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Identification of the enol tautomer of imidazolone propionate as the urocanase reaction product
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 220, No. 1, January, pp. 314-317, 1983
COMMUNICATION Identification of the Enol Tautomer of lmidazolone Propionate as the Urocanase Reaction Product’
LARRY
H. MATHERLY
AND
Biochemistry Program, Pennsylvania Laboratory, University Park, Received
July
ALLEN
T. PHILLIPS’
State University, Althouse Pennsylvania 16802
28, 1982, and in revised
form
October
18, 1982
A fluorescent
product was transiently formed during catalysis by urocanase from The fluorophore showed an emission maximum at 430 nm when excited at 330 nm, essentially identical to that exhibited by the enol tautomer of imidazolone propionate. The keto isomer was not fluorescent under these conditions. In aqueous acid solutions where imidazolone propionate is relatively stable, an equilibrium mixture of tautomeric forms contained approximately 1% of the enol isomer. In ethanolic solutions, the equilibrium concentration of enol tautomer increased to approximately 25%. The differing content of imidazolone propionate tautomers as a function of solvent conditions permitted a comparison of the keto and enol forms as substrates for the reverse reaction. This revealed an almost complete preference for the enol tautomer. These results are taken as direct proof that enol imidazolone propionate is the true urocanase reaction product. Pseudomonas
putida.
The second reaction in histidine catabolism involves the conversion of urocanic acid to imidazolone propionic acid (Fig. l), and a bound NAD’ is required for activity of urocanase (urocanate hydratase, EC 4.2.1.49) from Pseudomonas putida (1, 2). Evidence obtained from deuterium isotope labeling studies (3-5) is generally inconsistent with NAD+ functioning in its usual role of facilitating the direct transfer of a hydride ion via NADH formation. Furthermore, recent studies from this laboratory have provided evidence for a different role of NAD+, one in which it participates by formation of a covalent addition compound with urocanate (6, 7). In order to gain a better understanding of the mechanism for NAD+ function, it is important to establish whether the keto or the enol form of imidazolone propionate is the actual product of urocanase
action. The tautomeric identity of the product takes on a particular significance because NAD+ participation in an internal oxidation-reduction process can be most reasonably explained only if the keto tautomer of imidazolone propionate is formed initially (3). Retey and co-workers (4, 5) observed signals at intermediate times during ‘H-NMR monitoring of the urocanase reaction which they attributed to the enol tautomer of imidazolone propionate. These studies, however, did not permit a firm statement as to the origin of this tautomer, i.e., whether its formation preceded, followed, or accompanied that of the keto tautomer. Indeed, Gerlinger et al. (5) simply concluded that the enzymatic product was an equilibrium mixture of racemic keto tautomer and enol form roughly in a 2:l ratio. In this communication we present fluorescence and kinetic evidence that enol imidazolone propionate is the actual urocanase reaction product. Urocanic acid was obtained from Sigma Chemical Company. [%]Urocanic acid was prepared from L[U-“Clhistidine (New England Nuclear) by the method of Mehler et al. (8). N-Formylisoglutamine and 4oxoglutaramate were synthesized according to published procedures (9, 10).
i This investigation was supported by a grant from the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health (AM 13198). ‘To whom correspondence concerning this work should be addressed. 0003-9861/83/010314-04$03.00/O Copyright 0 198.3 by Academic Press, Inc. All rights of reproduction in any form reserved.
314
PRODUCT
FIG. 1. Reaction urocanic acid (I) pionic acid, shown tautomers.
OF
THE
UROCANASE
catalyzed by urocanase, in which is converted to imidazolone prohere as the keto (II) and enol (III)
Urocanase was isolated from P. putida ATCC 12633 as described by George and Phillips (11). The purified enzyme had a minimum specific activity of 1.9 wmol of urocanate consumed per minute per milligram of protein, equivalent to 90% purity or better. The activity assay for urocanase was based on the spectrophotometric measurement of urocanate disappearance at 277 nm, and was conducted as previously described (11). Protein was determined by the method of Groves et al. (12) with bovine serum albumin as the standard. Imidazolone propionate, both labeled and unlabeled, was prepared enzymatically and purified as described by Brown and Kies (13). To obtain solutions of different tautomeric composition, a solution of imidazolone propionate in HCl was evaporated in ~ocuo over PZOB and NaOH at 4°C. The residue was redissolved in either 0.5 M HCl or absolute ethanol, as appropriate, and allowed to equilibrate for approximately 6 h in the dark at 25°C under a Nz atmosphere. Estimation of the enol content of imidazolone propionate solutions was performed by the indirect bromine titration method of Meyer and Kappelmeier (14). Each sample of imidazolone propionate was cooled rapidly to 0°C and treated with 1 ml of 60 mM bromine in ethanol at 0°C. Within 10 s, 2 ml of 60 mM P-naphthol in ethanol was added to consume the excess bromine. The solution was acidified with 1 ml of 1 N HCl and 5 ml of 0.2 M KI was added. The iodine thus liberated was titrated with a standard sodium thiosulfate solution using a starch indicator. Total imidazolone propionate (enol plus keto) was determined from measurements of total nitrogen (15). A study of the reverse urocanase reaction was performed in a Thunberg tube containing 15 pg of purified urocanase in 1.95 ml of 0.1 M Tris-HCl, pH 7.5, with 2 rmol of [i4C]imidazolone propionate (2.14 X lo5 dpm wmol-‘) dissolved in 50 ~1 of 0.1 N HCl or absolute ethanol in the side arm. The buffer was previously evacuated and thoroughly purged with nitrogen. The Thunberg tube was evacuated and flushed with nitrogen prior to commencing the reaction by mixing the contents of the two compartments. After 15 min at 30°C the tube was opened and 1.0 ml of 2 N HCl was added. The mixture was fractionated on
315
REACTION
a column (1.2 X 32 cm) of Dowex 50-X8, H+ (200-400 mesh) with 2 N HCI. Fractions of 2 ml were collected at 7 min/fraction. Imidazolone propionate and its degradation products were eluted before fraction 70 while urocanate did not appear until approximately fraction 90. Samples of 1 ml were removed from each fraction and radioactivity was determined in a liquid scintillation counter using Tritosol scintillation cocktail (16). Efficiencies were calculated by internal standardization with [“Cltoluene. Fluorescence measurements were made in a Farrand Mark I spectrofluorometer. Samples were excited at 330 nm and emission spectra were scanned using lo-nm entrance and exit slits. Absorption measurements were recorded on a Cary Model 17 spectrophotometer. Fluorescence measurements during catalysis revealed the appearance of a material having an emission maximum at 430 nm when excited at 330 nm. This fluorescence increased as urocanate disappeared and gradually diminished upon depletion of substrate (Fig. 2). Since it seemed possible that the fluorescent species was the enol tautomer of imidazolone propionate which formed initially and then isomerized to give a mixture containing predominantly the keto tautomer, we sought to examine this by studying the properties of authentic imidazolone propionate. To test the possibility that the fluorescent material formed during catalysis was enol imidazolone propionate, we determined conditions for selectively stabilizing a particular tautomeric form of imidazolone propionate while minimizing its nonenzymatic decomposition. Imidazolone propionate undergoes an Op-dependent decomposition in neutral aqueous solution (13, 17), yet it can be preserved almost indef-
MINUTES
FIG. 2. Formation of a fluorescent intermediate during urocanase catalysis. Purified urocanase (0.5 mg) was added to a cuvette containing 25 Nmol urocanic acid in 2 ml of Tris-HCI, pH 7.5. Fluorescence emission was recorded continuously at 430 nm (25°C) while the substrate remaining was determined by absorbance at 277 nm, on samples diluted 200-fold into 0.1 M HCl.
316
MATHERLY
initely in acid (10) or in nonaqueous media if kept dark and cold. The absorption spectrum at pH 7.5 for imidazolone propionate stored in 0.5 M HCl solution is shown in Fig. 3. As has been reported previously (13), the compound exhibited an absorption maximum at 260 nm and only slight absorbance above 300 nm. Imidazolone propionate dissolved in absolute ethanol and held in the dark for 6 h under a Nz atmosphere exhibited quite different spectral properties upon dilution into pH 7.5 buffer (Fig. 3). There was strong absorbance at 260 nm but an additional absorption maximum at 330 nm was observed. These spectral changes were reversible, for evaporation of the ethanol, followed by redissolving of the residue in 0.5~ HCl, resulted in a return to the acid-type spectrum (Fig. 3). Indirect bromine titrations were performed on solutions of imidaxolone propionate to establish that the spectral changes were attributable to the reversible formation of the enol tautomer. Titration of samples taken from ethanolic solution revealed that an equilibrium content of 3.6 + 0.1 pmol of enol was present in a solution containing 14.6 pmol of total imidazolone propionate (i.e., 25% enol) while a sample of 14.8 pmol stored in 0.5 M HCl had 0.15 + 0.05 pmol of enol (i.e., 1% enol). Thus, from similar titrations for the solutions used in Fig. 3, we estimated the molar extinction coefficients for enol imidazolone propionate at pH 7.5 to be 8 X lo3 Me’ cm-i at 260 nm and 14 X lo3 M-’ cm-’ at 330 nm, while the keto tau-
,-
0 220
I 260
300 340 WAVELENGTH
380
420
FIG. 3. Absorption spectra for enol and keto imidazolone propionate. Spectra were recorded on samples of enol (- - -) and keto (. ’ -. * ) imidazolone propionate diluted lOO-fold into 50 mM potassium phosphate, pH 7.5. The original solutions, prepared as described in the text, contained 23 pmol of imidazolone propionate in 1 ml of ethanol or acid, of which 22 and l%, respectively, represented the enol tautomer. The spectrum of enol imidazolone propionate redissolved in 0.5~ HCl and allowed to equilibrate 10 h is also shown (-). Wavelength values are given in nm.
AND
PHILLIPS
WAVELENGTH
FIG. 4. Fluorescence emission spectra for enol imidazolone propionate and the fluorescent urocanase product. Spectra were recorded in 50 mM Tris-HCl, pH 7.5, on 0.52 rmol of imidazolone propionate (16% enol) and on a 5-fold-diluted sample from a reaction mixture prepared as described in the legend to Fig. 2. Wavelength values are given in nm.
tomer had values of 2 X 103 and < 100 M’ cm-’ at 260 and 330 nm, respectively. These latter values confirm previous observations (18). A comparison of the fluorescence emission spectra for imidazolone propionate containing 25% enol tautomer and the fluorescent enzymatic product is presented in Fig. 4. The spectra are very similar, both with a broad hand centered at 430 nm, in support of the conclusion that the two fluorescent species are identical. The keto tautomer of imidazolone propionate, obtained from 0.5 M HCl solution, was not fluorescent when examined under neutral pH conditions, nor were its common degradation products, Nformylisoglutamine and 4-oxoglutaramate. From data such as are shown in Figs. 2 and 4, it was calculated that only low levels of enol imidazolone propionate accumulated during catalysis, consistent with its rapid conversion to the nonfluorescent keto product (and also degradation products) at pH 7.5. To confirm this, the rates for the decomposition of the imidazolone propionate tautomers were directly examined. In the presence of oxygen at neutral pH, imidazolone propionate reportedly decomposes with a half-life of 24 min (13). Under anaerobic conditions at neutral pH the compound is more stable although it is still subject to nonenzymatic hydrolysis, generating N-formylisoglutamine (10). The oxygen-independent decomposition of keto imidazolone propionate at pH 7.5, as measured by loss of absorbance at 260 nm, had a half-life of 230 min. The halflife of the enol tautomer, as measured at pH 7.5 by loss of absorbance at 330 nm, was approximately 7 min.
PRODUCT
OF
THE
UROCANASE
If enol imidazolone propionate is the true product of urocanase catalysis, we reasoned that it should be the preferred substrate for the reverse reaction generating urocanate. The extent of urocanate formation was examined in two parallel anaerobic incubations of urocanase at pH 7.5, each with a total of 2 wmol of [“Climidazolone propionate. One reaction initially contained 0.4 pmol of enol tautomer (from ethanolic solution) and 1.6 pmol of keto form, while the other reaction initially contained 0.02 pmol of enol tautomer (from 0.5 N HCI solution), with the remainder being the keto isomer. The quantity of enzyme and reaction time were identical for the two incubations. Analysis of the quantity of urocanate produced in each reaction revealed the following results. When the imidazolone propionate preparation initially containing 0.4 pmol of enol tautomer served as substrate, 0.019 pmol of urocanate was formed in 15 min. This quantity represents roughly two-thirds of the equilibrium value (0.03 rmol) based on a Keq of 1.5 X 10-z for urocanate formation (18). When the imidazolone propionate preparation initially consisted of 0.02 rmol of enol tautomer but contained the same total amount of imidazolone propionate as the other reaction, urocanate was formed to the extent of 0.002 rmol in 15 min. This represents less than 10% of the equilibrium value and illustrates that urocanate production increased in response to an increased level of enol tautomer. An exact proportionality between urocanate formation and initial enol content was not noted, probably because the relatively rapid interconversion between enol and keto forms would alter the actual enol concentration toward the tautomeric equilibrium value, which is not known for pH 7.5 but which is likely to be lower than the ratio of 1 part enol to 4 parts keto used in the more favorable reaction. Data on the rate of tautomeric interconversion are required before a more detailed kinetic analysis of the reverse reaction can be undertaken, but the conclusion that the enol form of imidazolone propionate is the preferred substrate for the back reaction seems justified. The results presented in this report indicate that the enol tautomer of imidazolone propionate is the true urocanase product. Taken together with earlier studies which revealed that hydrogen addition to the urocanate side chain during catalysis is from solvent and not by intramolecular transfer (3-5), it appears very likely that the urocanase reaction does not involve an internal oxidation-reduction with NAD functioning to accept and transfer a hydride ion. We have previously shown that the substrate analog, imidazole propionate (dihydrourocanate), undergoes an enzyme-catalyzed exchange reaction with solvent protons and also reversibly forms a covalent complex with the NAD on urocanase (6). This complex involves addition of the 7 nitrogen of the im-
317
REACTION
idazole portion of imidazole propionate to position 4 on the pyridine ring of NAD. A similar compound appears to be an intermediate when urocanate is substrate as shown by transient-state kinetic studies (7). Although the fluorescent properties of such NAD-imidazole addition compounds are similar to those of imidazolone propionate enol tautomer, the adducts exist only on the enzyme, whereas fluorescence due to enol imidazolone propionate increases long after the time required to reach steady state (Fig. 2) and thus is easily distinguished from fluorescence due to adduct formation. Since reaction sequences which predict NADH as an intermediate on the basis of keto imidazolone propionate as the initial product would now seem to be excluded, it is necessary to direct attention to how the formation of an NAD-urocanate adduct facilitates water addition to the conjugated unsaturation system of urocanate. REFERENCES 1. EGAN, R. M., Chem. 252, 2. KEUL,
AND PHILLIPS, 5701-5707.
V., KAEPPELI,
F., GHOSH,
INSON, J. A., AND 254, 843-851. 3. EGAN,
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J. Biol.
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PHILLIPS,
20, 132-137. J. (1971)
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W. E., AND
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117,629-634.
6. MATHERLY, L. H., DEBROSSE, A. T. (1982) Biochemistry
C. W., AND 21,2789-2794.
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7. MATHERLY, L. H., JOHNSON, A. T. (1982) &o&em&y
K. A., AND 21,2795-2798.
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8. MEHLER,
A. H., TABOR,
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Chem. 10. 11.
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Chem 12.
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4,50-53.
Anal Biochem. 61, 623-627. Anal. B&hem. 63, 555-558.
L. H., AND PHILLIPS, 19, 5814-5818.
COHN, M. S., LYNCH, M. C., AND (1975) B&him. Biophys. Ada
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COMMUNICATION Identification of the Enol Tautomer of lmidazolone Propionate as the Urocanase Reaction Product’
LARRY
H. MATHERLY
AND
Biochemistry Program, Pennsylvania Laboratory, University Park, Received
July
ALLEN
T. PHILLIPS’
State University, Althouse Pennsylvania 16802
28, 1982, and in revised
form
October
18, 1982
A fluorescent
product was transiently formed during catalysis by urocanase from The fluorophore showed an emission maximum at 430 nm when excited at 330 nm, essentially identical to that exhibited by the enol tautomer of imidazolone propionate. The keto isomer was not fluorescent under these conditions. In aqueous acid solutions where imidazolone propionate is relatively stable, an equilibrium mixture of tautomeric forms contained approximately 1% of the enol isomer. In ethanolic solutions, the equilibrium concentration of enol tautomer increased to approximately 25%. The differing content of imidazolone propionate tautomers as a function of solvent conditions permitted a comparison of the keto and enol forms as substrates for the reverse reaction. This revealed an almost complete preference for the enol tautomer. These results are taken as direct proof that enol imidazolone propionate is the true urocanase reaction product. Pseudomonas
putida.
The second reaction in histidine catabolism involves the conversion of urocanic acid to imidazolone propionic acid (Fig. l), and a bound NAD’ is required for activity of urocanase (urocanate hydratase, EC 4.2.1.49) from Pseudomonas putida (1, 2). Evidence obtained from deuterium isotope labeling studies (3-5) is generally inconsistent with NAD+ functioning in its usual role of facilitating the direct transfer of a hydride ion via NADH formation. Furthermore, recent studies from this laboratory have provided evidence for a different role of NAD+, one in which it participates by formation of a covalent addition compound with urocanate (6, 7). In order to gain a better understanding of the mechanism for NAD+ function, it is important to establish whether the keto or the enol form of imidazolone propionate is the actual product of urocanase
action. The tautomeric identity of the product takes on a particular significance because NAD+ participation in an internal oxidation-reduction process can be most reasonably explained only if the keto tautomer of imidazolone propionate is formed initially (3). Retey and co-workers (4, 5) observed signals at intermediate times during ‘H-NMR monitoring of the urocanase reaction which they attributed to the enol tautomer of imidazolone propionate. These studies, however, did not permit a firm statement as to the origin of this tautomer, i.e., whether its formation preceded, followed, or accompanied that of the keto tautomer. Indeed, Gerlinger et al. (5) simply concluded that the enzymatic product was an equilibrium mixture of racemic keto tautomer and enol form roughly in a 2:l ratio. In this communication we present fluorescence and kinetic evidence that enol imidazolone propionate is the actual urocanase reaction product. Urocanic acid was obtained from Sigma Chemical Company. [%]Urocanic acid was prepared from L[U-“Clhistidine (New England Nuclear) by the method of Mehler et al. (8). N-Formylisoglutamine and 4oxoglutaramate were synthesized according to published procedures (9, 10).
i This investigation was supported by a grant from the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health (AM 13198). ‘To whom correspondence concerning this work should be addressed. 0003-9861/83/010314-04$03.00/O Copyright 0 198.3 by Academic Press, Inc. All rights of reproduction in any form reserved.
314
PRODUCT
FIG. 1. Reaction urocanic acid (I) pionic acid, shown tautomers.
OF
THE
UROCANASE
catalyzed by urocanase, in which is converted to imidazolone prohere as the keto (II) and enol (III)
Urocanase was isolated from P. putida ATCC 12633 as described by George and Phillips (11). The purified enzyme had a minimum specific activity of 1.9 wmol of urocanate consumed per minute per milligram of protein, equivalent to 90% purity or better. The activity assay for urocanase was based on the spectrophotometric measurement of urocanate disappearance at 277 nm, and was conducted as previously described (11). Protein was determined by the method of Groves et al. (12) with bovine serum albumin as the standard. Imidazolone propionate, both labeled and unlabeled, was prepared enzymatically and purified as described by Brown and Kies (13). To obtain solutions of different tautomeric composition, a solution of imidazolone propionate in HCl was evaporated in ~ocuo over PZOB and NaOH at 4°C. The residue was redissolved in either 0.5 M HCl or absolute ethanol, as appropriate, and allowed to equilibrate for approximately 6 h in the dark at 25°C under a Nz atmosphere. Estimation of the enol content of imidazolone propionate solutions was performed by the indirect bromine titration method of Meyer and Kappelmeier (14). Each sample of imidazolone propionate was cooled rapidly to 0°C and treated with 1 ml of 60 mM bromine in ethanol at 0°C. Within 10 s, 2 ml of 60 mM P-naphthol in ethanol was added to consume the excess bromine. The solution was acidified with 1 ml of 1 N HCl and 5 ml of 0.2 M KI was added. The iodine thus liberated was titrated with a standard sodium thiosulfate solution using a starch indicator. Total imidazolone propionate (enol plus keto) was determined from measurements of total nitrogen (15). A study of the reverse urocanase reaction was performed in a Thunberg tube containing 15 pg of purified urocanase in 1.95 ml of 0.1 M Tris-HCl, pH 7.5, with 2 rmol of [i4C]imidazolone propionate (2.14 X lo5 dpm wmol-‘) dissolved in 50 ~1 of 0.1 N HCl or absolute ethanol in the side arm. The buffer was previously evacuated and thoroughly purged with nitrogen. The Thunberg tube was evacuated and flushed with nitrogen prior to commencing the reaction by mixing the contents of the two compartments. After 15 min at 30°C the tube was opened and 1.0 ml of 2 N HCl was added. The mixture was fractionated on
315
REACTION
a column (1.2 X 32 cm) of Dowex 50-X8, H+ (200-400 mesh) with 2 N HCI. Fractions of 2 ml were collected at 7 min/fraction. Imidazolone propionate and its degradation products were eluted before fraction 70 while urocanate did not appear until approximately fraction 90. Samples of 1 ml were removed from each fraction and radioactivity was determined in a liquid scintillation counter using Tritosol scintillation cocktail (16). Efficiencies were calculated by internal standardization with [“Cltoluene. Fluorescence measurements were made in a Farrand Mark I spectrofluorometer. Samples were excited at 330 nm and emission spectra were scanned using lo-nm entrance and exit slits. Absorption measurements were recorded on a Cary Model 17 spectrophotometer. Fluorescence measurements during catalysis revealed the appearance of a material having an emission maximum at 430 nm when excited at 330 nm. This fluorescence increased as urocanate disappeared and gradually diminished upon depletion of substrate (Fig. 2). Since it seemed possible that the fluorescent species was the enol tautomer of imidazolone propionate which formed initially and then isomerized to give a mixture containing predominantly the keto tautomer, we sought to examine this by studying the properties of authentic imidazolone propionate. To test the possibility that the fluorescent material formed during catalysis was enol imidazolone propionate, we determined conditions for selectively stabilizing a particular tautomeric form of imidazolone propionate while minimizing its nonenzymatic decomposition. Imidazolone propionate undergoes an Op-dependent decomposition in neutral aqueous solution (13, 17), yet it can be preserved almost indef-
MINUTES
FIG. 2. Formation of a fluorescent intermediate during urocanase catalysis. Purified urocanase (0.5 mg) was added to a cuvette containing 25 Nmol urocanic acid in 2 ml of Tris-HCI, pH 7.5. Fluorescence emission was recorded continuously at 430 nm (25°C) while the substrate remaining was determined by absorbance at 277 nm, on samples diluted 200-fold into 0.1 M HCl.
316
MATHERLY
initely in acid (10) or in nonaqueous media if kept dark and cold. The absorption spectrum at pH 7.5 for imidazolone propionate stored in 0.5 M HCl solution is shown in Fig. 3. As has been reported previously (13), the compound exhibited an absorption maximum at 260 nm and only slight absorbance above 300 nm. Imidazolone propionate dissolved in absolute ethanol and held in the dark for 6 h under a Nz atmosphere exhibited quite different spectral properties upon dilution into pH 7.5 buffer (Fig. 3). There was strong absorbance at 260 nm but an additional absorption maximum at 330 nm was observed. These spectral changes were reversible, for evaporation of the ethanol, followed by redissolving of the residue in 0.5~ HCl, resulted in a return to the acid-type spectrum (Fig. 3). Indirect bromine titrations were performed on solutions of imidaxolone propionate to establish that the spectral changes were attributable to the reversible formation of the enol tautomer. Titration of samples taken from ethanolic solution revealed that an equilibrium content of 3.6 + 0.1 pmol of enol was present in a solution containing 14.6 pmol of total imidazolone propionate (i.e., 25% enol) while a sample of 14.8 pmol stored in 0.5 M HCl had 0.15 + 0.05 pmol of enol (i.e., 1% enol). Thus, from similar titrations for the solutions used in Fig. 3, we estimated the molar extinction coefficients for enol imidazolone propionate at pH 7.5 to be 8 X lo3 Me’ cm-i at 260 nm and 14 X lo3 M-’ cm-’ at 330 nm, while the keto tau-
,-
0 220
I 260
300 340 WAVELENGTH
380
420
FIG. 3. Absorption spectra for enol and keto imidazolone propionate. Spectra were recorded on samples of enol (- - -) and keto (. ’ -. * ) imidazolone propionate diluted lOO-fold into 50 mM potassium phosphate, pH 7.5. The original solutions, prepared as described in the text, contained 23 pmol of imidazolone propionate in 1 ml of ethanol or acid, of which 22 and l%, respectively, represented the enol tautomer. The spectrum of enol imidazolone propionate redissolved in 0.5~ HCl and allowed to equilibrate 10 h is also shown (-). Wavelength values are given in nm.
AND
PHILLIPS
WAVELENGTH
FIG. 4. Fluorescence emission spectra for enol imidazolone propionate and the fluorescent urocanase product. Spectra were recorded in 50 mM Tris-HCl, pH 7.5, on 0.52 rmol of imidazolone propionate (16% enol) and on a 5-fold-diluted sample from a reaction mixture prepared as described in the legend to Fig. 2. Wavelength values are given in nm.
tomer had values of 2 X 103 and < 100 M’ cm-’ at 260 and 330 nm, respectively. These latter values confirm previous observations (18). A comparison of the fluorescence emission spectra for imidazolone propionate containing 25% enol tautomer and the fluorescent enzymatic product is presented in Fig. 4. The spectra are very similar, both with a broad hand centered at 430 nm, in support of the conclusion that the two fluorescent species are identical. The keto tautomer of imidazolone propionate, obtained from 0.5 M HCl solution, was not fluorescent when examined under neutral pH conditions, nor were its common degradation products, Nformylisoglutamine and 4-oxoglutaramate. From data such as are shown in Figs. 2 and 4, it was calculated that only low levels of enol imidazolone propionate accumulated during catalysis, consistent with its rapid conversion to the nonfluorescent keto product (and also degradation products) at pH 7.5. To confirm this, the rates for the decomposition of the imidazolone propionate tautomers were directly examined. In the presence of oxygen at neutral pH, imidazolone propionate reportedly decomposes with a half-life of 24 min (13). Under anaerobic conditions at neutral pH the compound is more stable although it is still subject to nonenzymatic hydrolysis, generating N-formylisoglutamine (10). The oxygen-independent decomposition of keto imidazolone propionate at pH 7.5, as measured by loss of absorbance at 260 nm, had a half-life of 230 min. The halflife of the enol tautomer, as measured at pH 7.5 by loss of absorbance at 330 nm, was approximately 7 min.
PRODUCT
OF
THE
UROCANASE
If enol imidazolone propionate is the true product of urocanase catalysis, we reasoned that it should be the preferred substrate for the reverse reaction generating urocanate. The extent of urocanate formation was examined in two parallel anaerobic incubations of urocanase at pH 7.5, each with a total of 2 wmol of [“Climidazolone propionate. One reaction initially contained 0.4 pmol of enol tautomer (from ethanolic solution) and 1.6 pmol of keto form, while the other reaction initially contained 0.02 pmol of enol tautomer (from 0.5 N HCI solution), with the remainder being the keto isomer. The quantity of enzyme and reaction time were identical for the two incubations. Analysis of the quantity of urocanate produced in each reaction revealed the following results. When the imidazolone propionate preparation initially containing 0.4 pmol of enol tautomer served as substrate, 0.019 pmol of urocanate was formed in 15 min. This quantity represents roughly two-thirds of the equilibrium value (0.03 rmol) based on a Keq of 1.5 X 10-z for urocanate formation (18). When the imidazolone propionate preparation initially consisted of 0.02 rmol of enol tautomer but contained the same total amount of imidazolone propionate as the other reaction, urocanate was formed to the extent of 0.002 rmol in 15 min. This represents less than 10% of the equilibrium value and illustrates that urocanate production increased in response to an increased level of enol tautomer. An exact proportionality between urocanate formation and initial enol content was not noted, probably because the relatively rapid interconversion between enol and keto forms would alter the actual enol concentration toward the tautomeric equilibrium value, which is not known for pH 7.5 but which is likely to be lower than the ratio of 1 part enol to 4 parts keto used in the more favorable reaction. Data on the rate of tautomeric interconversion are required before a more detailed kinetic analysis of the reverse reaction can be undertaken, but the conclusion that the enol form of imidazolone propionate is the preferred substrate for the back reaction seems justified. The results presented in this report indicate that the enol tautomer of imidazolone propionate is the true urocanase product. Taken together with earlier studies which revealed that hydrogen addition to the urocanate side chain during catalysis is from solvent and not by intramolecular transfer (3-5), it appears very likely that the urocanase reaction does not involve an internal oxidation-reduction with NAD functioning to accept and transfer a hydride ion. We have previously shown that the substrate analog, imidazole propionate (dihydrourocanate), undergoes an enzyme-catalyzed exchange reaction with solvent protons and also reversibly forms a covalent complex with the NAD on urocanase (6). This complex involves addition of the 7 nitrogen of the im-
317
REACTION
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