THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 250, No. 13, Issue of Juiy 10, pp. 5033-5040, 1975

Printed in U.S.A.

Histidine Ammonia-lyase

THE USE OF 4-FLUOROHISTIDINE IN IDENTIFICATION OF THE RATE-DETERMINING STEP

(Received for publication, April 30, 1974)

CLAUDE B. KLEE,* KENNETH L. KIRK, LOUIS A. COHEN, AND PETER MCPHIE

From the Laboratory of Biochemical Pharmacology, the Laboratory of Chemistry, and the Laboratory of Bio- chemistry and Metabolism, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Insti- tutes of Health, Bethesda, Maryland 20014

SUMMARY

The (Y, 6 eliminations of NH3 from L-histidine and 4-fluoro- L-histidine by histidine ammonia-lyase appear to occur by similar mechanisms, although a large difference in V,,, for the two reactions was observed. Both reactions were shown to be reversible with an equilibrium constant of 4 to 5. The presteady state kinetics of the deamination of 4-fluoro- L-histidine indicates that the rate-determining step precedes the dissociation of ammonia from the enzyme. The isotope effect of 1.4 to 2.0 observed with 4-fluoro-DL-[fl-2Hz]histidine or DL-@-2Hz]histidine indicates that the C-H bond breakage is at least partially rate-determining for the deamination of both substrates.

Histidine ammonia-lyase effects au oc,/3 elimination of am-

monia from L-histidine to generate [runs-urocanic acid (l-6). It

has been postulated that the a-amino group of the substrate

adds covalcutly to a “dehydroalanyl” residue in the active site

of the enzyme (4, 7) ; removal of the p proton (4, 6) and rupture

of the L\I carbon to nitrogen bond result in the formation of uro-

canic acid and the amino enzyme postulated by Peterkofsky (6)

Histidine + enzyme F? NH%-enzyme + II+ + urocanic acid (1)

NH*-Enzyme + HOH ti enzyme + NH, (2)

(Equations 1 and 2). Reaction 2, initially found to be csperi-

mentally irreversible (6), was later shown to be reversible under

appropriate conditions (8) although at a \ery slow rate. The

existence of an amino-enzyme intermediate was deduced from

the observations that solvent tritium and [Ylurocanic acid were

incorporated into rcisolated I-+&dine, while ‘%Hs was not ill-

corporated (6). Xdtlitional support for the existence of this irl-

termediate is based on the noncompetitive kinetic behavior of

urocanate relative to histitline, as observed by Givot et al. (4).

The existence of the cschange reactions was taken as evidence

that the dissociation of the ammonia moiety from the enzyme

is the slowest step in net product formation. Because the exchange

* Present address, Section on Nucleic Acid Enzymology, Lab- oratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014.

reactions were similar in rate and, under some circumstances,

considerably slower than the over-all reaction, Rose suggested

that a common step, prior to the release of ammonia, was rate-

limiting in both exchanges (9). Furthermore, the stimulatory

effect of metal ions as well as the inhibitory effect of EI)TA on

the elimination reaction (1, 10-15) suggest that metal ions oper-

ate at the rate-determining step. It has also been proposed that

metals operate to “facilitate the deprotonation of the substrate”

(14). The evidence presented in this paper confirms the reversibility

of the over-all reaction and indicates that C-H bond cleavage

is at least partially rcsponsiblc for limiting the rate of deamina-

tion of histidine and of 4-fluorohistitline.

MATISRIALS .4SD MI~:THODS

Materials-L-fiistidine, urocanic acid, and L-[W%]histidine (312 mCi/mmol) were products of Schwarz/Mann. Silica Gel GF and G plates were obtained from Analtech. 2H20 (99.7%) was a product of Aldrich Chemical Co. 3H,0 (100 mCi/g) was purchased from New England Nuclear.

4.Fllloro-oL-histidine and 4-fluoro-L-histidine were synthesized as described previously (16, 17). To obtain 4-fluoro-nL-[3H]histi- dine, diethyl-~-acetamido[4-fluoro-5-imidazoylme~hyl]malor~a~e (17), 315 mg (1 mmol), in 0.3 ml of 3H20 (30 mCi), and 0.3 ml of concentrated hydrochloric acid were heated on a steam bath for 18 hours.’ The resulting 4-fluoro-oL-[a-3H]histidine was isolated and purified by the published procedure (17). The yield was 151 mg (Ucj,), the specific activity, 50 X lo3 cpm/pmol. DL-[~*H& Histidine and 4-fluoro-nr,-[p-2Hz]histidine were synthesized from the corresponding deuterated imidazole-j-methanols; the latter compounds were obtained by reduction of ethyl imidazole-5- carboxylate and of ethyl 4.flnoroimidazole-5-carboxylate with LiA12Ha. Mass spectral analysis demonstrated the presence of 2 deuterium atoms per molecule. The amino acid side chain was elaborated, and the products were purified by the published methods (17). Concentrations of the deuterated and nondeuterated nL-amino acids were measured with an amino acid analyzer, and the concentrations of the L enantiomers were measured enzy- matically with histidine ammonia-lyase (19) under the standard assay conditions at 0.03 and 0.06 mM nL-histidine and 0.2 mg of enzyme/ml or 0.04 and 0.07 rnM 4-fluoro-nL-histidine and 0.9 mg of enzyme/ml.

[U-‘VZ]Urocanic acid was obtained by treatment of L-[U-1X!]- histidine (specific activity 0.06 mCi/mmol) with histidine am- monia-lyase. At the end of the reaction, followed spectrophoto- metrically and by thin layer chromatography, the enzyme was inactivated by heating the mixture at 100” for 2 min, and the pre-

1 A similar method has been reported independently by Thanassi

(18).

5033

by guest on March 18, 2020

http://ww

w.jbc.org/

Dow

nloaded from

5034

cipitated protein was removed by filtration through a Millipore filter (0.45-u Dare size). The urocanic acid was freed of residual histidine on icolumn (1 X 15 cm) of Silica Gel 60 (30 to 70 mesh), eluted with butanol-l/ethyl acetate/acetic acid/water (l/l/l/l), and flash evaporated to dryness. With the solvent described above, all of the radioactive material co-chromatographed on thin layer plates with an authentic sample of urocanic acid. It,s specific ac- tivity was 83 x lo3 cpm/pmol. 4-Fluorourocanate and 4-fluoro[ol- 3H]urocanate were also obtained by treatment of 4-fluorohistidine and 4-fluoro[ol-3H]histidine, respectively, with histidine ammonia- lyase. The enzyme was removed at the end of the reaction by fil- tration through a Schleicher and Schuell collodion bag (No. loo), and the fluorourocanate was recrystallized from the filtrate. Its ultraviolet absorption spectrum at pH 8.0 has a maximum at 286 nm (& nm = 19.4 X 103). Under the same conditions urocanate

as amaximum at 277 nm ($7 “,,, = 18.8 X lo3 (20)). Chromatographic Separation of Histidine Derivatives-Thin

layer chromatography of histidine, 4-fluorohistidine, urocanic acid, and 4-fluorourocanic acid was performed on Silica Gel GF plates with butanol-l/acetic acid/ethyl acetate/water (l/l/l/l). The urocanic acid and its fluoro derivative were localized by their fluorescence, and histidine and 4.fluorohistidine were stained with ninhydrin. Their respective RF values were: urocanic acid, 0.72; 4-fluorourocanic acid, O.Q6; histidine, 0.38; and 4-fluorohistidine, 0.59. ltadioactive compounds were chromatographed in the pres- ence of carriers, and the various spots were scraped and cormted in a Triton toluene scintillation fluid (21). Quenching by the nin- hydrin reagent was between 5% and logo. The recoveries of radio- activity were between 75yo and 80%.

Enzyme Preparations-Histidine ammonia-lyase from Z’seu- domonas ATCC 1129913 was prepared as described previously (22);2 its specific activity was 12 to 14 units/mg. The protein concentra- tion was measured by its absorption at 279 nm with an extinction

17‘ coefficient of ~279 nm = 4.78 (23). Except when stated otherwise, the enzyme was reduced in the presence of 0.001 M dithiothreitol, and the excess thiol was removed by two dialyses against 150 vol- umes each of deionized water. The reduced enzyme was kept under nitrogen (O-4”); the enzyme could be kept in the reduced form for 1 to 2 weeks. Specific activity with and without added thiol was checked prior to the experiment.

Assays-The enzyme was assayed spectrophotometrically at 277 nm and 25” as previously described (10, 13, 20), in 0.1 M Tris- HCl buffer (pH 9.0) containing 0.1 mM MnClt or 0.1 mM CdCl, and 3.3 mM L-histidine, except when stated otherwise. One unit of enzyme catalyzes the formation of 1 pmol of urocanic acid per min under these conditions. Formation of 4-fluorourocanate was fol- lowed at 286 nm. Neither urocanic acid (10) nor 4.fluorourocanic acid exhibits significant changes in extinction coefficient between pH 7.5 and pH 9.0. When a carbonate buffer was used, an initial lag in enzyme activity was observed, and the rates used were ob- tained after 5 min of incubation.

Final estimates of the kinetic constants were obtained after fitting the data to Equation 3 for isotope effects experiments and Equation 4 for linear competitive inhibition by NHaHCO,. The

VA

V=KCA

VA v=

nomenclature used is that of Cleland (24). An interactive curve- fitting program, MLAB, was used with a PDP-10 digital computer

(25). An Aminco-Morrow stopped flow apparatus, mounted on a

Beckman DU monochrometer (26), was used for rapid kinetic experiments. Ultraviolet absorption spectra of the enzyme solu- tions were measured prior to the experiment. The second dialysis fluid was used to dilute the enzyme prior to steady state measure- ments. The substrate solutions contained 0.15 M N-2-hydroxy-

2 We wish to thank David L. Ilogerson, Jr., for growing large batches of cells and carrying out the first steps on the large scale purification.

ethylpiperazine-N’-2.ethanesulfonic acid-NaOH buffer (pH 7.3 or 8.5), 0.2 mM CdClz or 2 rnb% EDTA, and 2 mM L-histidine or 4- fluoro-L-histidine. The reaction was carried out at 27” and was followed at 286 nm for the formation of 4-fluorourocanate and 277 nm for the formation of urocanate. Steady state measurements were done immediately after stopped flow experiments and under the same conditions but at low enzyme concentrations.

Reversibility of Histidine Ammonia-lyase Reaction-lteversibil- ity of the histidine ammonia-lyase reaction was measured by con- version of [‘*C]urocanic acid and 4-fluoro[3H]urocanic acid to [‘4C]histidine and 4-fluoro[3H]histidine, respectively, in the pres- ence of enzyme and NH,HCO,. Incubation mixtures without en- zyme or NH,HCO, served as controls. Aliquots were taken at various times; the reaction was stopped by heating the samples at 100” for 2 min, and 5 ~1 of a 0.02 M solution of histidine or 4-fluoro- histidine was added then as carrier. These aliquots were subjected to thin layer chromatography, and the radioactivity present in the area corresponding to histldme or 4-fluorohistidine was measured. The amount of histidine or 4-fluorohistidine formed was calcu- lated from the known respective specific activities of the sub- strates and corrected for 75 to 85% recovery in order to calculate equilibrium constants. Formation of histidine and fluorohistidine was also measured directly with a Beckman Spinco model 120C amino acid analyzer (27). Protein that precipitated after the heat- ing step was removed by centrifugation, no carrier was added, and the supernatant fluid was lyophilized three times from water to remove NHdHCO,. The residue was suspended in 0.01 N HCl for amino acid analysis. 4-Fluorohistidine was eluted from the long column, using only the pH 3.5 buffer; it eluted at 106 to 107 min, and its color value was 80% that of histidine. The concentrations of urocanic acid and fluorourocanic acid were measured spectro- photometrically as well as by radioactivity. Long term reversibil- ity experiments were done in the presence of toluene, and the in- cubation mixtures were filtered through sterile Millipore filters prior to assays. At the end of these experiments a 50.~1 aliquot of the incubation mixture was placed on plates of supplemented nutrient broth (28) with 1.57, agar; no bacterial growth was de- tected after 24 hours. The specific activity of the enzyme was also measured and found to be between 80 and 9Oo/o of the starting ma- terial.

Exchange Reactions-Incorporation of [U-‘4Clurocanate into histidine and of 4-fluoro[3H]urocanate into 4-fluorohistidine was measured in the presence of 20 mM L-histidine or 4-fluoro-L-histi- dine, respectively, at 25” as indicated in the legend to Fig. 5. The initial rates of product formation and exchange reaction were de- termined as described below. The labeled histidine and 4.fluoro- histidine products were isolated by thin layer chromatography. The specific activity of the urocanate or 4-fluorourocanate during any interval was taken as the average of the specific activity at the beginning and the end of the interval. The urocanic or 4-fluoro- urocanic acids were purified by thin layer chromat,ography, and concentrations were measured spectrophotometrically. At low urocanate concentrations, samples were withdrawn at short time intervals, to avoid large changes in specific activity of the labeled compounds due to deamination of the substrate.

The incorporation of tritium from water into histidine or 4- fluorohistidine at 25” was measured in a complete histidine am- monia-lyase incubation mixture in the absence of added urocanate or 4-fluorourocanate. At various times, aliquots of 50 ~1 were di- luted with 0.5 ml of 50yo acetic acid and lyophilized to constant specific activity from 507, acetic acid (usually four successive lyophilizations). After the last lyophilization, the residue was diluted in the appropriate volume of 0.05 M potassium phosphate buffer (pH 7.8) for spectrophotometric determination of urocanate and 4-fluorourocanate concentrations by their respective absorp- tions at 277 nm and 286 nm. When these samples were subjected to thin layer chromatography, all of the radioact,ivity co-chromato- graphed with histidine or 4-fluorohistidine, respectively.

RESULTS

4-Fluoro-m-hi&line behaves as a strong competitive inhibitor

of hi&dine with a K, of 1.27 m&f, as compared with a K, for

m-hi&dine of 2.7 mM (Fig. 1). It can be seen from Fig. 2 that

4-fluoro-L-histidine is a very poor substrate. Since the imidazole

ring of 4-fluorohistidine is a weaker base than that of histidine,

by guest on March 18, 2020

http://ww

w.jbc.org/

Dow

nloaded from

08

06

I/[OL-HISTIDINEI (M-‘)

FIG. 1. Inhibition of deamination of nn-histidine by 4-fluoro- nn-histidine. The enzyme assays were carried out at 25” in 0.05 M

N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid-NaOH buffer (pH 8.0) containing 0.1 mM MnC12 and 0.67 fig/ml of reduced enzyme. V is expressed as micromoles of product formed per min per mg of protein. 0, no addition; 0, in the presence of 1.6 mM 4-fluoro-on-histidine; and A, in the presence of 3.2 mM 4-fluoro-nn- histidine. The increase in absorbance due to the deamination of 4-fluorohistidine was negligible (0.5 to 1%) compared to the in- creased absorbance due to the formation of urocanate under these conditions. Ki = 1.24 and 1.29 mM 4-fluoro-on-histidine. K, = 2.7 m&r nn-histidine.

PH

FIG. 2. Variation of K, and V,,, with pH. Assays were carried out at 25” in 0.05 M sodium carbonate-bicarbonate buffer. 0.1 mM Cd”+ (0 ), or in 0.05 M sodium carbonate-bicarbonate buffer, 10 mM EDTA (C), and 0.1 M N-2-hydroxyethylpiperazine-N’-2- ethanesulfonic acid (HEPES)-NaOH buffer, 0.1 mM Cd2+ (A), or in 0.1 M HEPES buffer, 10 mM EDTA (A). The indicated pH was measured at the end of each reaction. When L-histidine was used, the enzyme concentration was 0.15 to 0.4 pg/ml, and the reaction was followed at 277 nm. When 4-fluoro-n-histidine was used, the conditions were the same as above, but the enzyme concentrations were 4 to 13 pg/ml, and the reaction was followed at 286 nm. The V,., values are expressed as units per mg of protein, and the K, as millimolarity.

4-Fluoro-L Histldlne

0 3 6 9 12 15 IS

TIME keel

21 24 27 30 R 06

I

L-H,st,d,ne

0 I2 3 4 5 6 7 6 910 2

TIME (set) m

FIG. 3. Rapid kinetics of deamination of 4.fluoro-r-histidine and n-histidine by histidine ammonia-lyase measured by stopped flow technique. Top, deamination of 4-fluoro-r-histidine in 0.05 M N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES)- NaOH buffer (pH 7.3), 0.1 rnM CdCl,, in the presence of O.G3 mg/ml (3.3 nmol) of enzyme (Experiment 1, Table I). The details of the experiments are described in the text. Bottom, deamination of L-

histidine in 0.05 M HEPES-NaOH buffer (pH 7.3) and 1 rnM EDTA, in the presence of 0.38 mg/ml (1.8 nmol) of enzyme (Experiment 4, Table I). Right ordinate, the absorbance change was converted to nanomoles of 4-fluorourocanic or urocanic acid formed per nmol of enzyme tetramer. The zero values correspond to the absorbance of the enzyme at the given wavelength.

by 3.5 pK units,3 the Michaelis constants for both substrates

were measured as a function of $1, as shown in Fig. 2 (right).

The K,-pH curves for the two substrates are very similar in shape. Both Km values are similarly affected by the presence of EDTA, which results in a significant increase in Ii’,,, for both substrates without any change in the pH dependence. The Ti,,, values are shown on the left of Fig. 2; the V,,,, with 4-fluoro-n- histidine is 30 to 100 times smaller than that with L-histidine. The I’,,,,, values for the two substrates show a similar pH de- pendence and are similarly affected by EDTA.

Rapid Kinetic Experiments--The existence of both a hydrogen and a urocanate exchange reaction with histidine had previously been taken as evidence that the slow step in the formation of urocanate is the dissociation of NH3 from the enzyme (6, 9). Since this step is common to both substrates, the difference in V,,,,, slown above indicates that dissociation of ammonia from 1 the enzyme cannot be a common rate-limiting step; alternatively, the slow step in both cases may precede the loss of NHa, or the slow step may be different for the two substrates. The former hypothesis was tested by spectrophotometric measurement of presteady state kinetics, as shown in Fig. 3. At the top of the figure, a rapid kinetic experiment with 4-fluorohistidine is re- ported. No initial burst of activity is detected; if the rate-limiting step which controls the steady state rate followed the formation of 4-fluorourocanate, a burst of activity (29) should result in an increased absorbance of 0.08 to 0.3 at zero time. At all times the observed rate is linear with time and not significantly different from the steady state rate, as summarized in Table I. Further-

3 H. J. C. Yeh, K. L. Kirk, L. A. Cohen, and J. S. Cohen, J. Chem. Sot., in press.

by guest on March 18, 2020

http://ww

w.jbc.org/

Dow

nloaded from

5036

TABLE I

Evidence for absence oj initial burst of activity in deamination of 4.$uoro-L-histidine and L-histidine

Experi- ment No.

Conditions of experimenta

Fluorohistidine, pH 7.3, Cd2+

Fluorohistidine, pH 8.5, Cd2+

Histidine, pH 7.3, Cd2+

Histidine, pH 7.3, EDTA

Histidine, pH

8.5, EDTA

P

‘

roduct formed~

Steady Initial state rate

mol/min/mg

0.08

0.17

4.90

0.50

0.73

0.07

0.14

4.90

0.45

0.57

Ao-AeC Initial bun@

1.007-0.011

0.017

0.070

1.018-O. 05(

0.070

nmol producl/ nmol enzyme

tetremer

I.08 - 0.13

<0.3

2.0

0.5 - 1.5

2.0

a The incubation was carried out at 27” in the presence of the substrates indicated in the table at a concentration of 1 mM and

in 0.05 M N-2-hydroxyethylpiperazine-N’-2.ethanesulfonic acid- NaOH buffer at the pH indicated above. When present EDTA was 1 mM and Cd2+ 0.1 mM. Experiments 1 and 4 are the same as

described in Fig. 3. b The enzyme concentrations for initial rate measurements

were 0.70 to 0.93 mg/ml (3.3 to 4.3 nmol) (Experiments 1 and 2) and 0.38 mg/ml (1.8 nmol) (Experiments 3 to 5). The reaction was

followed for 1 to 30 s. The steady state rates were measured under identical conditions at concentrations of enzyme of 1 to 2 pg/ml. The micromoles of product formed were derived from the increase

in absorbance at 286 nm for fluorourocanate and at 277 nm for urocanate as indicated under “Materials and Methods.”

c Aa, absorbance extrapolated to zero time at 286 nm (Experi-

ments 1 and 2) or at 277 nm (Experiments 3 to 5). A,, measured absorbance due to the enzyme at 286 nm (Experiments 1 and 2) or at 277 nm (Experiments 3 to 5).

d The nanomoles of product formed during the putative initial burst was calculated for the difference of these values: A, - A,.

The concentration of enzyme is expressed in nanomoles of 215,000 molecular weight species.

more, extrapolation to zero time (Table I) clearly indicates that

no initial burst has occurred; the increase in absorption at 286

nm at zero time corresponds to less than 0.3 nmol of fluorouro- canate formed per nmol of enzyme tetramer at 2 pH values and in the presence of metal ions. In the case of fluorohistidine, there- fore, the rate-limiting step under these conditions precedes the dissociation of amrnonia from the enzyme.

The results of the experiments performed with L-histidine are not as conclusive. A representative experiment is shown at the

bottom of Fig. 3. At the earliest time measurable after mixing (25 ms at this electronic band pass setting), the absorbance change is occurring at the steady state rate (Table I), indicating the absence of any process with a rate constant greater than 100 s-i. However, extrapolation to zero time consistently shows an initial absorbance which could correspond to the very fast re- lease of as much as 2 nmol of urocanic acid per nmol of enzyme tetramer. The size of this initial absorbance is not very repro- ducible; at lower pH and in the presence of EDTA, both of which decrease the steady state rate, the zero time absorbance can be decreased to as little as 0.5 nmol of urocanic acid per nmol of enzyme tetramer. In the minimum time period between COW secutive experiments (15 to 20 s) the absorbance of the solution

FIG. 4. Isotope effect on the Michaelis constants of histidine ammonia-lyase. The assays were performed in 0.05 M N-2-hydroxy- ethylpiperazine-N’-2-ethanesulfonic acid-NaOH buffer (pH 8.0) containing 1 mM mercaptoethylamine. The enzyme was reduced by incubation for 10 min at 25“ prior to addition of substrate. The velocities are expressed as the increase in absorbance at 277 nm and 286 nm per min. Top, the substrates were nn-histidine (0) and uL-[P-2H>]histidine (0 ). The incubation mixture contained 0.1 mM CdCl, and 1.0 pg of enzyme/ml (left) and 1 mM EDTA and 2.5 pg of enzyme/ml (right). Bottom, the substrates were 4-fluoro- nn-histidine (0 ) and 4-fluoro-nL-[p-2Hz]histidine (0 ). The incu- bation mixtures contained 0.1 mM CdClz and 67 pg of enzyme/ml (left) and 1 mM EDTA and 120 rg of enzyme/ml (right).

will reach a high value (Fig. 3); therefore, we believe that the initial absorbance is an artifact, resulting from incomplete re- moval of product from the observation chamber, prior to the mixing of enzyme and substrate solutions for the next experi- ment. This objection is not true for 4-fluorohistidine experi- ments, because of the slow reaction rate.

Isotope E$ects-The absence of a demonstrable initial burst of activity in the presteady state kinetic experiments described above indicates that the rate-determining step in the deamina- tion of 4-fluorohistidine could correspond to the breaking of the C-N bond, the C-H bond, or to a concerted mechanism. In the latter pathways, substitution of the hydrogen in the fi posi- tion by a deuterium should decrease the rate of the over-all re- action. Lineweavcr-Uurk plots of the velocity of the reaction as a function of concentration of 4-fluoro-nn-histidinc and of 4- fluoro-un-[P-*Hz]histidine are shown in Fig. 4 (bottom). The Michaelis constants derived from these experiments are sum- marized in Table II. Isotopic replacement of the fl-hydrogens does not affect the K, values, but does decrease li,,, both in the presence of metal ions and of EDTA. Isotope effects of 1.7 and 2.0 were calculated. As showrl in Fig. 4 (top), isotope effects of 1.4 and 1.5 are observed with nn-histidine and its p deuterated analog. These findings suggest that the mechanism of the reac- tion is similar with both substrates and that breaking of the CO-

valent C-H bond determines at least partially the reaction rate. Since an exchange of 3H20 with histidine was observed pre-

viously for the deamination of histidine (6), the rate of deamina- tion of the deuterated substrate in *HZ0 was compared with that of nn-histidine in HZ0 as a function of pH and pD, in order to eliminate the possibility that some exchange of deuterium in the p position will decrease the isotope effect under the condi- tions described above. No significant change in the isotope effect was observed under these conditions (data not shown).

Exchange Reactiolzs-Peterkofsky has shown that solvent tritium and [i4C]urocanic acid can be incorporated into reisolated

by guest on March 18, 2020

http://ww

w.jbc.org/

Dow

nloaded from

5037

TABLE II

Michaelis constants with uL-histidine, oL-[P-2Hz]histidi12e, Q-~uoro-DL-histidilzc, and 4-~uoro-DL-[P-2H*]hiStidi?ze

EX- peri- merit NO.

Substrate Km"

mnrb

DL-Histidine 10.0 Zt 1.0 nL-[@-2H2]Histidine 11.2 f 2.0

DL-Histidine 2.7 + 0.2 nL-[fl-2Ht]Histidine 2.4 + 0.2

4-Fluoro-DL-histidine 6.4 + 1.0 4-Fluoro-o~-[/3-~H& 6.9 z!c 1.0

histidine

4-Fluoro-DL-histidine 1.25 f 0.1: 4.Fluoro-DL-[p-%I- 1.3 + 0.15

histidine

0

0

i

,

u?.ils/mg

3.04 + 0.13 2.07 & 0.20

11.60 + 0.26 8.30 f 0.20

.0068 z?z 0.0004 .0034 f 0.0003

0.092 It 0.003 0.055 z!c 0.002

,” s1 :@ fib

b

1.5

1.4

2.0

1.7

a The experimental conditions were as described in Fig. 4. The Michaelis constants were obtained by the curve-fitting method described under “Materials and Methods.”

* The concentrations correspond to that of the DL mixture. c Experiments 1 and 3 were done in the presence of 1 mM EDTA

and Experiments 2 and 4 in the presence of 0.1 mM CdC12.

hi&line by exchange reactions (6). The rate of the exchange reactions below pH 8 was found to exceed that of net product formation. A study of the initial rates of urocanate and 3Hz0 cxchauge was undcrtakcu to determine if proton retnoval prc- cedes, follows, or is simultaneous with C-N bond cleavage.

At p1-I 7.7, urocanatc cschangc is readily detectable (Fig. 5); under these csontlitions, both exchange and product formation are inhibited by high urocanate concentrations. The rate of fluorourocanate cschange is much lower (Fig. 5, right). 1Jntler conditions in which the rate of product formation is one-tenth that observed with hjstidiue, the rate of 4-fluorourocanate ex- chauge is 250 tirncs smaller thau [*Y,‘]urocanate exchange. Fur- thermore, the optimum concentratiou of 4-fluorourocanate for the exchange reaction (not reached) is higher than that of uro- canate (Fig. 5).4

The rate of the tritium exchange reaction is shown in Fig. 6 for both L-hi&dine and 4-fluoro-L-histidine. As in the urocanic acid exchange, the rate of tritium exchange is much smaller with 4-fluorohistidine than with histidine. For both exchanges, the reduced rates with the fluoro analog reflect the considerably lower value of V,,,. It can also be noted that the rate of tritium exchange is uot greatly affected by urocanate concetltration. At low pH, the rate of tritium eschange is linear, even at early times. When % exchange and [‘%]urocanate exchange into his- tidirle were tneasured simultaneously, the rate of 3H exchange exceeds slightly that of [YZ]urocanate exchange (Table III).

Reversibility of Reaction-The esistence of these exchange reactions and the lack of exchange of ammonia into histidiue had led to the conclusion that the reaction occurs in two steps (6), a

4 The net synthesis of [l%]histidine from [‘4C]urocanate and 4-fluorohistidine (as the source of NH3) was also demonstrated. Under the conditions described in Fig. 5, and in the presence of 20 mM 4-fluorohistidine and 1 mM [%]urocanate, the rate of [14C]- histidine was found to be 0.015 Mmol/min/mg of enzyme. Under the same conditions, no exchange was observed with 4-fluorouro- canate. This experiment provides further support for the existence of a common amino enzyme intermediate.

A. Htstldme

I I I I

2 4 6 0

1 A-I-*+--]f 2 4 6 0 IO 12

[UROCANATE~ (mM) w” % [4-FLUOROUROCANATE] (mM)

ii 3.

FIG. 5. A, dependence of the urocanic acid exchange reaction on urocanic acid concentration. The exchange reaction was carried out in 0.05 M N-2-hydroxyethylpiperaxine-N’-2-ethanesulfonic acid-NaOH buffer (pH 7.7) containing 0.1 mM Cd2+, 1 mM mercap- toethylamine, 20 mM L-histidine, and increasing concentrations of [‘%]urocanic acid as indicated in the figure, with 0.05 mg of enzyme/ml in a final volume of 25 ~1 for 0,3, and 5 min (1 and 2 mM urocanate); 0, 5, 10, and 20 min (4 and 8.5 mM urocanate). The re- action was stopped by heating for 3 min at 100”. The net formation of urocanic acid was measured by the increased absorption at 277 nm on a suitably diluted aliquot prior to boiling. The incorpora- tion of radioactivity into histidine was determined after thin layer chromatography as indicated under “Materials and Methods.” The initial rates were determined and are plotted in the ordinate. Urocanic acid formed per min per mg of enzyme, n ; [‘Glurocanate incorporated int,o histidine per min per mg of enzyme, 0. B, de- pendence of 4-fluorourocanic acid exchange on 4-fluorourocanic acid concentration. The experimental conditions were as described above, with the following exceptions. The enzyme concentration was 0.5 mg/ml, and the incubation times were 30 and 60 min (5 and 7.5 mM 4-fluorourocanate) and 60 and 120 min (8.5 and 11 mM 4-fluorourocanate). Increased absorption due to 4-fluorourocanate formation was measured at 286 nm. 4-Fluorourocanate formed per min per mg, A; 4-fluoro[3H]urocanate incorporated into 4-fluoro- histidine per min per mg, l

reversible formatiou of an amino enzyme intermediate and a slow irreversible dissociation of the amino enzyme, accounting for the apparent irreversibility of the over-all reaction (6). How- ever, it was subsequently shown that after prolonged incubation, the reaction is reversible with an equilibrium constant of 3 to 5 (8). To clarify this situation, the inhibition of urocanate for- mation by NH4HC03 in the presence of EDTAS was studied. The results could all be expressed as linear double reciprocal plots, compatible with Tc’Hs being a competitive inhibitor with respect to histidine. However, the nature of the inhibition could not be proved unequivocably, due to the high apparent Ki of NH4HC03 (0.2 M). We were able to confirm the reversibility of the reaction with urocanic acid as well as with 4-fluorourocanic acid within short incubation times by use of high concentrations of enzyme. The time course of such reversibility experiments is summarized in Table IV. The equilibrium constants derived from these ex- periments are similar for both substrates and are comparable to those published earlier for urocanic acid (8).

The dependence of the rate of the reverse reactiou on the con-

5 In view of the ability of NHaHCO, to bind the metal activators (37) and thereby inhibit the enzyme noncompetitively, the reac- tion was carried out in the presence of EDTA.

by guest on March 18, 2020

http://ww

w.jbc.org/

Dow

nloaded from

III 4-Fluor0[~H]uro-

FIG. 6. Incorporation of solvent tritium into histidine and 4- canate (0.8 mM

fluorohistidine during the histidine ammonia-lyase reaction. The incubation mixture was 0.05 M N-2-hydroxyethylpiperazine-N’-2- ethanesulfonic acid-NaOH buffer (pH 7.8) (top), or 0.05 M potas- sium phosphate buffer (pH 7.2) (bottom), 0.1 mM Cd”+, 1 mM mer- IV 4-Fluoro[3H]uro- captoethylamine, 0.08 mg of enzyme/ml for histidine deamination canate (12 mM) or 0.8 mg/ml for 4.fluorohistidine deamination, and 1 mCi of 3Hz0 in a final volume of 0.2 ml. The enzyme was incubated for 10 min at 25” prior to addition of either substrate at a concentration of 40 mM. The net formation of products, urocanate (0, 0) and . . ^..

TABLE IV

Reversibility of histidine ammonia-lyase reaction

Substrate

I [W]Urocanate (9 mM)

II Urocanate” (60 mM)

fiuorourocanate (w, q ), and the incorporation of trltlum Into histidine (A, A) and 4-fluorohistidine (v, v) was measured as described under “Materials and Methods.”

TABLE III

Comparison of tritium and urocanate exchange reactions

Incubation time? Urocanic acid Urocanic acid Tritium concentration incorporationb incorporation”

p?tZOl/?d

Zero time. 4.00 0.15 25min............. 5.10 2.05 Differences.. 1.10

~ ~

0.10 2.50

1.90 2.40 -

a The incubation mixture was 0.05 M potassium phosphate buffer (pH 7.2), 0.1 mM Cd %+, 1 rnM mercaptoethylamine, 20 mM L-histi- dine, and 4 mM [Wlurocanic acid and contained 0.016 mg of en- zyme and 1 mCi of 3H,0 in a final volume of 0.2 ml. The reaction was carried out as described in Fig. 6 and under “Materials and Methods.”

b Urocanic acid incorporation and tritium incorporation into histidine were measured after separation by thin layer chroma- tography. The specific activity of urocanic acid after 25 min of incubation was corrected for the increase in concentration and the decrease in radioactive material, and the specific activity used to calculate the micromoles per ml at 25 min was the average of the specific activities at zero time and at 25 min.

centration of urocanic acid and of 4-fluorourocanic acid was also measured. In contrast to the forward reaction, where the K, of histidine is higher than that of 4-fluorohistidine, the estimated K, for 4-fluorourocanate (5 mM) is higher than that for urocanate (3 md. vm,, values for the back reaction with these substrates (0.002 and 0.007 uriits/mg, respectively) are much smaller than those of the forward reaction. Furthermore, the rate of forma- tion of histidine is not significantly affected bJ- IGDTA, but the formation of 4-fluorohistidine is reducctl to undetectable levels in the presence of the metal-binding agent. Since both rcvcrse reactions were not performed in the presence of saturating corn

centrations of NH3, these values of V max arc not true constants.

Inc;libma:ion

hrs

0

1 6

20

pmol/ml

0.046 0.058 0.100 0.061 0.293 0.056 0.270 0.074

36 2.920

0 0.008 2 0.019 7 0.026

24 0.031

0 0.030 2 0.168 7 0.288

24 0.360 36 0.372

Product formed“

+ Enzyme - Enzyme

0.031

0.002 0.004 0.004

<0.002

0.02 <0.02

0.02 <0.02 <0.02

4.8 5.2

5.1

4.0

5.0

a The assay mixture containing 0.03 M N-2-hydroxyethylpiper- azine-N’-2-ethanesulfonic acid-NaOH buffer (pH 7.7),6 0.1 mM Cd2+, 0.16 M NHaHCOs, and urocanate or [Wlurocanate (specific activity 83,000 cpm/pmol) or 4-fluoro[3H]urocanate (specific ac- tivity 50,000 cpm/Mmol) as indicated, was incubated at room tem- perature in the presence of 2 ~1 of toluene/0.16 ml with and with- out enzyme. The enzyme concentrations were 0.2 mg/ml (I and II), 0.4 mg/ml (III), and 1 mg/ml (IV). Histidinc or 4-fluorohis- tidine were determined as indicated under “Materials and Meth- ods,” 15.‘to 75-J aliquots for each time point.

b The concentration of NHdHC03 was 0.26 M; histidine content was determined with an amino acid analyzer on a 3OOJ aliquot.

As argued below, their difference should be far greater than ob- served.

DISCUSSIOS

Substitution in various positions of the imidazole ring of L-

histidine yields analogs with substrate properties markedly dif- ferent from those of the natural substrate for hi&dine ammonia- lyase. For example, 2-fluorohistidinc is a substrate, with K, =

0.02 M, L-NT-methylhistidine is a weak competitive inhibitor, Ki = 0.1 nz, and L-N’T-meth.lhistidilie is ncithcr a substrate nor an inhibitor. In contrast, 4-fluorohistidiue is a competitive irl- hibitor with a Ki lower than the K, of histidine (Fig. 1). The 4-fluoro compound is alTo a substrate for the cnzymc; since its rate of deamination is 30. to 100.fold smaller than that of histi- dine, this analog provides a useful tool for study of the mecha- nism of the enzymic reaction.

It is conceivable that the low rate of deamination in the case of the 4-fluoro compound might result from an inhomogeneity in the enzyme preparation, only a small fraction of the popula- tion being active with the analog. However, it seems unlikely that the I<, of the analog would give such good agreement with its Ki with regard to histidine, if heterogeneity were the case. Such agreement is to be expected, of course, if it is both substrate and inhibitor for the same enzyme. Other results also argue against this notion.

6 After addition of NHaHCO, the observed pH was 8.1.

by guest on March 18, 2020

http://ww

w.jbc.org/

Dow

nloaded from

The absence of a detectable initial burst of activity in the rapid kinetic experiments (Table I) indicates that the rate-de- termining step, in the case of 4-fluorohistidine, camlot follow the formation of 4-fluorourocanic acid (29) and probably involves C-H or C-N bond cleavage, or both, in a concerted mecha- nism. The stopped flow data with histidine are less conclusive because the fast rection rate makes the measurement of zero time absorbance less accurate. However, since the deamination of both substrates is stimulated by metal ions (11-15) and 1’,,,, values are reduced by EDTA to a similar extent for each, even at high pH, formation of product (the cx ,fl elimination reaction) is most likely the common rate-limiting step.

The observation that both substrates show small but repro- ducible P-deuterium isotope effects, 1.4 to 2.0, suggests that cleavage of a C-H bond determines at least partially the reac- tion rate in each case (30). However, the small size of the effects, particularly for histidine, indicates that secondary isotope ef- fects should also be considered (31). Thus, in a carbonium ion mechanism, the rate of C-N bond cleavage could be reduced by the two fi-2H atoms through hyperconjugation; C-H bond cleavage may also be reduced by the nonleaving 2H. An equi- librium effect, due to the presence of this atom in the urocanate produced, could also be postulated. Secondary isotope effects arising from such causes are usually even srnaller than those re- ported here, particularly for 4-fluorohistidine (31). In the deami- nation of a mixture of L-[U-14C]histidine and (P-S)-L-[3Hl]histi- dine, a small but reproducible isotope discrimination (1.1 to 1.3) was observed. This would be expected to be even larger in the absence of the tritium exchange reaction. A similar isotope discrimination was reported in the deamination of (&!+L-

[3Hl]phenylalanine and of (/KS-I-[3Hl]tyrosine by phenylalanine ammonia-lyase. Hanson et al. suggested a concerted elimination mechanism for both substrates (5, 32, 33). Such results, together with those obtained with 4-nitro-L-histidine7 are more compati- ble with a primary isotope effect. A final caveat, in the inter- pretation of all isotope effects in enzymic reactions, is that loss of a substrate proton from some group on the enzyme may be kinetically important; in such a case, the size of the isotope ef- fect might be expected to be independent of the substrate used.

If the reaction is considered to follow an “ordered uni-bi” mechanism (in the terminology of Cleland (5, 24)), it is possible to estimate V,,,, for the reverse reactions, using the Haldane relationship

K,, = vZ;,;;Ki,h-~a

2 h,s

where K,, is the equilibrium constant, 8, and 112 are the maxi- mum velocities for the forward and back reactions, and the other K values are the Michaelis constants for each substrate. From the data in Fig. 2 and Table IV, the values obtained are 0.04 and 0.0025 units/mg for urocanate and 4-fluorourocanate, respec- tively. The experimental results are in proper order and indicate that formation of an amino enzyme intermediate, while relatively slow, is probably not the rate-limiting step of the reverse reac- tion for the two substrates. Whereas EDTA markedly inhibits both forward reactions, it inhibits the reverse reaction with fluorourocanate much more than with urocanate. This result is puzzling and is being explored further.

In the rate-lirniting LY ,p elimination, the slow step may be rupture of the C-H or C-N bond, or concerted rupture of both.

7 No isotope effect was observed in the deamination of 4-nitro- L-[fl-2Hz]histidine, in which the p proton is labilized by the sub- stituent (34).

5

6. 7. 8.

9. 10.

11.

12.

13. 14.

15. 16.

17.

5039

i2s shown in Table III, tritium exchange into histidine is some- what faster than urocanic exchange. If isotope discrimination were taken into account, the difference could be even greater. This result, together with the facts that tritium exchange can be observed under initial rate conditions which do not support significant urocanate exchange (Figs. 5 and 6), and that the rate of tritium exchange is not significantly dependent on the uro- canate concentration , suggests that C-H bond cleavage may precede C-N bond cleavage slightly (9). Separation of the steps may not be sufficient to invoke a carbanion mechanism, as postu- lated by Bright (35) for the deamination of P-methylaspartate. In the latter case, C-N bond cleavage is considered rate-limit- ing, and a large tritium exchange is inhibited by the elimination product, mesaconate. Alternatively, if the rate of urocanate dis- sociation is not significantly faster than the rate of the preceding elimination reaction, a difference in the rates of the urocanate and tritium exchanges could also be observed in a concerted mechanism.

It is not clear why 4-fluorohistidine is such a poor substrate for the enzyme. Although the fluorine atom has a marked effect in depressing the basicity of the imidazole ring, it has little effect 011 that of the primary amino group.3 The absence of a signifi- cant isotope effect or1 the K, values of both substrates suggests that both may be essentially dissociation constants. If so, the analog seems to bind to the enzyme more strongly than does histidinc. Similarities in isotope effects and in the inhibitory ef- fect of EDTA suggest that the mechanism of the reaction is the same in both cases. Perhaps the differences in V,,, result from the inability of the analog to force the enzyme into an active form, as required by the “induced-fit” theory (36).

Acknowledgments-We wish to thank Lawrence A. LaJohn and Lasava S. Tidwell for their skillful assistance in carrying out some of the experiments, George Poy, who kindly performed the amino acid analyses, and William R. Landis for measurements of the mass spectra. We are especially grateful to John E. Folk for his critical reading of the manuscript.

REFERENCES

T~BOR, H. (1954) Pharmacol. Rev. 6, 299-343 KUROGOCHI, V., FUKUI, Y., NaKAGAWA, T., AND YAMAMOTO,

I. (1957) JaD. J. Pharmacol. 6. 147-152 RET&, J., F&z, H., AND ZEY&MAKER, W. P. (1970) FEBS

Lett. 6, 203-205 GIVOT, I. L., SMITH, T. A., AND ABELES, R. H. (1969) J. Biol.

Chem. 244, 6341-6353 HANSON, K. R., AND HAVIR, E. A. (1972) in The Enzymes

(BOYER, P. I)., ed) 3rd Ed, Vol. 7, pp. 75-166, Academic Press, New York

PETER&SKY, A. (1962) J. Biol. Chem. 237, 787-795 WICKNER. R. B. (1969) J. Biol. Chem. 244, 6550-6552 WILLISM~, V. R.. AND ‘HIROMS, J. M. (196i) Biochim. Biophys.

Acta 139, 214-il6 ROSE. I. A. (1966) Annu. Rev. Biochem. 36. 47-52 ME&R, A. H., A&D TABOR, H. (1953) J. Biol. Chem. 201,775-

784 PETERKOFSKY, A., AND MEHLER, L. N. (1963) Biochim. Bio-

phys. Acta 73, 159-162 CORNELL, N. W., AND VILLEE, C. A. (1968) Biochim. Biophys.

Acta 167, 172-178 RECHLER, M. M. (1969) J. Biol. Chem. 244, 551-559 GIVOT, I. L., MILDVAN, A. S., AND ABPLES, R. H. (1970) Fed.

Proc. 29, 1590 KLEE, C. B. (1972) J. Biol. Chem. 247, 1398-1406 KIRK, K. L., AND COHEN, L. A. (1971) J. Amer. Chem. Sot. 93,

3060 KIRK, K. L., AND COHEN, L. A. (1973) J. Amer. Chem. Sot.

96, 4619-4624; J. Org. Chem. 38, 3647-3648

by guest on March 18, 2020

http://ww

w.jbc.org/

Dow

nloaded from

5040

18. THANASSI, J. W. (1971) J. Org. Chem. 36, 3019-3021 19. HASSALL, H. (1970) Anal. Biochem. 36, 335-345 20. TABOR, H., AND MEHLER, A. H. (1955) Methods Enzymol. 2,

228-233 21. PATTERSON, M. S., AND GREENE, R. C. (1965) Anal. Chem. 37,

854-857 22. KLEE, C. B. (1970) J. Biol. Chem. 246, 31433152 23. KLEE, C. B., AND GLADNER, J. A. (1972) J. Biol. Chem. 247,

8051-8057 24. CLELAND, W. W. (1963) Biochim. Biophys. Acta 67, 104-137 25. KNOTT, G. I)., AND REECE, D. K. (1971) Modellab User Docu-

mentation, Division of Computer Research and Technology Report, National Institutes of Health, Bethesda, Maryland

26. SUMMERS, M. R., AND MCPHIE, P. (1972) Biochem. Biophys. Res. Commun. 47, 831-837

27. SPACKMAN. D. H. (1967) Methods Enzumol. 11. 3-15 28. HANSON, &. S., B&IA&A, J., AND”%XJLMA;STER, J. (1964)

Biochem. Biophys. Res. Commun. 17, l-11

29. HARTLEY, B. S., AND KILBY, B. A. (1954) Biochem. J. 66, 288- 297

30. WALSH, C., LOCKRIDGE, O., MASSEY, V., AND ABELES, R. (1973) J. Biol. Chem. 248, 7049-7054

31. JENCKS, W. P. (1969) in Catalysis in Chemistry and Enzymology, pp. 243-281, McGraw-Hill Book Co., New York

32. WIGHTMAN, R. H., STAUNTON, J.. B~TTI~;~~sBY, A. It., AND HAN- SON, K. It. (1972) J. Chem. Sot. 2355-2364

33. STRANGE. P. G.. STAUNTON. J.. WILTSHIRE. H. It.. BATTERSBY. A. R., HANSON, K. R., AND HAVIR, E. A.‘(1972) >. Chem. Sot: 2364-2372

34. KLEE, C. B., KIRK, K. L., MCPHIE, P., AND COHEN, L. A. (1974) Fed. Proc. 33, 1318

35. BRIGHT, H. J. (1964) J. Biol. Chem. 239, 2307-2315 36. KOSHLAND. D. E. (1960) Adv. Enzumol. 22.45-97 37. MARTELL, ‘A. E., END ‘CALVIN, Ik (1952) in Chemistry of the

Metal Chelute Compounds, pp. 189-190, Prentice-Hall, Engle- wood Cliffs, New Jersey

by guest on March 18, 2020

http://ww

w.jbc.org/

Dow

nloaded from

C B Klee, K L Kirk, L A Cohen and P McPhierate-determining step.

Histidine ammonia-lyase. The use of 4-fluorohistidine in identification of the

1975, 250:5033-5040.J. Biol. Chem. 

  http://www.jbc.org/content/250/13/5033Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/250/13/5033.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on March 18, 2020

http://ww

w.jbc.org/

Dow

nloaded from