonensis G2T glucose isomerase (AgoGI) (wild type) were immobilized with ionic and covalent binding on DEAE-sepharose matrix. Afterward, kinetic and biochemical parameters of the immobilized enzymes were evaluated. The pH and temperature parameters, in which the ionic and covalent immobilized enzymes showed the best activity, were determined as 6.50 and 85 °C, respectively. The kinetic data (Vmax and Km) of ionic bound AgoGI on DEAE-sepharose were 4.85±2.09 μmol/min/mg protein and 130,57±5,42 mM, as covalent immobilized AgoGI on the same matrix were 40.51± 0.81 μmol/min/mg protein µmol/min and 127,28±2,96 mM, respectively. Consequently, the usage of DEAE-sepharose for both covalent and ionic immobilization as immobilization matrix did not exhibit any negative effects on biochemical and kinetic parameters of glucose isomerase. Therefore, immobilized AgoGI on DEAE-sepharose was an excellent and promising tool for HFCS production.

Inclusion of mPRISM potential for polymer-induced protein interactions enables modeling of second osmotic virial coefficients in aqueous polymer-salt solutions.

The downstream processing of therapeutic proteins is a challenging task. Key information needed to estimate applicable workup strategies (e.g. crystallization) are the interactions of the proteins with other components in solution. This information can be deduced from the second osmotic virial coefficient B22 , measurable by static light scattering. Thermodynamic models are very valuable for predicting B22 data for different process conditions and thus decrease the experimental effort. Available B22 models consider aqueous salt solutions but fail for the prediction of B22 if an additional polymer is present in solution. This is due to the fact that depending on the polymer concentration protein-protein interactions are not rectified as assumed within these models. In this work, we developed an extension of the xDLVO model to predict B22 data of proteins in aqueous polymer-salt solutions. To show the broad applicability of the model, lysozyme, γ-globulin and D-xylose ketol isomerase in aqueous salt solution containing polyethylene glycol were considered. For all proteins considered, the modified xDLVO model was able to predict the experimentally observed non-monotonical course in B22 data with high accuracy. When used in an early stage in process development, the model will contribute to an efficient and cost effective downstream processing development.

The non-oxidative pentose phosphate pathway controls the fermentation rate of xylulose but not of xylose in TMB3001

Saccharomyces cerevisiae is able to ferment xylose, when engineered with the enzymes xylose reductase (XYL1) and xylitol dehydrogenase (XYL2). However, xylose fermentation is one to two orders of magnitude slower than glucose fermentation. S. cerevisiae has been proposed to have an insufficient capacity of the non-oxidative pentose phosphate pathway (PPP) for rapid xylose fermentation. Strains overproducing the non-oxidative PPP enzymes ribulose 5-phosphate epimerase (EC 5.1.3.1), ribose 5-phosphate ketol isomerase (EC 5.3.1.6), transaldolase (EC 2.2.1.2) and transketolase (EC 2.2.1.1), as well as all four enzymes simultaneously, were compared with respect to xylose and xylulose fermentation with their xylose-fermenting predecessor S. cerevisiae TMB3001, expressing XYL1, XYL2 and only overexpressing XKS1 (xylulokinase). The level of overproduction in S. cerevisiae TMB3026, overproducing all four non-oxidative PPP enzymes, ranged between 4 and 23 times the level in TMB3001. Overproduction of the non-oxidative PPP enzymes did not influence the xylose fermentation rate in either batch cultures of 50 g l−1 xylose or chemostat cultures of 20 g l−1 glucose and 20 g l−1 xylose. The low specific growth rate on xylose was also unaffected. The results suggest that neither of the non-oxidative PPP enzymes has any significant control of the xylose fermentation rate in S. cerevisiae TMB3001. However, the specific growth rate on xylulose increased from 0.02–0.03 for TMB3001 to 0.12 for the strain overproducing only transaldolase (TAL1) and to 0.23 for TMB3026, suggesting that overproducing all four enzymes has a synergistic effect. TMB3026 consumed xylulose about two times faster than TMB30001 in batch culture of 50 g 1−1 xylulose. The results indicate that growth on xylulose and the xylulose fermentation rate are partly controlled by the non-oxidative PPP, whereas control of the xylose fermentation rate is situated upstream of xylulokinase, in xylose transport, in xylose reductase, and/or in the xylitol dehydrogenase.

The non-oxidative pentose phosphate pathway controls the fermentation rate of xylulose but not of xylose in Saccharomyces cerevisiae TMB3001.

Saccharomyces cerevisiae is able to ferment xylose, when engineered with the enzymes xylose reductase (XYL1) and xylitol dehydrogenase (XYL2). However, xylose fermentation is one to two orders of magnitude slower than glucose fermentation. S. cerevisiae has been proposed to have an insufficient capacity of the non-oxidative pentose phosphate pathway (PPP) for rapid xylose fermentation. Strains overproducing the non-oxidative PPP enzymes ribulose 5-phosphate epimerase (EC 5.1.3.1), ribose 5-phosphate ketol isomerase (EC 5.3.1.6), transaldolase (EC 2.2.1.2) and transketolase (EC 2.2.1.1), as well as all four enzymes simultaneously, were compared with respect to xylose and xylulose fermentation with their xylose-fermenting predecessor S. cerevisiae TMB3001, expressing XYL1, XYL2 and only overexpressing XKS1 (xylulokinase). The level of overproduction in S. cerevisiae TMB3026, overproducing all four non-oxidative PPP enzymes, ranged between 4 and 23 times the level in TMB3001. Overproduction of the non-oxidative PPP enzymes did not influence the xylose fermentation rate in either batch cultures of 50 g l(-1) xylose or chemostat cultures of 20 g l(-1) glucose and 20 g l(-1) xylose. The low specific growth rate on xylose was also unaffected. The results suggest that neither of the non-oxidative PPP enzymes has any significant control of the xylose fermentation rate in S. cerevisiae TMB3001. However, the specific growth rate on xylulose increased from 0.02-0.03 for TMB3001 to 0.12 for the strain overproducing only transaldolase (TAL1) and to 0.23 for TMB3026, suggesting that overproducing all four enzymes has a synergistic effect. TMB3026 consumed xylulose about two times faster than TMB30001 in batch culture of 50 g l(-1) xylulose. The results indicate that growth on xylulose and the xylulose fermentation rate are partly controlled by the non-oxidative PPP, whereas control of the xylose fermentation rate is situated upstream of xylulokinase, in xylose transport, in xylose reductase, and/or in the xylitol dehydrogenase.

Short Enzymatic Synthesis ofL-Fucose Analogs

A short enzymatic route for the synthesis of L-fucose analogs modified at the nonpolar terminus is reported. In particular, fucose derivatives bearing extended linear (1b) and branched (1e) saturated, or various unsaturated (1c, 1d) aliphatic chains have been prepared, in order to increase hydrophobic contacts. The rather general approach involves a sequential application of the recombinant enzymes L-fuculose 1-phosphate aldolase (FucA) and L-fucose ketol isomerase (FucI) from E.coli. Enantiomerically pure L-fucose analogs have been prepared in up to 30% overall yield starting from the appropriate hydroxyaldehyde precursors and dihydroxyacetone phosphate as readily available components. Unsaturated 2-hydroxyaldehydes have been efficiently prepared by alk(en/yn)yl Grignard addition to cinnamaldehyde followed by controlled ozonolysis of the styrene fragment.

Structure and mechanism of l-fucose isomerase from Escherichia coli

The three-dimensional structure of l-fucose isomerase from Escherichia coli has been determined by X-ray crystallography at 2.5 Å resolution. This ketol isomerase converts the aldose l-fucose into the corresponding ketose l-fuculose using Mn 2+ as a cofactor. Being a hexamer with 64,976 Da per subunit, l-fucose isomerase is the largest structurally known ketol isomerase. The enzyme shows neither sequence nor structural similarity with other ketol isomerases. The hexamer obeys D 3 symmetry and forms the crystallographic asymmetric unit. The strict and favorably oriented local symmetry allowed for a computational phase extension from 7.3 Å to 2.5 Å resolution. The structure was solved with an l-fucitol molecule bound to the catalytic center such that the hydroxyl groups at positions 1 and 2 are ligands of the manganese ion. Most likely, l-fucitol mimics a bound l-fucose molecule in its open chain form. The protein environment suggests strongly that the reaction belongs to the ene-diol type.

Evidence for a mannitol cycle in Orobanche ramosa and Orobanche crenata

Summary The activities of various enzymes associated with mannitol metabolism in The activities of various enzymes associated with mannitol metabolism in Orobanche ramosa L. and Orobanche crenata Forsk. were measured both in the crude extracts of plant tissue and callus. The detection of D-mannose-6-phosphate ketol isomerase (EC 5.3.1.8), D-mannitol-l-phosphate: NADP oxidoreductase (EC 1.1.1.nn) and D-mannitol-l-phosphate phosphohydrolase (EC 3.1.3.22) activities revealed a similar pathway as postulated by Rumpho et aI. (1983) and Loescher et aI. (1992) for celery and privet, while the appearance of D-mannitol-l-phosphate:NAD oxidoreductase (EC 1.1.1.17) activity in O. callus suggests the possibility of an alternative pathway. To exclude artefacts, the above mentioned enzymes were partially purfied and further characterized. The occurrence of a low affinity D-mannitol: NAD oxidoreductase (EC 1.1.1.67)gives evidence for a mannitol cycle in the two Orobanche species. L. and Orobanche crenata Forsk . were measured both in the crude extracts of plant tissue and callus. The detection of D-mannose-6-phosphate ketol isomerase (EC 5.3.1.8), D-mannitol-1-phosphate: NADP oxidoreductase (EC 1.1.1.nn) and D-mannitol-1-phosphate phosphohydrolase (EC 3.1.3.22) activities revealed a similar pathway as postulated by Rumpho et al. (1983) and Loescher et al. (1992) for celery and privet, while the appearance of D-mannitol-1-phosphate: NAD oxidoreductase (EC 1.1.1.17) activity in O. callus suggests the possibility of an alternative pathway. To exclude artefacts, the above mentioned enzymes were partially purfied and further characterized. The occurrence of a low affinity D-mannitol: NAD oxidoreductase (EC 1.1.1.67) gives evidence for a mannitol cycle in the two Orobanche species.

Phosphorylation-dependent regulation of amidotransferase during the development of Blastocladiella emersonii

The enzyme amidotransferase [2-amino-2-deoxy- d-glucose-6-phosphate ketol isomerase (amino-transferring); EC 2.6.1.16] catalyzes the first step in the hexosamine biosynthetic pathway. In Blastocladiella emersonii the sensitivity of the enzyme to the inhibitor uridine-5′-diphospho- N-acetylglucosamine (UDP-GlcNAc) is developmentally regulated. The inhibitable form of amidotransferase activity present in the zoospore is converted to a noninhibitable form during germination. The latter form is present throughout the growth phase and sensitivity to UDP-GlcNAc gradually returns to the zoospore level during sporulation [C. P. Selitrennikoff, N. E. Dalley, and D. R. Sonneborn (1980) Proc. Natl. Acad. Sci. USA 77, 5998–6002] . The following evidence suggests that a phosphorylation/dephosphorylation mechanism underlies this interconversion: (i) Both the vegetative and zoospore enzymes have the same molecular weight of 140,000, but the vegetative enzyme elutes significantly earlier on a DEAE-cellulose column than does the zoospore enzyme. (ii) The increased sensitivity to UDP-GlcNAc occurring in vivo and in vitro correlates with increased phosphorylation of a polypeptide of apparent M r 76,000. This component copurifies with amidotransferase activity through ion-exchange chromatography and sucrose density gradient centrifugation. (iii) Desensitization and concurrent dephosphorylation of sensitive amidotransferase can be observed in vitro after treatment with a partially purified magnesium-dependent phosphoprotein phosphatase from zoospores.

Sulfhydryl groups of glucosamine-6-phosphate isomerase deaminase from Escherichia coli

Glucosamine-6-phosphate isomerase deaminase (2-amino-2-deoxy- d-glucose-6-phosphate ketol isomerase (deaminating), EC 5.3.1.10) from Escherichia coli is an hexameric homopolymer that contains five half-cystines per chain. The reaction of the native enzyme with 5′,5′-dithiobis-(2-nitrobenzoate) or methyl iodide revealed two reactive SH groups per subunit, whereas a third one reacted only in the presence of denaturants. Two more sulfhydryls appeared when denatured enzyme was treated with dithiothreitol, suggesting the presence of one disulfide bridge per chain. The enzyme having the exposed and reactive SH groups blocked with 5′-thio-2-nitrobenzoate groups was inactive, but the corresponding alkylated derivative was active and retained its homotropic cooperativity toward the substrate, d-glucosamine 6-phosphate, and the allosteric activation by N-acetyl- d-glucosamine 6-phosphate. Studies of SH reactivity in the presence of enzyme ligands showed that a change in the availability of these groups accompanies the allosteric conformational transition. The results obtained show that sulfhydryls are not essential for catalysis or allosteric behavior of glucosamine-6-phosphate deaminase.

Purification, molecular and kinetic properties of glucosamine-6-phosphate isomerase (deaminase) from Escherichia coli

Glucosamine-6-phosphate isomerase (deaminase), (2-amino-2-deoxy- d-glucose-6-phosphate ketol isomerase (deaminating), EC 5.3.1.10) has been purified to homogeneity from Escherichia coli B as judged by several criteria of purity. The procedure included ammonium sulfate fractionation, anion-exchange chromatography and a biospecific affinity chromatography step with N-ϵ- amino-n- caproyl- d-glucosamine 6-phosphate bound to agarose as the ligand, the elution being performed with GlcNAc6 P. The enzyme appears to be an hexamer of about 178 kDa, composed of six subunits of 29 700 ± 300 Da; the isoelectric point was 6.0–6.1 and the sedimentation constant 9.0 S. The amino-acid composition of the enzyme was determined and a value for E 275 1% of 4.55 was calculated. The molecular activity was 1800 s −1 for the deamination reaction and 455 s −1 for the reaction of GlcN6 P formation. A positive homotropic cooperativity was found for both sugar substrates; it was stronger for GlcN6 P in the deamination reaction (Hill number 2.7 at pH 7.7). Ammonia behaved as a Michaelian substrate. Cooperativity was abolished by 0.1 mM GlcNAc6 P; this allosteric modulator activated the reaction in both directions, with a positive K-effect upon both sugar phosphates, but had no effect on K m for ammonia. The initial velocity patterns for the amination reaction were obtained under conditions of hyperbolic kinetics produced by GlcNAc6 P; the K m values for the allosteric substrates were determined under the same conditions, and their dependence upon pH was studied.

Development of phosphogluco‐isomerase isozymes in perennial ryegrass (Lolium perenne)

The development of isozymes of phosphogluco‐isomerase (PGI; D‐glucose‐6‐phosphate ketol isomerase EC 5.3.1.9.) in perennial ryegrasses was followed from dry seed through to leaf senescence using starch gel electrophoretic separations. Root isozymes were also examined. Two forms of the enzyme were found, one (PGI/2) being present in all tissues and at all stages of the life cycle. The other (PGI/1) had two zones of activity, one of which was detected only in light‐exposed tissue. Normal development of this form could be inhibited by growing seedlings on distilled water. Some alleles of PGI/2 not previously reported for ryegrasses are also described.

Instability during storage of phosphogluco‐isomerase isoenzymes from ryegrasses (Lolium spp.)

The stability during storage of phosphogluco‐isomerase (D‐glucose‐6‐phosphate ketol isomerase EC 5.3.1.9) isoenzymes coded for at the PGI/2 locus has been examined. Extracts were prepared from leaves of several diploid and tetraploid Italian (Lolium multiflorum Lam.) and perennial (Lolium perenne. L.) ryegrass cultivars, as well as from interspecific hybrids. It was clearly demonstrated that extracts from plants homozygous for a specific PGI/2 allele could quickly generate new band forms upon storage. The novel forms were not due to aggregation or disintegration of the original enzyme molecule, and some of the generated bands electrophoresed to gel positions characteristic of other alleles of the same locus. An assessment was also made of the effects of a range of compounds added to the storage buffer. The most likely explanation was that the observed changes were due to the action of proteases, and the implications, especially for those using isoenzymes as genetic markers, are discussed.

The effect of trans-stilbene oxide and other structurally related inducers of drug-metabolizing enzymes on the pentose phosphate pathway and other enzymes of carbohydrate metabolism

trans-Stilbene oxide has been found earlier to be a new type of inducer of drug-metabolizing systems. Here we demonstrate that treatment of rats with this xenobiotic results in an increase in the activity of the cytosolic glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, the first and third enzymes in the pentose phosphate pathway, to 350% and 170% of the control values, respectively. At the same time microsomal glucose 6-phosphate dehydrogenase activity was unaffected by administration of trans-stilbene oxide or benzil. The time course and dose-response of the increases in glucose 6-phosphate and 6-phosphogluconate dehydrogenase activities have been characterized. The activities of ribulose 5-phosphate 3-epimerase and ribose 5-phosphate ketol isomerase, enzymes further along in the pentose phosphate pathway, were not significantly affected by trans-stilbene oxide or benzil. An investigation of the effect of treating rats with different metabolites of stilbene and with other structurally related compounds on hepatic cytosolic glucose 6-phosphate dehydrogenase activity revealed the structural features which are important for increasing this activity. Finally, it was found that administration of trans-stilbene oxide did not affect the activities of glucokinase and phosphoglucose isomerase, the two glycolytic enzymes which can produce glucose 6-phosphate, the link between glycolysis and the pentose phosphate shunt.

Immobilization of glucose isomerase

AbstractThe immobilization of glucose isomerase (D‐xylose ketol isomerase, EC 5.3.1.5) by covalently bonding to various carriers and by adsorption to ion exchange resins was attempted in order to obtain a stable immobilized enzyme which can be used for continuous isomerization of glucose in a column.Of the covalent bonding methods, the colloidal silica‐glutaraldehyde method showed the highest binding capacity and gave the stablest immobilized glucose isomerase. The LUDOX HS‐30 bound glucose isomerase column showed a half‐life of 24 days and an enzyme usage of 0.07 units per gram of isomerized sugar (d.s., fructose 45%).Of the resins used, the macromolecular type or porous type strongly basic anion exchange resins showed the highest binding capacity and gave the stablest immobilized glucose isomerase. The Amberlite IRA‐904 resine‐bound glucose isomerase showed a half‐life of 23 days and an enzyme usage of 0.06 units per gram of isomerized sugar (d. s., fructose 45%).Based on the ease of the immobilization process, the possibility of carrier ruse the extensive use already achieve by ion exchange resins in the sugar industry, IRA‐904 resin was selected as the candidate for commercialization.

Immobilization of glucose isomerase.

The immobilization of glucose isomerase (D-xylose ketol isomerase, EC 5.3.1.5) by covalently bonding to various carriers and by adsorption to ion exchange resins was attempted in order to obtain a stable immobilized enzyme which can be used for continuous isomerization of glucose in a column. Of the covalent bonding methods, the colloidal silica-glutaraldehyde method showed the highest binding capacity and gave the most stable immobilized glucose isomerase. The Ludox HS-30 bound glucose isomerase column showed a half-life of 24 days and an enzyme usage of 0.07 units per gram of isomerized sugar (d.s, fructose 45%). Of the resins used, the macromolecular type or porous type strongly basic anion exchange resins showed the highest binding capacity and gave the most stable immobilized glucose isomerase. The Amberlite IRA-904 resine-bound glucose isomerase showed a half-life of 23 days and an enzyme usage of 0.06 units per gram of isomerized sugar (d.s., fructose 45%). Based on the ease of the immobilization process, the possibility of carrier reuse and the extensive use already achieved by ion exchange resins in the sugar industry, IRA-904 resin was selected as the candidate for commercialization.

Immunofluorescence and histochemical localization of glucosephosphate isomerase in neural tissues.

The distribution of glucosephosphate isomerase (GPI, D-glucose-6-phosphate ketol isomerase) in mouse nervous tissue has been determined at the light microscopic level by immunofluorescence and histochemical procedures. The fluorescence procedure, which utilizes anti-GPI antibodies, detected lower levels of GPI than the histochemical procedure, which relies upon the catalytic activity of the enzyme. The distribution of GPI in nervous tissue is very similar to that of hexokinase. High levels of GPI were found in the Purkinje cells, the molecular layer, and the glomeruli of the granular layer in the cerebellar cortex; the pontine nuclei and the inferior olivary nuclei of the pons and medulla; the neurons of the thalamus and hypothalamus; the pyramidal cells, the dentate nuclei, and Ammons' horn of the cerebral cortex; the ventral horn cells of the spinal cord; and ventricular cells, choroid plexus cells, and the leptomeninges. The neuropil throughout the central nervous system (CNS) stained uniformly with moderately high levels of GPI. No GPI was observed in the myelin sheaths of the CNS.

Three forms of Candida utilis ribose 5-phosphate ketol isomerase.

Three forms (I, II, and III) of R 5-P ketol icomerase (EC 5.3,1.6) were separated and purified from Candida utilis. The Kms for R 5-P by I, II, and III were 0.77 mM, 0.67 mM and 0.16 mM, respectively. The isoelectric points of the forms, I, II, and III were 3.79, 3.87 and 3.99, respectively.The activation energies of the forms, I, II, and III were 10,800, 12,000 and 6360 cal/mole, respectively. The forms, I, II, and III were all inhibited with chelating reagent, EDTA (1 mM), but not affected with thiol reagents such as p-CMB, ICH2COOH, NEM, and DTNB. These properties are quite different from the other known R 5-P ketol isomerases, all of those are not affected with EDTA, but inhibited with the thiol reagents as described above.

Physico-chemical and enzymatic properties of purified glucose isomerases from Streptomyces olivochromogenes and Bacillus stearothermophilus.

The physico-chemical properties of the purified glucose isomerases [d-xylose ketol isomerase, EC 5.3.1.5] of Streptomyces olivochromogenes and Bacillus stearothennophilus were examined. The molecular size and shape of both enzymes were similar. The molecular weights, sedimentation coefficients, partial specific volumes, diffusion constants and Stokes’ radii of the Streptomyces and Bacillus enzymes were determined to be 120,000 and 130,000, 7.55 S and 9.35 S, 0.725 and 0.736 ml/g, 5.87 × 10-7 and 6.82 × 10-7 cm2/sec, and 51 and 53 A, respectively. The Streptomyces glucose isomerase was found to consist of two subunits, each having a molecular weight of 56,000. Large differences were found in the amino acid compositions of these two enzymes, especially in their serine, proline, tyrosine, lysine and arginine contents. The enzymatic properties of both these purified glucose isomerases were also examined, and it was seen that they both displayed activity on d-xylose, d-xylulose, d-glucose, d-fructose, d-arabin...

Purification and properties of D-xylose isomerase from Lactobacillus xylosus.

d-Xylose isomerase (d-xylose ketol isomerase, EC 5.3.1.5) was purified from d-xylose-grown cells of homofermentative Lactobacillus xylosus and compared with d-xylose isomerase of heterofermentative Lactobacillus brevis. The molecular weight was estimated to be 183,000 by thin layer chromatography on Sephadex G-200. The enzyme was composed of four subunits each having a molecular weight of 45,000. The optimum pH was 7.5, and the enzyme was more stable at the alkaline pH region than L. brevis enzyme. The former was stable at pH from 6.5 to 11.0, but the latter was stable in a narrow neutral to slightly acidic pH (5.7 to 7.0). Other properties resembled those of L. brevis enzyme. The enzyme was active on d-xylose and d-glucose. Michaelis constant for d-xylose was 5.3 mm. The enzyme activity was inhibited competitively by xylitol with an inhibition constant (Ki) of 7 mm.

Purification, properties and structure of ribose 5-phosphate ketol isomerase from Candida utilis.

Candida utilis Ribose 5-phosphate ketol isomerase (EC 5.3.1.6) was purified. The enzyme is homogeneous by ultracentrifugal analysis and disc electrophoresis. The isoelectric point of the enzyme is 3.8, Km for R 5-P is 0.108 mM and Kis by 5′-AMP and 6-P-G are 0.16 mM and 0.61 mM, respectively. The thermodynamic parameters, ΔH, ΔG (35°C), ΔS (35°C) and E are 4336 cal/mole, — 5629 cal/mole, 32.15 cal/deg-mole and 6360 cal/mole, respectively. The molecular weight of the enzyme is 183,000 and the enzyme is assumed to consist of three subunits, one with a molecular weight of 75,000 and two identical subunits each with a molecular weight of 54,000.