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[22] Synthesis of urea with urease in water-organic solvent mixtures
234
IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS
[22]
[22] S y n t h e s i s o f U r e a w i t h U r e a s e in W a t e r - O r g a n i c Solvent Mixtures B y LARRY G . B U T L E R
The synthesis of urea in animals involves several enzymes and is driven by hydrolysis of 3 mol of ATP per mole of urea synthesized. 1 Urea can be readily synthesized nonenzymatically from the inorganic salt ammonium cyanate; this original synthesis by W6hler in 1828 was a landmark in the history of chemistry. 2 It has now become possible to demonstrate the synthesis of urea from an inorganic salt in a reaction catalyzed by a single enzyme, independent of hydrolysis of ATP or any other high energy compound. The inorganic salt is ammonium carbonate; the enzyme is urease, which catalyzes urea synthesis by reversal of urea hydrolysis2 Reversal of hydrolytic reactions was originally considered as a biosynthetic route; the first claim of an enzyme-catalyzed synthesis reaction was by reversal of the hydrolytic reaction catalyzed by maltase (a-glucosidase). 4 Because water is not only the solvent but is a reactant at high concentration, the equilibrium for hydrolytic reactions lies far toward hydrolysis when carried out in aqueous media. Methods for overcoming this thermodynamic constraint have been developed so that reversal of peptide bond hydrolysis has become a useful method for semisynthesis of proteins 5 and is being extended to large-scale synthesis of specialized materials such as aspartame. 6 Hydrolysis of urea is equivalent to hydrolysis of two peptide bonds, so the equilibrium lies even farther toward hydrolysis than for comparable reactions such as peptide bond hydrolysis. 7 Nevertheless, synthesis of urea from ammonium carbonate can readily be detected and quantitated. Replacement of part of the solvent water by water-miscible organic solvents dramatically increases the yield of urea. 3 This increase was presumed to be due to the corresponding decrease in water activity, shifting the equilibrium toward urea synthesis, 3 but an effect of the organic solJ H. 2 F. 3 L. 4 C. 5 R. 6 K. 7 A.
A. Krebs and K. Henseleit, Hoppe-Seyler's Z. Physiol. Chem. 210, 33 (1932). W#hler, Poggendorff's Annalen 12, 253 (1828). G. Butler and F. J. Reithel, Arch. Biochem. Biophys. 178, 43 (1977). Hill, J. Chem. Soc. 73, 634 (1898). E. Offord, "Semisynthetic Proteins." Wiley, New York, 1980. Oyama and K. Kihara, CHEMTECH 14, 100 (1984). Kapune and V. Kasche, Biochem. Biophys. Res. Commun, 80, 955 (1978).
METHODS IN ENZYMOLOGY, VOL. 136
Copyright © 1987 by Academic Press, Inc. AI! rights of reproduction in any form reserved.
[22]
UREASE-CATALYZED UREASVNTHEStS
235
vent on ionization a may also be involved. Several problems associated with synthesis by reversal of enzymatic hydrolysis reactions are illustrated by investigation of the effects of reaction conditions on the rate and extent of urea synthesis as catalyzed by urease. Source of Enzyme. Jack bean urease extracted and purified by the standard acetone crystallization procedure 9 with minor modifications ~° was used in demonstrating urease-catalyzed urea synthesis) Utilization of urease from other sources should be possible, although conditions for good reaction rates would have to be established for the particular enzyme used. Conditions which determine the position of equilibrium of the urea hydrolysis reaction (see below) are of course independent of the source of the enzyme. Assays
Urea Synthesis. In order to obtain readily measurable amounts of urea it is necessary to use relatively high concentrations of substrate. Typical conditions are 0.75 M (NH4)zCO3, 5 mM EDTA, and 5 mM 2-mercaptoethanol (no additional buffer is necessary). The urease concentration should be about I Sumner Unit 11 per milliliter. Under these conditions Jack bean urease catalyzes urea synthesis at about 2% of the rate at which it hydrolyzes urea (conditions for urea hydrolysis specified below). Urea concentration is determined spectrophotometrically by a modification ~2of the Fearon reaction.13 Incubated samples, zero time blanks, or urea standards containing up to 1/zmol urea in a total volume of 0.2-1.0 ml in screw-capped tubes are quenched by addition of 5 ml of 52% H3PO4 (600 ml of 85% H3PO4 diluted with water to 1 liter). Samples can be kept at room temperature at this stage for up to an hour without significant loss of urea, so that several samples can be run together through the remainder of the procedure. To each quenched sample is added 1.0 ml of an aqueous solution containing 6 g of 2,3-butanedione monoxime and 0.3 g thiosemicarbazide (both from Aldrich Chem. Co.) per liter. After heating for 30 min on a boiling water bath, samples are cooled to room temperature and their absorbance at 530 nm is determined immediately. The assay is not affected by high concentrations of ( N H 4 ) 2 C O 3 . At smaller sample volumes (0.2-0.5 ml) the apparent extinction coefficient for urea (approxi8 G. A. Homandberg, J. A. Mattis, and M. Laskowski, Jr., Biochemistry 17, 5220 (1978). 9 G. Mamiya and G. Gorin, Biochim. Biophys. Acta 105, 382 (1965). ~0C. M. Kaneshiro, Ph.D. Dissertation, University of Oregon, Eugene (1975). N j. B. Sumner, this series, Vol. 2, p. 378. ~2 M. K. Schwarz, this series, Vol. 17, p. 857. 13 W. R. Fearon, Biochem. J. 33, 902 (1939).
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IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS
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mately 2 × l06 M -~ c m - 0 is relatively insensitive to the presence of organic solvents in the sample. For samples in which the equilibrium concentration of urea is being determined, it is advisable, after measuring the urea concentration, to add fresh urease and incubate again until the urea concentration is constant. Urea Hydrolysis. For comparison to urea synthesis, urea hydrolysis can be measured by an adaptation of the same assay using 2 mM urea as substrate, in the presence of 0.2 mM EDTA, 5 mM 2-mercaptoethanol, and 20 mM imidazole acetate, pH 7.0. This urea concentration is below the Km for urea 14 so the rate decreases continually throughout the incubation. Data points at four different reaction times are plotted semilogarithmically; the first-order rate constant is determined from the slope. Effect of Organic Solvents on Enzyme Stability The use of organic solvents to enhance the yield of urea is possible only if urease activity can be maintained in the presence of the solvents. Fortunately, Jack bean urease is known to be rather stable in organic solvents such as glycerol.~5 Moreover, it is extracted and crystallized in 32% acetone. 9 In connection with these experiments it was observed that urease in 50% aqueous dimethylformamide, ethanol, or pyridine irreversibly lost most of its activity in a period of a few hours at room temperature. However, activity was recovered in high yield from urease dissolved in 50% aqueous solutions of acetone or polyhydroxylic alcohols such as glycerol and 1,2-propanediol. In an attempt to stabilize Jack bean urease against denaturation and loss of catalytic activity by high concentrations of organic solvents, the enzyme was immobilized and cross-linked by two independent methods. In neither case, however, did immobilized cross-linked urease have improved stability in the presence of organic solvents) Subsequent work was done only with soluble urease. Effect of Organic Solvents on Reaction Rate Organic solvents affect urea hydrolysis in much the same way that they affect urea synthesis .3 The effect of organic solvents on reaction rate can thus be conveniently determined by measuring urea hydrolysis, which requires less enzyme than urea synthesis. The effects are strongly dependent on the nature of the organic solvent, as well as its concentra~4 K. R. L y n n , Biochim. Biophys. Acta 146, 205 (1967). ~5 C. C. Contaxis and F. J. Reithel, J. Biol. Chem. 246, 677 (1971).
[22]
U R E A S E - C A T A L Y Z E D UREA SYNTHESIS
237
tion. The solvents were reagent grade but not redistilled; further purification of the solvents might be warranted. When compared at the same concentration (50%), the rate of urea hydrolysis ranged from undetectable (in ethanolamine) up to equal activity in acetone to that observed in strictly aqueous solution. 3 Rates in 50% aqueous glycerol, dimethylformamide, ethanol, and pyridine were 15, 49, 56, and 65% of the rate in water. In acetone and glycerol, in which urease is stable indefinitely, the activity as a function of organic solvent concentration shows rather different patterns, with most of the activity retained up to 75% acetone and much less activity observed at relatively low glycerol concentrations) Solvent selection for proposed urea synthesis processes wouldrequire optimization of the particular solvent for enzyme stability, reaction rate, and yield of urea (see later section). Effect of Organic Solvent, Substrate, and Hydrogen Ion Concentration on Yield of Urea The equilibrium concentration of urea, determined in solvents in which urease is stable so that achievement of equilibrium can be assured, increases as the water activity (related to concentration) diminishes. The yield of urea at equilibrium is approximately a second-order function of water concentration (activity) as predicted by the equilibrium constant. 3 The yield of urea as a function of (NH4)2CO3 concentration, at constant solvent composition, strongly increases as the substrate concentration increases. 3 In 50% glycerol, the observed urea yield was greater than the theoretical yield (which assumes concentration and activity are equivalent) at low substrate concentrations and lower than the theoretical yield at high substrate concentrations. The yield of urea is greatly enhanced in alkaline conditions) H + is a by-product of urea synthesis from HCO3-, according to Eq. (1). 2 NH4 + + H C O 3 - --0 u r e a + 2 H 2 0 + H +
(1)
Effect of the Nature of the Organic Solvent on Yield of Urea The equilibrium concentration of urea differs greatly depending upon which organic solvents are utilized to replace part of the water. Compared at a concentration of 50%, the equilibrium concentration of urea in aqueous acetone is over 12-fold higher than in aqueous glycerol. 3 Other organic solvents tested gave yields between these values, with hydroxylic solvents generally showing the lowest yields) As determined by measurement of the solubility of urea and ammonium carbonate, the primary effect of organic solvents is on the activity of the salt, rather than urea.
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IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS
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Use of Organic Solvents to Study the Reaction Mechanism It has been reported that the products of urease-catalyzed urea hydrolysis are ammonium and carbamate ions. 16 Measuring urea synthesis rather than hydrolysis, it is possible to show that maximum initial rates are obtained only if the substrate ammonium bicarbonate has been dissolved for an hour before urease is added; freshly dissolved substrate gives much slower initial rates of urea synthesis s This difference is likely due to a requirement for synthesis from carbamate, which forms nonenzymatically at a slow rate after ammonium bicarbonate is dissolved. In order to prove this point, carbamate concentration should be measured in both fresh and aged substrate solutions, and the rate of synthesis from ammonium carbamate should be directly compared with the rate of urea synthesis from ammonium carbonate. Significance The small amounts of urea synthesized (approximately O. 1% yield) by reversal of the urease reaction are not likely to warrant consideration of this process for large-scale urea production. Urease does, however, provide an interesting model for investigation of the reversal of hydrolytic enzyme reactions, both because of the historical significance of urea synthesis, and because urease illuminates several factors which limit utilization of the technology of reversal of enzymatic hydrolysis. Perhaps most important is the realization that no single set of conditions is optimal for all the parameters affecting urea synthesis. For example, conditions which give high urea yields also give slow rates of synthesis, and conditions in which synthesis is rapid give poor yields. Enzyme efficiency and stability and substrate solubility likewise may be affected differently by organic solvents. Some of these problems may be partially overcome by using mixtures of different organic solvents.
~6R. L. Blakely, J. A. Hinds, H. E. Kunze, E. C. Webb, and B. Zerner, 1991 (1969).
Biochemistry 8,
IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS
[22]
[22] S y n t h e s i s o f U r e a w i t h U r e a s e in W a t e r - O r g a n i c Solvent Mixtures B y LARRY G . B U T L E R
The synthesis of urea in animals involves several enzymes and is driven by hydrolysis of 3 mol of ATP per mole of urea synthesized. 1 Urea can be readily synthesized nonenzymatically from the inorganic salt ammonium cyanate; this original synthesis by W6hler in 1828 was a landmark in the history of chemistry. 2 It has now become possible to demonstrate the synthesis of urea from an inorganic salt in a reaction catalyzed by a single enzyme, independent of hydrolysis of ATP or any other high energy compound. The inorganic salt is ammonium carbonate; the enzyme is urease, which catalyzes urea synthesis by reversal of urea hydrolysis2 Reversal of hydrolytic reactions was originally considered as a biosynthetic route; the first claim of an enzyme-catalyzed synthesis reaction was by reversal of the hydrolytic reaction catalyzed by maltase (a-glucosidase). 4 Because water is not only the solvent but is a reactant at high concentration, the equilibrium for hydrolytic reactions lies far toward hydrolysis when carried out in aqueous media. Methods for overcoming this thermodynamic constraint have been developed so that reversal of peptide bond hydrolysis has become a useful method for semisynthesis of proteins 5 and is being extended to large-scale synthesis of specialized materials such as aspartame. 6 Hydrolysis of urea is equivalent to hydrolysis of two peptide bonds, so the equilibrium lies even farther toward hydrolysis than for comparable reactions such as peptide bond hydrolysis. 7 Nevertheless, synthesis of urea from ammonium carbonate can readily be detected and quantitated. Replacement of part of the solvent water by water-miscible organic solvents dramatically increases the yield of urea. 3 This increase was presumed to be due to the corresponding decrease in water activity, shifting the equilibrium toward urea synthesis, 3 but an effect of the organic solJ H. 2 F. 3 L. 4 C. 5 R. 6 K. 7 A.
A. Krebs and K. Henseleit, Hoppe-Seyler's Z. Physiol. Chem. 210, 33 (1932). W#hler, Poggendorff's Annalen 12, 253 (1828). G. Butler and F. J. Reithel, Arch. Biochem. Biophys. 178, 43 (1977). Hill, J. Chem. Soc. 73, 634 (1898). E. Offord, "Semisynthetic Proteins." Wiley, New York, 1980. Oyama and K. Kihara, CHEMTECH 14, 100 (1984). Kapune and V. Kasche, Biochem. Biophys. Res. Commun, 80, 955 (1978).
METHODS IN ENZYMOLOGY, VOL. 136
Copyright © 1987 by Academic Press, Inc. AI! rights of reproduction in any form reserved.
[22]
UREASE-CATALYZED UREASVNTHEStS
235
vent on ionization a may also be involved. Several problems associated with synthesis by reversal of enzymatic hydrolysis reactions are illustrated by investigation of the effects of reaction conditions on the rate and extent of urea synthesis as catalyzed by urease. Source of Enzyme. Jack bean urease extracted and purified by the standard acetone crystallization procedure 9 with minor modifications ~° was used in demonstrating urease-catalyzed urea synthesis) Utilization of urease from other sources should be possible, although conditions for good reaction rates would have to be established for the particular enzyme used. Conditions which determine the position of equilibrium of the urea hydrolysis reaction (see below) are of course independent of the source of the enzyme. Assays
Urea Synthesis. In order to obtain readily measurable amounts of urea it is necessary to use relatively high concentrations of substrate. Typical conditions are 0.75 M (NH4)zCO3, 5 mM EDTA, and 5 mM 2-mercaptoethanol (no additional buffer is necessary). The urease concentration should be about I Sumner Unit 11 per milliliter. Under these conditions Jack bean urease catalyzes urea synthesis at about 2% of the rate at which it hydrolyzes urea (conditions for urea hydrolysis specified below). Urea concentration is determined spectrophotometrically by a modification ~2of the Fearon reaction.13 Incubated samples, zero time blanks, or urea standards containing up to 1/zmol urea in a total volume of 0.2-1.0 ml in screw-capped tubes are quenched by addition of 5 ml of 52% H3PO4 (600 ml of 85% H3PO4 diluted with water to 1 liter). Samples can be kept at room temperature at this stage for up to an hour without significant loss of urea, so that several samples can be run together through the remainder of the procedure. To each quenched sample is added 1.0 ml of an aqueous solution containing 6 g of 2,3-butanedione monoxime and 0.3 g thiosemicarbazide (both from Aldrich Chem. Co.) per liter. After heating for 30 min on a boiling water bath, samples are cooled to room temperature and their absorbance at 530 nm is determined immediately. The assay is not affected by high concentrations of ( N H 4 ) 2 C O 3 . At smaller sample volumes (0.2-0.5 ml) the apparent extinction coefficient for urea (approxi8 G. A. Homandberg, J. A. Mattis, and M. Laskowski, Jr., Biochemistry 17, 5220 (1978). 9 G. Mamiya and G. Gorin, Biochim. Biophys. Acta 105, 382 (1965). ~0C. M. Kaneshiro, Ph.D. Dissertation, University of Oregon, Eugene (1975). N j. B. Sumner, this series, Vol. 2, p. 378. ~2 M. K. Schwarz, this series, Vol. 17, p. 857. 13 W. R. Fearon, Biochem. J. 33, 902 (1939).
236
IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS
[22]
mately 2 × l06 M -~ c m - 0 is relatively insensitive to the presence of organic solvents in the sample. For samples in which the equilibrium concentration of urea is being determined, it is advisable, after measuring the urea concentration, to add fresh urease and incubate again until the urea concentration is constant. Urea Hydrolysis. For comparison to urea synthesis, urea hydrolysis can be measured by an adaptation of the same assay using 2 mM urea as substrate, in the presence of 0.2 mM EDTA, 5 mM 2-mercaptoethanol, and 20 mM imidazole acetate, pH 7.0. This urea concentration is below the Km for urea 14 so the rate decreases continually throughout the incubation. Data points at four different reaction times are plotted semilogarithmically; the first-order rate constant is determined from the slope. Effect of Organic Solvents on Enzyme Stability The use of organic solvents to enhance the yield of urea is possible only if urease activity can be maintained in the presence of the solvents. Fortunately, Jack bean urease is known to be rather stable in organic solvents such as glycerol.~5 Moreover, it is extracted and crystallized in 32% acetone. 9 In connection with these experiments it was observed that urease in 50% aqueous dimethylformamide, ethanol, or pyridine irreversibly lost most of its activity in a period of a few hours at room temperature. However, activity was recovered in high yield from urease dissolved in 50% aqueous solutions of acetone or polyhydroxylic alcohols such as glycerol and 1,2-propanediol. In an attempt to stabilize Jack bean urease against denaturation and loss of catalytic activity by high concentrations of organic solvents, the enzyme was immobilized and cross-linked by two independent methods. In neither case, however, did immobilized cross-linked urease have improved stability in the presence of organic solvents) Subsequent work was done only with soluble urease. Effect of Organic Solvents on Reaction Rate Organic solvents affect urea hydrolysis in much the same way that they affect urea synthesis .3 The effect of organic solvents on reaction rate can thus be conveniently determined by measuring urea hydrolysis, which requires less enzyme than urea synthesis. The effects are strongly dependent on the nature of the organic solvent, as well as its concentra~4 K. R. L y n n , Biochim. Biophys. Acta 146, 205 (1967). ~5 C. C. Contaxis and F. J. Reithel, J. Biol. Chem. 246, 677 (1971).
[22]
U R E A S E - C A T A L Y Z E D UREA SYNTHESIS
237
tion. The solvents were reagent grade but not redistilled; further purification of the solvents might be warranted. When compared at the same concentration (50%), the rate of urea hydrolysis ranged from undetectable (in ethanolamine) up to equal activity in acetone to that observed in strictly aqueous solution. 3 Rates in 50% aqueous glycerol, dimethylformamide, ethanol, and pyridine were 15, 49, 56, and 65% of the rate in water. In acetone and glycerol, in which urease is stable indefinitely, the activity as a function of organic solvent concentration shows rather different patterns, with most of the activity retained up to 75% acetone and much less activity observed at relatively low glycerol concentrations) Solvent selection for proposed urea synthesis processes wouldrequire optimization of the particular solvent for enzyme stability, reaction rate, and yield of urea (see later section). Effect of Organic Solvent, Substrate, and Hydrogen Ion Concentration on Yield of Urea The equilibrium concentration of urea, determined in solvents in which urease is stable so that achievement of equilibrium can be assured, increases as the water activity (related to concentration) diminishes. The yield of urea at equilibrium is approximately a second-order function of water concentration (activity) as predicted by the equilibrium constant. 3 The yield of urea as a function of (NH4)2CO3 concentration, at constant solvent composition, strongly increases as the substrate concentration increases. 3 In 50% glycerol, the observed urea yield was greater than the theoretical yield (which assumes concentration and activity are equivalent) at low substrate concentrations and lower than the theoretical yield at high substrate concentrations. The yield of urea is greatly enhanced in alkaline conditions) H + is a by-product of urea synthesis from HCO3-, according to Eq. (1). 2 NH4 + + H C O 3 - --0 u r e a + 2 H 2 0 + H +
(1)
Effect of the Nature of the Organic Solvent on Yield of Urea The equilibrium concentration of urea differs greatly depending upon which organic solvents are utilized to replace part of the water. Compared at a concentration of 50%, the equilibrium concentration of urea in aqueous acetone is over 12-fold higher than in aqueous glycerol. 3 Other organic solvents tested gave yields between these values, with hydroxylic solvents generally showing the lowest yields) As determined by measurement of the solubility of urea and ammonium carbonate, the primary effect of organic solvents is on the activity of the salt, rather than urea.
238
IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS
[22]
Use of Organic Solvents to Study the Reaction Mechanism It has been reported that the products of urease-catalyzed urea hydrolysis are ammonium and carbamate ions. 16 Measuring urea synthesis rather than hydrolysis, it is possible to show that maximum initial rates are obtained only if the substrate ammonium bicarbonate has been dissolved for an hour before urease is added; freshly dissolved substrate gives much slower initial rates of urea synthesis s This difference is likely due to a requirement for synthesis from carbamate, which forms nonenzymatically at a slow rate after ammonium bicarbonate is dissolved. In order to prove this point, carbamate concentration should be measured in both fresh and aged substrate solutions, and the rate of synthesis from ammonium carbamate should be directly compared with the rate of urea synthesis from ammonium carbonate. Significance The small amounts of urea synthesized (approximately O. 1% yield) by reversal of the urease reaction are not likely to warrant consideration of this process for large-scale urea production. Urease does, however, provide an interesting model for investigation of the reversal of hydrolytic enzyme reactions, both because of the historical significance of urea synthesis, and because urease illuminates several factors which limit utilization of the technology of reversal of enzymatic hydrolysis. Perhaps most important is the realization that no single set of conditions is optimal for all the parameters affecting urea synthesis. For example, conditions which give high urea yields also give slow rates of synthesis, and conditions in which synthesis is rapid give poor yields. Enzyme efficiency and stability and substrate solubility likewise may be affected differently by organic solvents. Some of these problems may be partially overcome by using mixtures of different organic solvents.
~6R. L. Blakely, J. A. Hinds, H. E. Kunze, E. C. Webb, and B. Zerner, 1991 (1969).
Biochemistry 8,