Báo cáo Y học: Characterization of the active site of histidine ammonia-lyase from Pseudomonas putida pptx

Characterization of the active site of histidine ammonia-lyase fromPseudomonas putida Dagmar Ro¨ther1, La´szlo´ Poppe2, Sandra Viergutz1, Birgid Langer1and Ja´nos Re´tey1 1 Institute for

Characterization of the active site of histidine ammonia-lyase from

Pseudomonas putida

Dagmar Ro¨ther1, La´szlo´ Poppe2, Sandra Viergutz1, Birgid Langer1and Ja´nos Re´tey1

1 Institute for Organic Chemistry, University of Karlsruhe, Germany;2Institute for Organic Chemistry, Budapest University of Technology and Economics, Hungary

Elucidation of the 3D structure of histidine ammonia-lyase

(HAL, EC 4.3.1.3) from Pseudomonas putida by X-ray

crystallography revealed that the electrophilic prosthetic

group at the active site is

3,5-dihydro-5-methylidene-4H-i-midazol-4-one (MIO) [Schwede, T.F., Re´tey, J., Schulz,

G.E (1999) Biochemistry, 38, 5355 – 5361] To evaluate the

importance of several amino-acid residues at the active site

for substrate binding and catalysis, we mutated the

following amino-acid codons in the HAL gene: R283,

Y53, Y280, E414, Q277, F329, N195 and H83 Kinetic

measurements with the overexpressed mutants showed that

all mutations resulted in a decrease of catalytic activity The

mutants R283I, R283K and N195A were < 1640, 20 and

1000 times less active, respectively, compared to the single

mutant C273A, into which all mutations were introduced

Mutants Y280F, F329A and Q277A exhibited < 55, 100 and

125 times lower activity, respectively The greatest loss of

activity shown was in the HAL mutants Y53F, E414Q,

H83L and E414A, the last being more than 20 900-fold less

active than the single mutant C273A, while H83L was

18 000-fold less active than mutant C273A We propose that the carboxylate group of E414 plays an important role as a base in catalysis To investigate a possible participation of active site amino acids in the formation of MIO, we used the chromophore formation upon treatment of HAL with

L-cysteine and dioxygen at pH 10.5 as an indicator All mutants, except F329A showed the formation of a 338-nm chromophore arising from a modified MIO group The UV difference spectra of HAL mutant F329A with the MIO-free mutant S143A provide evidence for the presence of a MIO group in HAL mutant F329A also For modelling of the substrate arrangement within the active site and protonation state of MIO, theoretical calculations were performed Keywords: histidine ammonia-lyase; HAL 3,5-dihydro-5-methylidene-4H-imidazol-4-one; MIO; site-directed mutagenesis

Histidine ammonia-lyase (HAL, EC 4.3.1.3) is the first

enzyme in the nonoxidative degradation pathway of

L-histidine The enzymic catalysis begins with a Friedel –

Crafts-type reaction, which helps to transformL-histidine to

trans-urocanate (reviewed in [1]) An analogous mechanism

was proposed for the reaction catalysed by the homologous

enzyme phenylalanine ammonia-lyase (PAL, EC 4.3.1.5)

which convertsL-phenylalanine into trans-cinnamic acid, a

precursor of a great variety of phenylpropanoids [2]

Approximately 30 years ago it was postulated that a

dehydroalanine residue at the active site of both enzymes

acted as electrophilic prosthetic group [3 – 5] Mutagenesis

experiments showed that this dehydroalanine is

post-translationally formed from serines 143 and 202 of HAL

and PAL, respectively [6,7] More recently, the X-ray

structure of HAL was solved at 2.1 A˚ resolution [8] It was shown that the prosthetic group is not dehydroalanine but a 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) It was proposed that this group is formed by cyclization of

an intramolecular 142ASG144 tripeptide followed by subsequent elimination of two molecules of water (Fig 1)

A similar mechanism was proposed for the formation of the p-hydroxy-benzylidene-imidazol-5-one fluorophore

of the green fluorescent protein from Aequorea victoria [9] Co-crystallization was possible neither with the substrate nor with an inhibitor and therefore the exact binding mode forL-histidine could not be solved by crystal structure analysis In this work we describe the preparation

of HAL mutants in which a number of active site amino-acid residues have been changed to evaluate their importance in substrate binding or catalysis Irreversible inhibition with

L-cysteine and formation of a 338-nm chromophore [10,11] and UV difference spectra [12] were also measured to see whether a MIO group is present at the active sites of the enzyme variants

M A T E R I A L S A N D M E T H O D S

Bacterial strains and plasmids HAL was overexpressed in E coli BL21 (DE3) cells The gene for HAL from Pseudomonas putida was subcloned in the expression vector pT7-7 [6]

Correspondence to J Re´tey, Institute of Organic Chemistry, University

of Karlsruhe, Richard-Willsta¨tter-Allee, D-76128 Karlsruhe.

Fax: 1 49 721 6084823, Tel 1 49 721 6083222,

E-mail: biochem@ochhades.chemie.uni-karlsruhe.de

Enzymes: histidine ammonia-lyase (EC 4.3.1.3; Swiss-Prot accession

no P21310); phenylalanine ammonia-lyase (EC 4.3.1.5; Swiss-Prot

accession no P24481).

Dedication: dedicated to Professor Wolfgang Buckel on the occasion of

his 60th birthday.

(Received 28 June 2001, accepted 5 September 2001)

Abbreviations: HAL, histidine ammonia-lyase; PAL, phenylalanine

ammonia-lyase; MIO, 3,5-dihydro-5-methylidene-4H-imidazol-4-one.

Site-directed mutagenesis

Mutagenesis was carried out in a C273A mutated gene for

HAL from Pseudomonas putida to permit a subsequent

crystallization without forming polymeric forms of enzyme

[13]

HAL mutants R283I, R283K, H83L, N195A, E414A,

E414Q, Q277A and F329A were performed following

the QuickChangeTM site-directed mutagenesis system

(Stratagene) [14]

The oligonucleotides used in the mutagenesis reactions

were:

HAL-R283I(1): 50

-CGTACTCGCTGATCTGCCAGCCG-30; HAL-R283I( – ): 50-CGGCTGGCAGATCAGCGAGTA

CG-30; HAL-R283K(1): 50-CGTACTCGCTGAAATGC

CAGCCG-30; HAL-R283K( – ): 50-CGGCTGGCATTTCA

GCGAGTACG-30; HAL-H83L(1): 50-GTGCTGTCCC

TGGCCGCTGG-30; HAL-H83L( – ): 50-CCAGCGGCCA

GGGACAGCAC-30; HAL-N195A(1): 50-GCCCTGCTCG

CCGGCACCCAG-30; HAL-N195A( – ): 50-CTGGGTGCC

GGCGAGCAGGGC-30; HAL-E414A(1): 50-GCCAA

CCAGGCAGACCACGTATCG-30; HAL-E414A( – ): 50

-CGATACGTGGTCTGCCTGGTTGGC-30

HAL-E414Q(1): 50-GCCAACCAGCAAGACCACGT

ATCG-30; HAL-E414Q( – ): 50-CGATACGTGGTCTTG

CTGGTTGGC-30; HAL-Q277A(1): 50-CGACAAGGT

CGCGGACCCGTACTCG-30; HAL-Q277A( – ): 50-CGA

GTACGGGTCCGCGACCTTGTCG-30; HAL-F329A(1):

50-CGGTGGCAACGCCCACGCAGAACC-30;

HAL-F329A( – ): 50-GGTTCTGCGTGGGCGTTGCCACCG-30

HAL mutants Y53F and Y280F were constructed

following a method described by Olsen et al [15]

The oligonucleotides used in these mutagenesis reactions

were: HAL-Y53F: 50-CGCACTGCCTTCGGCATCAAC-30;

HAL-Y280F: 50-CCAGGACCCGTTCTCGCTGCGC-30

The mutations were checked by sequence analysis using

the dideoxynucleotide chain-termination method [16]

Protein expression and purification

E coli BL21 (DE3) cells carrying the plasmids with the

genes for wild-type HAL and HAL mutants were cultured

and HAL was purified as described previously [6]

SDS/PAGE and Western blot analysis SDS/PAGE was carried out according to Laemmli [17] using 10% polyacrylamide gels The gels were stained with Coomassie Brillant Blue R250 Western Blot analysis was performed following a previously described method using nitrocellulose blotting filters [18,19] Wild-type HAL and mutants were detected with rabbit polyclonal antibodies raised against HAL from Pseudomonas putida (the antibody was a generous gift from G Mu¨nscher, Behringwerke AG, Marburg, Germany)

Enzyme assay and protein determination HAL activity was measured spectrophotometrically at 25 8C following the formation of trans-urocanate at 277 nm The assay was performed in 1-cm quartz cuvettes by modifi-cation of the method described in [20] with enzyme concentrations varying between 1 and 25 mg for active enzymes and between 0.1 and 1 mg for less active mutants The enzyme was preincubated at 25 8C for 5 min in 950 mL 0.1M sodium pyrophosphate pH 9.3 supplemented with

10 mM ZnCl2 and 2 mM glutathione Reaction was started

by adding 50 mL of a 0.5-M L-histidine solution Wild-type enzyme and moderately active mutant enzymes were measured

in intervals of 1 min for 5 min, less active enzyme mutants were measured in intervals of 5 min for 20 min For deter-mination of Km and Vmax L-histidine concentrations were varied from 0.5 to 35 mM Kinetic parameters (Km, Vmax) were determined using a double reciprocal plot [21] Because we used pure enzyme fractions it was possible to measure the turnover numbers (kcatvalues) accounting for a molecular mass of Mr¼ 214.372 of the tetrameric HAL Determination of the protein concentration was carried out according to Warburg & Christian [22,23], Murphy & Kies [24] and Groves et al [25] and Smith et al [26] As a reference protein for the measurements we used bovine serum albumin (BSA)

Irreversible inactivation withL-cysteine Irreversible inactivation by L-cysteine was carried out in

1 cm quartz cuvettes as described earlier [10,11,27] A total

Fig 1 Mechanism for the formation of MIO by cyclization of an intramolecular 142ASG144 tripeptide.

of 0.75 mg (3.5 nmol) enzyme was dissolved in 1.0 mL

50 mM NaHCO3/Na2CO3 buffer pH 10.5 Inhibition was

started by addition ofL-cysteine to a final concentration of

10 mM Inactivation was controlled spectrophotometrically

in a Cary 3E spectrophotometer (Varian), following the

increase in absorbance at 338 nm during 50 min in intervals

of 10 min to get repetitive overlays of the absorption spectra

For determining the time dependence of the inactivation

by activity measurements 12 nmol enzyme was dissolved in

2 mL 50 mMNaHCO3/Na2CO3buffer pH 10.5 Inactivation

of the enzyme was started by addition ofL-cysteine to a final

concentration of 10 mM Every 10 min, we used a 200-mL

aliquot of the reaction mixture for the enzyme assay After

60 min the residual mixture was dialysed against 50 mM

NaHCO3/Na2CO3 buffer pH 10.5 to remove L-cysteine

followed by an enzyme assay

UV difference spectroscopy

UV difference spectra were measured at enzyme

concen-trations of 2 mg (9 nmol) in 1 mL 10 mMTris/HCl pH 7.2

from 240 to 360 nm using 1-cm quartz cuvettes [12] First a

blank with the MIO-free HAL mutant S143A was measured

followed by a scan of the wild-type enzyme and various

active site mutants of HAL

Substrate fit and optimization within the active area

Calculations were performed on 300 – 500 MHz Pentium II

computers running underWINDOWS 95 orWINDOWS98 For

molecular mechanics, a switched smoothing function which gradually reduced nonbonding interactions to zero from

10 A˚ inner radius to 14 A˚ outer radius, was applied Otherwise, all calculations were performed by using default settings of the program packages

Analysis of the X-ray structure of the HAL homotetramer (PDB code: 1B8F) showed that Ser143 is fully covered by residues of three monomer subunits within a global area of

25 A˚ radii This part (representing 475 amino-acid residues,

a number which is comparable to the 509 amino-acid resi-dues size of a monomeric HAL unit, together with structure waters, a glycerol molecule and a sulfate anion) was cut off from the full HAL homotetramer structure and used for modelling the substrate free and substrate incorporating states of the active site by MM1 calculations of the

HYPERCHEM package [28] All the mutated residues were found within 12 A˚ radii around the methylene carbon of MIO formed from Ser143 Therefore the outside sphere between 12 and 25 A˚ of the whole 25 A˚ radii globe was kept

‘frozen’ during the calculations The calculations were performed on 1232 atoms within the 12 A˚ inside area Conformational analysis ofL-histidine in its zwitterionic state was performed byPM3 calculations in thePC SPARTAN PROpackage [29] similarly as reported for the zwitterionic form ofL-phenylalanine [30] The appropriate zwitterionic

L-histidine structure was docked to the substrate-free X-ray structure of HAL by applying the following considerations (a) The C5 position of theL-histidine imidazole ring should

be close enough to the methylene of the MIO to perform the nucleophilic addition to the C¼C double bond (b) The NH31

Fig 2 Calculated models for the zwitterionic L -histidine binding (A), for the cationic intermediate containing (B), and for the trans-urocanate/ammonia binding (C) state of HAL’s active site.

and the pro-R b-H should be nearly antiperiplanar (c) The

best zwitterionic conformation ofL-histidine fulfilling these

requirements was aligned by RMS fit over two water

mol-ecules (hydrogen bonded to the imidazole N of H83 and to

the carbonyl O of Asn195) and the sulfate anion, all of

which are present in the experimental X-ray structure of

HAL in the close vicinity of the MIO methylidene moiety

The atomic pairs used for this fit were: H83 coordinating

water O , imidazolyl-N1 of the histidine, Asn195

coordinating water O , NH31 of histidine, and S atom

of the sulfate anion , carboxylate C of histidine After

docking, the sulfate ion and the two water molecules were

deleted and the structure containing the zwitterionic

L-histidine substrate was optimized using theMM1 method

of theHYPERCHEM[28] program (Fig 2A)

The cationic intermediate state was obtained by con-structing a single bond between theL-histidine imidazole C5 and MIO methylidene C atoms, correcting the atom and bond types and orders, and relaxing the structure byMM1 optimization (Fig 2B)

The trans-urocanate/ammonia binding model was obtained from the cationic intermediate model by breaking the appropriate bonds, correcting the atom and bond types and orders, and optimizing the structure by theMM1 method (Fig 2C)

Calculations of electronic spectra of different forms of a truncated MIO model

Full PM3 geometry optimization on a truncated MIO model compound and on its possible protonated forms (Fig 3) followed by single point calculations for their excited states were performed in theHYPERCHEM[28] package

R E S U L T S A N D D I S C U S S I O N

Construction, overproduction and purification of active site mutants

On the basis of the recently elucidated X-ray structure of HAL [8], several active site amino-acid residues can be identified These are R283, Y53, E414, Y280, N195, H83, Q277 and F329 (see Fig 4) To evaluate the importance

of these residues, HAL mutants were constructed at the corresponding sites using the QuickChangeTMsite-directed mutagenesis kit and the method of Eckstein [14,15] The results of the mutagenesis experiments were verified by sequence analysis Overproduction and purification of the HAL mutants were carried out as described by Langer et al [6] Crude extracts of bacterial cells producing wild-type enzyme and the mutated variants were separated by SDS/ PAGE to compare the expression rates and the sizes of the recombinant proteins In all cases, high quantities of recombinant enzyme were produced showing the same

Fig 3 Calculated UV absorptions of a truncated MIO model

compound and of its protonated forms Calculations were performed

by PM3 method Symbols indicate the calculated relative oscillator

strengths: (s), strong; (m), medium; (w), weak.

Fig 4 Active site of HAL.

monomeric size Western Blot analysis showed that all

enzyme variants were detected by the anti-HAL Ig

E coli BL21 (DE3) used as host did not show any HAL

activity A search in the Swiss-Prot data bank for sequences

homologous to HAL from various sources was negative

Purification of wild-type HAL and HAL mutants resulted

in yields varying from 5 to 80 mg pure enzyme per L cell

culture After purification the turnover number or kcat of

recombinant wild type HAL was 86 s21 which is in

agreement with a specific activity of 24 U:mg21previously

reported [6]

Characterization of the mutants by kinetic measurements

Steady state kinetic parameters of HAL mutants were

measured at substrate concentrations varying from 0.5 to

35 mM L-histidine Comparison of the Km values revealed

that all mutants have similar affinities forL-histidine This

indicates that several residues are responsible for binding of

the substrate and mutagenesis of a single residue affects

the Km value very little HAL mutant C273A which was

constructed to achieve better crystals [13] and the double

mutant C273A/R283I showed a somewhat higher Km

(18 mM) than other mutants or wild-type enzyme

(Km¼ 3 – 8 mM), pointing to the relative importance of

R283 for substrate binding HAL mutant C273A showed a

fivefold lower kcat compared to wild-type HAL as was

recently described [13] In Table 1 the Kmand kcatvalues of

the HAL variants and the factors kcatC273A: kcatmut(kcatof

single mutant C273A divided by kcat of HAL double

mutants) are listed The factors show to what extent the

double mutants are less active in relation to the single

mutant C273A HAL mutant C273A/R283K shows < 20

times lower activity compared to HAL mutant C273A,

whereas a substitution of this arginine by isoleucine leads to

a larger decrease of activity (1640 times less active than

mutant C273A) This indicates that a noncationic residue at

that position results in a more severe decrease of activity

Substitution of Y53, which is positioned in the

neighbour-hood of R283 at the active site, leads to more dramatic

effects Exchange to phenylalanine results in a 2650-fold

less activity compared to the single mutant C273A These data indicate that this region in the active site may be responsible for coordination of a cationic group of

L-histidine that is located near an anionic group of the substrate We propose therefore that the neighbouring resi-dues R283 and Y53 coordinate the carboxylic and amino group of the substrateL-histidine, respectively Based on the X-ray structure of HAL (Fig 4) in Fig 2, models for binding ofL-histidine (Fig 2A), the cationic intermediate formed by attack of C5 of the imidazole moiety of

L-histidine at the methylidene carbon of MIO (Fig 2B), and trans-urocanate and ammonia (Fig 2C) at the active site of HAL are shown which explain possible functions of some active site residues Residues Q277 and F329 (see Fig 4) were both converted into alanine which resulted in a 125 and 100 times lower activity, respectively (Table 1) The cationic intermediate-binding model (Fig 2B) indicates

Fig 5 Model for the binding of L -histidine at the active site of HAL.

Table 1 Kinetic constants of wild-type HAL and active site mutants HAL activity was measured by following the formation of trans-urocanate at

277 nm in the presence of purified enzyme The enzyme was preincubated at 25 8C in 0.1 M sodium pyrophosphate (pH 9.3) supplemented with

10 m M ZnCl 2 and 2 m M glutathione Reaction was started by addition of a 0.5- M L -histidine solution The L -histidine concentrations were varied from 0.5 to 35 m M The kinetic constants K m (m M ) and V max (U:mg21or mmol:min21:mg 21

) were determined using a double reciprocal plot [21] Turnover numbers or k cat values (s21) were determined with the molecular mass M r ¼ 53 593 for one subunit of the tetrameric HAL Determination of protein concentration was carried out according to Warburg and Christian [22,23], Murphy and Kies [24], Groves et al [25] and Smith et al [26].

K m (m M ) k cat (s 21 ) k catC273A /k catmut ratio Wild-type HAL 3.9 ^ 0.9 86 ^ 6

C273A/E414A HAL 6.1 ^ 0.7 0.00086 ^ 0.00007 20 930 C273A/E414Q HAL 1.7 ^ 0.9 0.053 ^ 0.0025 339

C273A/H83L HAL 1.2 ^ 0.4 0.001 ^ 0.0002 18 000

the p-stacking role of F329 This residue may stabilize the

s-complex-like intermediate and prevents abstraction of the

proton of the C5 carbon by excluding any basic group A

dramatic loss in activity was achieved by substitution of

N195 and E414 The measured kcat values were 1000 –

20 930 times, respectively, lower than that of the wild-type

enzyme HAL mutant H83L showed almost no activity We

propose that these residues have important functions in the

enzyme HAL which is shown in Fig 5 H83 is possibly

involved in binding and orienting of the imidazolyl moiety

ofL-histidine at the active site (Fig 2A) and stabilization of

the cationic intermediate arising from the imidazolyl moiety

of the substrate (Fig 2B) Because of the larger distance

(4.01 A˚ , Table 2) the interaction of H83 with the substrate

imidazole group could be mediated by a H3O1molecule or, alternatively, by coordination with a metal ion such as Mn21

or Zn21 (Fig 6) The calculated steric arrangement of the cationic intermediate suggests that the imidazolyl N1-H

of the substrate may polarize the MIO group by partial protonation of its carbonyl oxygen (Fig 2B) This partial protonation facilitates the electrophilic attack at the methyl-ene moiety of MIO by decreasing of the electron density in the p system of the C¼C double bond (Fig 5)

Calculation of the UV spectra of MIO and the energy of putative intermediate states

For estimating the degree of polarization of the MIO moiety

in the substrate free state of HAL by partial protonation of

Fig 6 Arrangement of H83 and substrate imidazole in the cationic

intermediate model of HAL compared to experimental Zn 21

com-plex found in human carbonic anhydrase II (PDB code: 1CRA) [31].

Table 2 Distances in models of the HAL active site Selected distances (measured in A ˚ ) in models for the zwitterionic L -histidine binding (a), for the cationic intermediate containing (b), and for the trans-urocanate/ammonia binding (c) state of HAL’s active site are listed.

Atomic pairs Model a Model b Model c S142 C3 – His C4 0 4.43 1.53 3.89 S142 O1 – His N1 0 4.79 2.61 3.21 H83 N1 0 – His N3 0 4.01 4.15 5.25 E414 O1 – His C3 5.54 3.66 3.22 N195 O1 – His N2 2.89 3.55 3.04 Y53 OH – His N2 4.85 4.05 3.72 Y53 OH – His O1 2.84 2.87 4.55 Q277 N4 – His O1 4.31 4.04 6.32 R283 NH1 – His O 0 1 4.39 4.04 4.79 R283 NH2 – His O 0 1 3.68 3.16 3.00

Fig 7 Mechanism for the formation of the

338 nm chromophore by irreversible inactivation of HAL with -cysteine.

polar amino-acid residues, the electronic spectrum of this

chemically unprecedented chromophore was calculated in a

truncated model at thePM3 level of theory (Fig 3) Spectra

for the protonated MIO structures (fully protonated at the

carbonyl O, at N1 and at N3, respectively) were calculated

similarly The calculated absorption maximum at 303 nm

for the nonprotonated MIO model is in good agreement with

the experimentally determined maximum around 302 nm

in the UV difference spectra obtained by substracting the

spectra of lacking mutants from spectrum of

MIO-containing HAL On the other hand, all the three fully

protonated MIO models gave significantly different

calculated UV spectra This indicates that the MIO moiety

in the substrate free state of HAL is not substantially

protonated Consequently, the substrate itself activates the

MIO by partial protonation upon approaching it The fact,

that methylation either at N1 or N4 of the imidazolyl

moiety of L-histidine resulted in compounds which are

neither substrates nor inhibitors of HAL (S Viergutz &

J Re´tey, unpublished results), indicates the importance of

the orienting effect of H83 (N1 methylated analogue) and

the partial protonation effect on MIO (N4 methylated

analogue)

The carbonyl group of N195 may be involved in hydrogen

bonding to the ammonium function of the substrate (Fig 2C)

and also to the leaving ammonia molecule (Fig 2C) The

active site residue E414 should have a key role in catalysis

and with the assistance of Y280 may provide the enzymic

base designed to abstract the activated b proton of the

substrate Y53, R283 and Q277 might be involved in

anchoring the carboxylate moiety of the substrate or the

cationic intermediate (Fig 2A,B) Inspection of the whole

HAL tetramer reveals that residue Y53, which showed the

most dramatic effect on the reaction rate among these three

residues, is located at the edge of a channel through which

the substrate can enter into or the product can be released from the active site

The calculated total energies for the zwitterionic

L-histidine binding (Fig 2A) and cationic intermediate binding models (Fig 2A) were similar, whereas

< 46 kJ:mol21 lower total energy was obtained for

Fig 8 Inactivation of HAL with L -cysteine to a final concentration

of 10 m M , and an enzyme concentration of 0.75 mg (3.5 nmol) in

1 mL For further details see Experimental procedures Inactivation of

HAL mutant C273A and repetitive scans between 0 and 50 min after

supplementation of L -cysteine (A) Inactivation of HAL mutant C273A/

Y280F and repetitive scans between 0 and 50 min (B) Inactivation of

HAL mutant C273A/Y280F and C273A/H83L 20 and 48 h after

supplementation of L -cysteine, respectively (C).

Fig 9 Inactivation with L -cysteine and enzyme assay between 0 and 60 min after supplementation of L -cysteine For experimental details see Legend to Fig 8 and Experimental procedures After 60 min the residual reaction mixture was dialysed to remove unbound cysteine (A) Inactivation of HAL mutant C273A/F329A with L -cysteine (B) Inactivation of HAL mutant S143A with L -cysteine.

Fig 10 UV difference spectra of HAL mutants S143A and C273A (solid line) and HAL mutants S143A and C273A/F329A (dotted line) These were measured at enzyme concentrations of 2 mg (9 nmol) per ml in 10 m M TrisHCl buffer pH 7.2 For further details see Experimental procedures.

the relaxed trans-urocanate/ammonia binding structure

(Fig 2C) In this structure, as a consequence of a < 608

flop of the carboxylate group of the urocanate caused by its

conjugation, i.e coplanarity to the p system of the forming

C¼C double bond, the carboxylate moiety is displaced from

the vicinity of the Y53, R283 and Q277 triad The calculated

distances between particular atoms of the substrate histidine

(His) and of important active site amino acids are listed in

Table 2

Identification of the MIO group by inhibition with

L-cysteine and UV difference spectroscopy

To determine whether the various mutant enzymes have a

prosthetic MIO group at their active sites, we performed

irreversible inhibition with L-cysteine to a final

concen-tration of 10 mM in slightly basic solution and in the

presence of O2 Under these conditions, both wild-type HAL

and HAL mutant C273A show an increase in absorbance at

338 nm during 50 min as previously described [11] The

chromophore is generated by nucleophilic attack of the

thiolate anion of cysteine at the MIO group followed by

oxidation and intramolecular S-to-N rearrangement as

recently proposed (Fig 6) [32,33] In Fig 8A repetitive

scans of the single mutant C273A following the inactivation

with L-cysteine are shown During 50 min, an absorbance

maximum develops that is located around 338 nm Some

double mutants showed different behaviour upon treatment

withL-cysteine The mutant C273A/F329A did not show an

absorbance maximum upon treatment withL-cysteine even

after 24 h of incubation In the case of the HAL mutant

C273A/Y280F and mutant C273A/H83L there was a slower

increase in absorbance but after 20 and 48 h, respectively, a

chromophore around 338 nm appeared also in these cases

(Fig 8C) These results indicate the presence of a MIO at

their active sites, but in a less reactive form Concomittant

with the formation of a new chromophore the activity of the

enzyme decreases irreversibly After addition ofL-cysteine,

the activity of the enzymes dropped very quickly and in

most cases there was no activity at all after 60 min Removal

of excess cysteine by dialysis did not restore activity

(Fig 9A) The MIO-free HAL mutant S143A also showed

inactivation, but 60 min after treatment withL-cysteine the

remaining solution regained almost 100% of its original

activity upon dialysis (Fig 9B) In this caseL-cysteine does

not bind irreversibly to the enzyme

With HAL mutants C273A, C273A/F329A and C273A/

H83L we carried out UV difference spectroscopic

measure-ments, because this method is an excellent means to show

the presence of the MIO group [12] (Fig 10) HAL mutants

C273A and C273A/F329A both showed a maximum around

302 nm in the UV difference spectra with HAL mutant

S143A which lacks an intact MIO group (Fig 9) This result

indicates that HAL mutant C273A/F329A contains a MIO

group at their active site but is not able to form a 338-nm

chromophore withL-cysteine and dioxygen

It is noteworthy that reduced glutathione of high purity,

present in the enzyme essay, does not inhibit HAL However

under certain conditions glutathione may releaseL-cysteine

which is an inhibitor It may be better to use dithiothreitol to

keep some cysteine residues in the reduced form The role

of Zn21and other metal ions carrying two positive charges

has been thoroughly investigated [34] The decrease of the

enzymatic activity by removal of such ions and the slight activation at their presence might be explained by assuming their interaction with H83 and the substrate histidine (Fig 6) In contrast no metal-ion effect has ever been observed on the PAL reaction This is in agreement with the lack of histidine in a similar position in all PAL sequences

A C K N O W L E D G E M E N T S

We thank Prof G E Schulz and Dr T F Schwede (University of Freiburg, Germany) for the cooperation in the work on HAL and PAL and the production of two HAL mutants The work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie D R thanks the Land Baden-Wu¨rttemberg for a scholarship for graduate students L P thanks the Hungarian OTKA (T-033112) for financial support We thank A Sigrist for help with the figures and

S Vollmer for technical assistence.

R E F E R E N C E S

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