Ketol Isomerase Research Articles

14] Assay for d-ribose-5-phosphate ketol isomerase and d-ribulose-5-phosphate 3-epimerase

Publisher Summary This chapter discusses the assay for D-ribose-5-phosphate ketol isomerase and D-ribulose- 5-phosphate 3-epimerase. The assay for the isomerase is based upon the increase in absorbance observed at 290 nm, when ribose-5-phosphate ketol isomerase acts upon ribose 5-phosphate to form ribulose 5-phosphate. When the reaction has reached equilibrium, the addition of epimerase gives rise to a further increase in absorbance as xylulose 5-phosphate is formed. In the presence of an excess of the isomerase, the second rate of absorbance increase is proportional to the activity of the epimerase. In assay of ribose-5-phosphate ketol isomerase, the reaction mixture contains 1.78 ml 50 mM triethanolamine, HC1 buffer and 0.20 ml 100 mM ribose 5-phosphate (20 μmoles). It is placed in the cell compartment at 37° of a double-beam spectrophotometer connected to a recorder, and read against a control cuvette containing an equal volume of buffer. In the assay of ribulose-5-phosphate 3-epimerase, the assay mixture is set up as before, and 0.01 ml (2 units) of the isomerase is added. The absorbance increases rapidly at first and then levels off after 12–15 min. If small amounts of epimerase are present in the isomerase the recorder trace shows a slight upward slope. The calculation of isomerase and epimerase activity is presented. The isomerase assay has been used to follow the purification of the enzyme from spinach and other sources, in the determination of Michaelis constants.

A spectrophotometric procedure for following the d-ribulose 5-phosphate 3-epimerase reaction in the direction of ribulose 5-phosphate formation

A continuous spectrophotometric procedure for following the conversion of d-xylulose 5-phosphate to d-ribulose 5-phosphate by d-ribulose 5-phosphate 3-epimerase is described. Transketolase, ribose 5-phosphate ketol isomerase, glycerol 3-phosphate dehydrogenase, and triose phosphate isomerase were used as coupling enzymes and both practical and theoretical criteria for the validity of a coupled assay were satisfied. The initial velocity of the reaction was determined at a number of d-xylulose 5-phosphate concentrations and K m and V values of 0.15 ± 0.02 (SEM) m m d-xylulose 5-phosphate and 10.5 ± 0.6 (SEM) μmoles/min/mg protein were calculated from a reciprocal plot.

The detection and occurrence of multiple forms of D-ribose 5-phosphate ketol isomerase

A procedure is described for detecting D -ribose 5-phosphate ketol isomerase after electrophoresis, using ribose 5-phosphate as a substrate and alkaline 3:4-dinitrobenzoic acid to reveal the ribulose 5-phosphate formed. The enzyme was assayed by two different methods in the supernatant fraction of rat tissues and two electrophoretic forms were found in spleen, small intestine, uterus, heart muscle, and brain. Only one form was detected in lung, blood, kidney, and liver, while three forms were found in an extract of spinach. A partial separation of the two forms in rat spleen was achieved by chromatography on DEAE-Sephadex A-50. However, both these forms and the single form in rat liver had similar K m values lying between 0.28 and 0.39 m M D -ribose 5-phosphate.

Phosphoglucose Isomerase from Human Erythrocyte: PREPARATION AND PROPERTIES

Abstract Phosphoglucose isomerase (d-glucose 6-phosphate ketol isomerase, EC 5.3.1.9) of the human erythrocyte was resolved into a major and two minor components. The major enzyme form, isomerase a, was purified to constant specific activity and appeared homogeneous by chromatography, electrophoresis, and ultracentrifugation. The minor enzyme forms, isomerases b and c, were also prepared in high purity in limited quantity. Isomerases a, b, and c differed slightly in their charge properties (isoelectric points at pH 9.2, 9.1, and 9.0, respectively) but were indistinguishable on the basis of molecular size. Sedimentation velocity and partial specific volume measure of isomerase a yielded values of s20, w = 7.0 S and 0.745 cm3 per g. Molecular weight of the native enzyme was 125,000 by sedimentation equilibrium analysis with dissociation induced by denaturing agents yielding two subunits of identical or similar size. Isomerase a was distinguishable from b and c on the basis of a lesser Km for fructose 6-phosphate. Isomerases a, b, and c were identifiable with the major and two minor enzyme forms obtained on electrophoresis of crude hemolysate in starch gel.

Induction and repression of L-arabinose isomerase in Salmonella typhimurium.

As with other inducible enzymes, the induced synthesis of l-arabinose isomerase (l-arabinose ketol isomerase, EC 5.3.1.4) in Salmonella typhimurium is subject to catabolite repression. Of the three catabolite repressors tested, glucose produces maximum repression. Analogues of catabolite repressors like 2-deoxy-d-glucose and d-fucose also inhibit the synthesis of the enzyme. The catabolite repression is completely reversed in the presence of 1.5 x 10(-3)m cyclic 3',5'-adenosine monophosphate (AMP). The maximum repression is produced in glucose-grown cells in glucose-containing induction medium. Cyclic 3',5-AMP reverses this repression provided that the cells are treated with ethylenediaminetetraacetic acid (EDTA). In normal cells, cyclic 3',5'-AMP has no effect on the induction but in EDTA-treated cells the cyclic nucleotide enhances synthesis of the enzyme. The inhibition produced by d-fucose cannot be reversed by cyclic 3',5'-AMP. d-Fucose competes with the inducer l-arabinose in some step(s) involved in the process of induction.

Allosteric Control of Glucosamine Phosphate Isomerase from the Adult Housefly and Its Role in the Synthesis of Glucosamine 6-Phosphate

Abstract Glucosamine 6-phosphate synthesis has been examined in young adult houseflies (Musca domestica Linnaeus) at a time of active feeding and chitin production and shown to be dependent upon fructose 6-phosphate and inorganic ammonia in a reaction catalyzed by glucosamine phosphate isomerase (2-amino-2-deoxy-d-glucose 6-phosphate ketol isomerase (deaminating), EC 5.3.1.10). The 580-fold purified enzyme is specific for fructose 6-phosphate and some form of inorganic ammonia, and the product has been identified as glucosamine 6-phosphate. The molecular weight of the enzyme is about 154,000. The pH optimum of the enzyme for amination of fructose 6-phosphate is 7.1 to 7.3 in Tris-HCl and Tris-maleate buffers, between 7.7 and 8.2 in Tris-HCl containing inorganic phosphate, and 8.3 in Tris-HCl containing glucose 6-phosphate. The shift in the pH optimum in the presence of inorganic phosphate and glucose 6-phosphate accompanies the activation of the enzyme in the direction of amination by these two compounds. The pH optimum for the deamination of glucosamine 6-phosphate appears to occupy a broad region between pH 6.8 and 8.3 and is almost unaffected by either inorganic phosphate or glucose 6-phosphate, as is the rate of the reaction. The Km values for fructose 6-phosphate, ammonia, and glucosamine 6-phosphate are 36 mm, 25 mm, and 17 mm, respectively. In the presence of 5 mm glucose 6-phosphate, the Km values for fructose 6-phosphate and ammonium chloride are 3 mm and 3.3 mm, respectively, the Km for glucosamine 6-phosphate remaining unchanged. The activation constant for glucose 6-phosphate is 9.5 mm and the data indicate binding at one allosteric site only. N-Acetylglucosamine 6-phosphate activates the enzyme in both directions, the Km for glucosamine 6-phosphate being less than 0.5 mm. The equilibrium constant is in the range of 0.096 to 0.18 m for Keq = (fructose 6-phosphate) (ammonium chloride)/ (glucosamine 6-phosphate). In the presence of saturating concentrations of substrates and activators, the initial rate of amination in the presence of N-acetylglucosamine 6-phosphate is 3.8 times the rate of deamination. The initial rate of amination in the presence of glucose 6-phosphate is 3.1 times the rate of deamination in the presence of N-acetylglucosamine 6-phosphate, glucose 6-phosphate not altering the rate of deamination. Mannose 6-phosphate inhibits the amination of fructose 6-phosphate, but this is partially reversed by glucose and N-acetylglucosamine 6-phosphates. In the presence of N-acetylglucosamine 6-phosphate, glucose 6-phosphate has no effect on amination of fructose 6-phosphate. It is not clear whether glucose and N-acetylglucosamine 6-phosphates bind at the same site. Inorganic phosphate, itself an activator, partially inhibits activation by glucose 6-phosphate. Glucosamine 6-phosphate has no effect on the enzyme other than as a substrate. The absence in these animals of an enzyme which catalyzes formation of glucosamine 6-phosphate from l-glutamine and fructose 6-phosphate, the activation of the enzyme by glucose 6-phosphate only in the direction of amination of fructose 6-phosphate, and the higher initial rates of amination (3.8 times) than of deamination are evidence that the enzyme is a link in the synthetic pathway leading to chitin.

Purification of a D-mannose isomerase from Mycobacterium smegmatis.

An enzyme, d-mannose ketol isomerase, catalyzing the isomerization of d-mannose and d-fructose was purified approximately 60-fold from cells of Mycobacterium smegmatis grown on mannose as the sole carbon source. This enzyme was shown to catalyze the conversion of d-mannose and d-lyxose to ketoses. The ketose produced from mannose was identified as fructose by chemical and chromatographic methods. The reaction was shown to be reversible, the equilibrium ratio of fructose to mannose being approximately 65 to 35. The pH optimum was about 7.5, and the K(m) for mannose was estimated to be 7 x 10(-3)m. Mannose isomerase activity was greatest in cells grown on mannose, whereas cells grown on fructose had about 30% as much activity. Very low levels of activity were detected in cells grown on other substrates. There was an immediate increase in enzyme activity on transfer of cells from nutrient broth to a mannose mineral salts medium.

Pentose Metabolism in Candida utilis

L-Arabinose ketol isomerase (EC 5.3.1.4) was extracted from cells of Candida utilis which were grown in sulfite pulp mill waste. The enzyme was partially purified 135-fold by means of fractionation with ammonium sulfate, on DEAE-cellulose and DEAE-Sephadex A-50 column chromatographies and gel filtration on Sephadex G-200. Optimal pH and temperature for the activity of the enzyme were 7.0 and 60°C, respectively. The enzyme reaction was activated by Co2+ and Mn2+, and was inhibited by Na-pyrophosphate. The Michaelis-Menten's constant for L-arabinose was 1.07 × 10-1M, and the thermodynamic quantities, ΔH, ΔG (35°C), ΔS (35°C) and E were 8914 cal/mole, -1397 cal/mole, 33.5 cal/deg. mole and 9588 cal/mole, respectively.

Purification and characterization of human phosphoglucose isomerase

1. 1. The glycolytic enzyme phosphoglucose isomerase ( d-glucose-6-phosphate ketol isomerase, EC 5.3.1.9) has been purified from human muscle. This preparation was homogeneous by several criteria. 2. 2. The molecular weight of the enzyme obtained by the sedimentation-equilibrium method was 134 000. Following dissociation in guanidine hydrochloride the molecular weight was 61 000, indicating a dimeric structure for the enzyme. The isoelectric point of the enzyme was determined by electrofocusing and found to be pH 8.9. 3. 3. The amino acid composition of the enzyme was determined.

The purification of transketolase from Candida utilis.

Transketolase (EC 2.2.1.1) was purified 12-fold from an extract of dried Candida utilis by two fractionations with acetone followed by chromatography on DEAE-Sephadex. The fractions from the column were free from D-ribose 5-phosphate ketol isomerase and D-ribulose 5-phosphate 3-epimerase and could be used for the assay of D-xylulose 5-phosphate and of the epimerase. Three peaks of transketolase activity were eluted from the chromatography column; the material from each peak had a Michaelis constant for D-xylulose 5-phosphate close to the mean value for the three peaks of 6.8 × 10−5 M and gave the same two-banded pattern on cellulose acetate electrophoresis. When transaldolase was present in the material placed on the column, it appeared with transketolase in each of the three peaks. The enzyme could be stored for several months, in solution frozen at −20°. Dialysis against water caused a loss of activity which could be restored by the addition of thiamine pyrophosphate. The enzyme catalyzed the condensation of hydroxypyruvate with D-glyceraldehyde phosphate to form D-xylulose 5-phosphate.

Pentose Metabolism in Candida utilis

l-Arabinose ketol isomerase (EC 5.3.1.4) was extracted from cells of Candida utilis which were grown in sulfite pulp mill waste.The enzyme was partially purified 135-fold by means of fractionation with ammonium sulfate, on DEAE-cellulose and DEAE-Sephadex A-50 column chromatographies and gel filtration on Sephadex G-200.Optimal pH and temperature for the activity of the enzyme were 7.0 and 60°C, respectively. The enzyme reaction was activated by Co2+ and Mn2+, and was inhibited by Na-pyrophosphate.The Michaelis-Menten’s constant for l-arabinose was 1.07×10−1m, and the thermodynamic quantities, ΔH, ΔG (35°C), ΔS (35°C) and E were 8914 cal/mole, −1397 cal/mole, 33.5 cal/deg mole and 9588 cal/mole, respectively.

The purification and properties of D-allosephosphate isomerase of Aerobacter aerogenes.

Aerobacter aerngenes produces an inducible D-allose 6-phosphate ketol isomerase that nonspecilically isomerizes ribose 5-phosphate. The isomerase was separated from other isomerases present in extracts of cells grown on D-allose by repeated precipitation with protamine. Eighty percent of the activity of the purified preparation was lost on storage at 0 °C for 48 hours. The enzyme was most active and most stable at pH 8.5 and most active at 40 °C. Cobalt, manganese, magnesium, and phosphate ions inhibited at 0.05 M. Mercuric chloride, p-chloromer-curiphenylsulfonate, and iodoacetate also inhibited the isomerase but this was prevented by the addition of cysteine or reduced glutathione. These thiols also reactivated the enzyme when activity was lost during dialysis. 6-Phosphoallonate was a strong inhibitor and to a lesser degree 6-phosphogluconate, 6-phosphoglucosamine, 5-phosphoribonate, and erythrose 4-phosphate. For allose 6-phosphate Kswas 1.2 × 10−3 M and for ribose 5-phosphate, 4.5 × 10−3 M.

Intracellular pH effect upon phosphoglucose isomerase in Escherichia coli

Abstract 1. 1. Whole cells of Escherichia coli hydrolyze fructose 1,6-diphosphate to fructose 6-phosphate, and when the cells are incubated in an unbuffered medium the product is not isomerized to glucose 6-phosphate. The reason for this lack of isomerization has been investigated. 2. Ubder specified conditions phosphoglucose isomerase ( d -glucose-6-phosphate ketol isomerase, EC 5.3.1.9) is inhibited, probably because of a low pH in the cells which is unfavourable for the phosphoglucose isomerase reaction, but which permits other enzymes in the glycolytic sequence to act. 3. 3. Aerobically incubated cells of E. coli contain more acids than what is found under anaerobic conditions. This may result in a pH inhibition of the phosphoglucose isomerase which is dependent upon the oxygen pressure. 4. 4. In suspensions of E. coli buffered at pH 5.8, hydrolysis in the 1 position is competing favourably woth glycolytic breakdown of fructose 1,6-diphosphate. 5. 5. The specific radioativity of mannitol 1-phosphate observed in experiments with 32 P-labelled orthophosphate, strongly suggests that E. coli contains an active mannitol kinase.

Competitive shunt mechanism

6-Phosphogluconate, the first product of the “hexone monophosphate shunt”, has been shown to be a competitive inhibitor of phosphoglucoisomerase (d-glucose-6-phosphate ketol isomerase, EC 5.3.1.9). Similarly aspartate has been shown to be a competitive inhibitor of fumarase (l-malate-hydro-lyase, EC 4.2.1.2). This competitive inhibition of a main metabolic sequence, i.e. glucose 6-phosphate to fructose 6-phosphate on the one hand, and fumarate to l-malate on the other, by the initial product of a shunt relation has been given the name “Competitive Shunt Mechanism”.