An active site homology model of phenylalanine ammonia-lyaseDagmar Ro¨ther1, La´szlo´ Poppe2, Gaby Morlock1, Sandra Viergutz1and Ja´nos Re´tey1 1 Institute for Organic Chemistry, Univers
Trang 1An active site homology model of phenylalanine ammonia-lyase
Dagmar Ro¨ther1, La´szlo´ Poppe2, Gaby Morlock1, Sandra Viergutz1and Ja´nos Re´tey1
1
Institute for Organic Chemistry, University of Karlsruhe, Germany;2Institute for Organic Chemistry, Budapest University of Technology and Economics, Hungary
The plant enzyme phenylalanine ammonia-lyase (PAL,
EC 4.3.1.5) shows homology to histidine ammonia-lyase
(HAL) whose structure has been solvedby X-ray
crystal-lography Basedon amino-acidsequence alignment of the
two enzymes, mutagenesis was performedon amino-acid
residues that were identical or similar to the active site
resi-dues in HAL to gain insight into the importance of this
residues in PAL for substrate binding or catalysis We
mutatedthe following amino-acidresidues: S203, R354,
Y110, Y351, N260, Q348, F400, Q488 andL138
Deter-mination of the kinetic constants of the overexpressedand
purifiedenzymes revealedthat mutagenesis ledin each case
to diminished activity Mutants S203A, R354A and Y351F
showeda decrease in kcatby factors of 435, 130 and235,
respectively Mutants F400A, Q488A andL138H showeda 345-, 615- and14-foldlower kcat, respectively The greatest loss of activity occurredin the PAL mutants N260A, Q348A andY110F, which were 2700, 2370 and75 000 times less active than wild-type PAL To elucidate the possible func-tion of the mutatedamino-acidresidues in PAL we built a homology model of PAL based on structural data of HAL andmutagenesis experiments with PAL The homology model of PAL showed that the active site of PAL resembles the active site of HAL This allowedus to propose possible roles for the corresponding residues in PAL catalysis Keywords: phenylalanine ammonia-lyase; PAL; MIO; site-directed mutagenesis; homology model
Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) is a very
important plant enzyme that catalyses the conversion of
L-phenylalanine into E-cinnamic acidwhich is the precursor
of a great variety of phenylpropanoids, such as lignins,
flavonoids and coumarins [1,2] Because of its central role in
plant metabolism, PAL is a potential target for herbicides
[2]
The relatedenzyme histidine ammonia-lyase (HAL;
EC 4.3.1.3) catalyses a very similar reaction, converting
L-histidine into E-urocanic acid Amino-acid sequence
comparison of histidine and phenylalanine ammonia-lyases
from different organisms revealed that there are several
homologous regions indicating that their active sites are
very similar [3] For about 30 years it has been believedthat
a dehydroalanine acts as electrophilic prosthetic group at
the active site of both HAL and PAL [4–6] Recently the
three-dimensional structure of HAL was solved by X-ray
crystallography revealing that the electrophilic prosthetic
group 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO)
is the catalytically essential moiety rather than the dehydro-alanine (Fig 1) [7] It has been proposedthat this MIO group is generatedby autocatalytic cyclization of the A142-S143-G144 moiety of HAL This process resembles the fluorophore formation of the green fluorescent protein [8] More recently, we provided spectroscopic evidence for the presence of a prosthetic MIO group at the active site of PAL [9]
Here we report the exchange of several amino-acid residues in PAL that are identical or similar to active site residues of HAL and evaluation of their importance in substrate binding and catalysis by enzyme kinetic behaviour
of the mutants andby a homology model of PAL
M A T E R I A L S A N D M E T H O D S
Bacterial strains and plasmids Wild-type PAL andPAL mutants were overexpressedin
E coliBL21(DE3) cells The gene coding for phenylalanine ammonia-lyase from P crispum was changedto the codon-usage of E coli andclonedin vector pT7-7 followedby a transformation in E coli BL21(DE3) cells containing vector pREP4-GroESL [10]
Site-directed mutagenesis Phenylalanine ammonia-lyase mutants were produced by following the instruction manual of the QuickChange(tm) Site-Directedmutagenesis kit (Stratagene) [11] The oligo-nucleotides used in the mutagenesis reactions were: S203A(+): 5¢-catcactgctgccggcgacctgg-3¢, S203A(–): 5¢-cca ggtcgccggcagcagtgatg-3¢; Q488A(+): 5¢-cagcacaacg ctgacgt taac-3¢, Q488A(–): 5¢-gttaacgtcagcgttgtgctg-3¢; Q488E(+):
Correspondence to J Re´tey, Institute of Organic Chemistry,
University of Karlsruhe, Richard-Willsta¨tter-Allee,
D-76128 Karlsruhe, Germany.
Fax: + 49 721 6084823, Tel.: + 49 721 6083222,
E-mail: biochem@ochhades.chemie.uni-karlsruhe.de
Abbreviations: PAL, phenylalanine ammonia-lyase; HAL, histidine
ammonia-lyase; MIO, 3,5-dihydro-5-methylidene-4H-imidazol-4-one.
Enzymes: histidine ammonia-lyase (EC 4.3.1.3); phenylalanine
ammonia-lyase (EC 4.3.1.5).
Note: in this paper, the numbering of amino acids for PAL from
P crispum is consistent with the SWISS-PROT database (P24481)
recordbut not with the numbering usedin previous PAL papers.
(Received15 February 2002, accepted8 May 2002)
Trang 25¢-cagcacaacgaagacgttaac-3¢, Q488E(–): 5¢-gttaacgtctcgttg
tgctg-3¢; Y351F(+): 5¢-caggaccgttttgctctgcg-3¢, Y351F(–):
5¢-cgcagagcaaaacggtcctg-3¢; Y110F(+): 5¢-ccgactcctttggcg
ttacc-3¢, Y110F(–): 5¢-ggtaacgccaaaggagtcgg-3¢; R354A(+):
5¢-cgttatgctctggctacctctcc-3¢, R354A(–): 5¢-ggag aggtagccag
agcataacg-3¢; N260A(+): 5¢-gcactggttgctggtaccgctg-3¢,
N260A(–): 5¢-cagcggtaccagcaaccagtgc-3¢; Q348A(+): 5¢-aa
acgaaagcggaccgttat-3¢, Q348A(–): 5¢-ataacggtccgctttcgg
ttt-3¢; F400A(+): 5¢-ggtggtaacgcccaggggac-3¢, F400A(–):
5¢-gtc ccctgggcgttaccacc-3¢; L138H(+): 5¢-gatccgcttccacaacg
ctg-3¢, L138H(–): 5¢-cagcgttgtggaagcggatc-3
The mutations were verifiedby sequence analysis using
the dideoxynucleotide chain-termination method [12]
Protein expression and purification
E coli BL21 (DE3) cells carrying the plasmids with the
genes for wild-type PAL and PAL mutants were cultured
andPAL was purifiedas describedpreviously [10]
SDS/PAGE and Western blot analysis
SDS/PAGE was carriedout according to Laemmli [13]
using 10% polyacrylamide gels The gels were stained with
Coomassie Brillant Blue R250 Western Blot analyses were
performedfollowing a previously describedmethodusing
nitrocellulose blotting filters [14,15] Wild-type PAL and
mutants were detected with rabbit polyclonal antibodies
raisedagainst PAL from P crispum (the antibody was a generous gift of N Amrhein, Eidgeno¨ssische Technische Hochschule, Zu¨rich)
Enzyme assay and determination of protein concentration
PAL activity was measuredspectrophotometrically at 30C following the formation of E-cinnamate at 290 nm The assay was performedin 1-cm quartz cuvettes by modifica-tion of the methoddescribedin [16] with enzyme concen-trations varying between 10 and20 lg for active enzymes andbetween 0.3 and0.4 mg for less active mutants The enzyme was preincubatedat 30C for 5 min in 750 lL of 0.1MTris/HCl pH 8.8 The reaction was started by adding
250 lL of a 20-mM L-phenylalanine solution Wild-type enzyme andmoderately active mutants were measuredin intervals of 1 min for 5 min, less active enzyme mutants were measuredin intervals of 5 min for 20 min For determination of Kmand Vmax,L-phenylalanine concentra-tions were variedfrom 0.01 to 5 mM Kinetic constants (Km,
Vmax) were determined using a double reciprocal plot [17] The isolatedenzymes were electrophoretically pure as verifiedby staining with Coomassie Brillant Blue R250 andtherefore it was possible to measure the turnover numbers (kcat) with the relative molecular mass of 311.313 for the tetrameric PAL Determination of protein concen-tration was carriedout according to Warburg & Christian [18,19], Murphy & Kies [20] andGroves et al [21] BSA was usedas reference protein for the measurements
Sequence comparison Amino-acidsequences of HAL from P putida [22], HAL from Homo sapiens [23] andPAL from P crispum [24] were extractedfrom the SWISS-PROT database Sequence alignment was carriedout using the computer program
MALIGN (HUSAR, DKFZ Heidelberg) MALIGN is a HUSAR adaptation of the programMAP[25]
Homology modelling Model of PAL 64–531 monomer The sequence of PAL (Swiss-Prot: P24481) was submittedto SWISS-MODEL (AutomatedProtein Modelling Server) [26,27] The PAL structure homology model resulting from this first approach, which was folded over the HAL structure (PDB: 1B8F), containedamino acids 64–531 [28] This partial model was optimized by different molecular me-chanics force fields usingCHARMM[29], Amber-95 [30,31] andMM3Prot [32] force fieldimplementations in the
TINKER [33] package; Amber3 andMM+ force field implementations in HYPERCHEM [34] package; andthe GROMOS 96 43B1 [35] implementation inSWISS-
PDBVIEW-ER Calculations were performedon 300–850 MHz Pen-tium III computers running under WINDOWS95, WIN-DOWS98 or LINUX (REDHAT 6.2) A switchedsmoothing function, which gradually reduced nonbonding interactions
to zero from a 10-A˚ inner radius to a 14-A˚ outer radius, was generally applied Otherwise, all the calculations here and later were performedby using default settings of the program packages These optimizedstructures were comparedby single point energy calculations and
Fig 1 The MIO moiety in HAL from P putida The mechanism of
the PAL reaction through Friedel–Crafts type attack of MIO on the
phenyl ring of L -phenylalanine.
Trang 3Ramachandran plot analyses using the SWISS-PDBVIEWER
3.6 package [27] For further modelling, the PAL 64–531
monomer optimizedby MM+ force-fieldofHYPERCHEM
[34] was used During the MM+ optimization, the
A202-S203-G204 triadwas replacedby MIO in this structure
Model of PAL 64–531 homotetramer The raw PAL
64–531 fragment homotetramer was built by the SWISS
-PDBVIEWER 3.6 [27] package using the MM+ optimized
PAL 64–531 fragment model and the cell parameters of the
HAL structure (space group: I222) [7] The MIO structures
were rebuilt by MM+ calculation andkept frozen in the
homotetramer during Amber3 optimization performed by
HYPERCHEM[34]
Model of PAL 1–63 fragment: The 1–63 fragment of PAL
was built using secondary structure prediction data
(obtainedby using PHDsec, version 5.94–317 [36,37], and
3D-PSS Psi-Pred [38,39]) Raw three-dimensional models
corresponding to the predicted secondary structure were
built andoptimizedin the HYPERCHEM [34] and SWISS
-PDBVIEWER3.6 [27] packages Evaluation of the raw
three-dimensional models as described for the PAL 64–531
fragment resultedin the best foldof the 1–63 fragment
Model of PAL 532–716 fragment Swiss-MODEL [26,27]
has not founda template eligible for modelling the
terminal fragment (532–716) of the PAL sequence
(Swiss-Prot: P24481) Although several reasonable hits were
foundwhen the 532–716 fragment was submittedto
3D-PSS search [38,39] (e.g 15% identity over 184 amino
acids with 1FPW chain A; 19% identity over 184 amino
acids with 1JBA chain A; 22% identity over 172
amino acids with 2FHA; 18% identity over 153 amino
acids with 1BG7; 17% identity over 179 amino acids
with 1AKE chain A; 22% identity over 162 amino acids
for 1GGQ chain A), the first approach models built
from these templates by SWISS-PDBVIEWER 7.02 [27]
showedno proper contacts with the core 64–531
fragment The secondary structure prediction data for
the whole PAL sequence (by using PHDSEC [36,37])
showed 14% identity over 184 residues between the PAL
532–716 sequence fragment andthe 91–275 fragment of
chain B in the aspartase structure (PDB accession no
1JSW) [40,41] Because aspartase is also an
ammonia-lyase andits structure shows substantial structural
similarity to that of the template HAL (long parallel
helices anda quasi tetrameric structure), the PAL
532–716 fragment was modelled using chain B of the
aspartase structure as template by the SWISS-PDBVIEWER
7.02 [37] package providing the 532–716 model fitting
best to the core 64–531 fragment
Model of the full PAL 1–716 monomer The full PAL
1–716 monomer was obtainedfrom the optimizedPAL
1–63, 64–531 and532–716 fragments by manual ad
just-ments and rigid-body approach optimizations using by the
TINKERpackage [33] The rms fit of four full PAL 1–716
monomers over the Amber3-optimizedPAL 64–531
frag-ment homotetramer indicated the necessity of modifications
in the 621–640 loop of the full PAL model avoiding spatial
overlap The necessary loop modifications in the full PAL
package, followedby Amber3 optimization of the outside area (fragments 1–70 and525–716 in each monomer chain)
of the reconstitutedfull PAL homotetramer by the HYPER-CHEM[34] program
Quality assessment of the model Analysis of the full PAL homotetramer was performedby the SWISS-PDBVIEWER 7.02 [27] package No significant clashes were foundin the contact regions between the unique chains The aspherical nature of the active homo-tetramer form of PAL is supportedby the sedimentation constant and the Stokes’ radius data found by sucrose density gradient centrifugation of PAL from potato [42]
Of the 716 amino acids in the chain A of the model, 43 were outside of the likely Phi/Psi combinations in the Ramachandran plot (including Gly and Pro) Most of the
deviations were foundin the 64–531 andthe 532–716 fragments (29 and14, respectively), whereas only one difference was found in the 1–63 fragment Further quality assessments were made by WHAT IF v4.99 [43,44] [e.g bondlengths Z score 0.528 ± 0.012; 27 bumps between atomic pairs over 0.1 A˚; four Gly residues have unusual backbone oxygen position; the rms Z score for all improper dihedrals (1.253) was within normal ranges; packing Z scores less than )2.50 were foundfor I141: )3.68, H309: )2.93, R52: )2.80, F621:)2.73, E291: )2.51, P699: )2.50] andPROCHECKV3.5 [45,46] through EMBL the Biotech Validation Suite, Heidelberg [47] Although some deviations in bond lengths, angles, side chain planarities and hydrogen bonding environments were foundby these checks, mainly in the terminal 532–716 fragment, thePROCHECK overall average G factor for the protein ()0.35) was acceptable Because the model gave reliable assembly aroundthe active site, for which a good match of residues from three chains is necessary, it seems
to be trustworthy, at least in this region
Optimization and substrate fit within the active site area
Analysis of the full PAL homotetramer showedthat S203 is fully coveredby residues of three monomer subunits within
a global area of 25 A˚ radii This part was cut from the full PAL homotetramer model, and used for modelling the substrate-free andsubstrate binding states of the active site
by MM+ calculations [34] within 15-A˚ radii around S203 The outside sphere between 15 and 25 A˚ of the whole 25-A˚ radii globe was kept frozen during the calculations, which were performedon 1949 atoms within the 15-A˚ inside area Conformational analysis of phenylalanine in its zwitter-ionic state by PM3 calculations of PC SPARTAN PRO [48] package was performed[28], andthe lowest energy confor-mation was usedas starting structure of the substrate The zwitterionic L-Phe structure was docked to the substrate-free active site model by applying the following consider-ations: (a) the C2position of the phenyl ring ofL-Phe should
be close enough to the methylene of the MIO to perform the nucleophilic addition to the C¼ C double bond; and (b) the
NH3+ andthe pro-S b-H shouldbe antiperiplanar [28] Several, slightly different arrangements satisfied these requirements These starting structures containing the zwitterionic - substrate were optimizedby MM+
Trang 4methodof theHYPERCHEM[34] program, andthe one of the
lowest energy was considered as the best fit (Fig 5A)
The r-complex-like intermediate state was obtained by
constructing a single bondbetween theL-Phe C2andMIO
methylidene C atoms, correcting the atom and bond types
andorders, andrelaxing the structure by MM+
optimiza-tion (Fig 5B) The E-cinnamate/ammonia binding model
was obtainedfrom the r-complex model by breaking the
appropriate bonds, correcting the atom andbondtypes and
orders, and optimizing the structure by MM+ method
(Fig 5C)
Overlay of the models on the HAL structure
The substrate-free active site model (25 A˚ radius around
the MIO) for PAL was overlaidonto the similar portion
of the experimental structure of HAL containing a
sulfate ion [7] by the SWISS-PDBVIEWER 3.7 [27] package
(Fig 4A) The models were visualized using the
WEBLABVIEWER [49] program The two models for the
active sites containing the cationic intermediate state of
the substrate for PAL andHAL [50] were aligned
similarly (Fig 4B)
Calculation of the charge distribution in theL-Phe – MIO
r-complex intermediate
The [L-Phe-MIO] r-complex model was cut off from the
whole r-complex containing the active site model
Semiempirical (CNDO, MNDO, AM1, PM3, ZINDO/1,
ZINDO/S) and ab initio (STO-3G) calculations for atomic
charge distributions were performed on this truncated
r-complex model by using the built-in methods of the
HYPERCHEM[34] program No change/reversal in
polariza-tion order of hydrogen atoms (see PM3 results in Fig 6B)
calculated by the different methods was found
R E S U L T S A N D D I S C U S S I O N
Amino-acid sequence comparison and site-directed
mutagenesis
Amino-acidsequence comparison of HAL andPAL from
different organisms showed a sequence identity of about 40
and20% when comparing different sequences of HAL
among one another andcomparing sequences of HAL and
PAL, respectively [3]
Recently, the X-ray structure of HAL from P putida was
solvedby Schwede et al., who discovered a new electrophilic
prosthetic group at the active site, namely MIO (Fig 1) [7]
The four active sites of HAL are constructedby the
assembly of two subunits of the tetrameric enzyme Active
site amino-acidresidues in the case of HAL from P putida
are S143, the predominant precursor of MIO group, as well
as R283, Y53, E414, Y280, Q277, F329, N195 andH83
These active site residues may be important for substrate
binding, catalysis or MIO formation [7,50]
All active site residues in HAL have an analogous residue
in the PAL protein sequence except residues H83 and E414
Figure 2A shows the amino-acidcomparison of HAL from
P putida[22], HAL from H sapiens [23] andPAL from
P crispum [24] Active site residues of HAL and the
respective residues in the PAL sequence are shown in red
Figure 2B shows the predictions for the secondary structure
of PAL using four different methods The more reliable predictions are marked with blue colour, the less reliable ones with grey
To examine the importance of this residues for PAL activity, we constructedenzyme mutants at the correspond-ing sites uscorrespond-ing the QuickChangeTM Site-Directedmutagen-esis system [11] We carriedout mutagenSite-Directedmutagen-esis reactions in residues S203, R354, Y110, Y351, N260, Q348, F400 of the PAL amino-acidsequence andalso in residues L138 and Q488 that replace residues H83 and E414 in HAL, respectively
The result of the mutagenesis experiments were verified
by sequence analysis [12]
Protein expression and purification The plasmids containing the mutated genes were trans-formedin cells of E coli BL21(DE3) that were previously transformedwith the chaperonin-expressing plasmid pREP4-groESL [10] After expression andpurification of wild-type PAL and the various PAL mutants SDS/PAGE andWestern-Blot analysis of crude extracts were performed
to check expression levels andmonomeric size of the enzyme variants In all cases similar quantities of recombinant enzymes were produced showing the same monomeric size Western-Blot analysis revealedthat all enzyme variants were detected with anti-PAL antibodies After purification
of wild-type PAL and the different PAL mutants yields between 5 and30 mg pure enzyme per litre cell culture were obtained
Kinetic characterization of the enzyme mutants Steady state kinetic parameters of wild-type PAL and the PAL mutants were measuredat substrate concentrations varying from 0.01 to 5 mM L-phenylalanine Table 1 shows the kinetic constants of wild-type PAL and the constructed andmeasuredPAL mutants The factor kcat wtPAL/kcat
mutPAL is the kcat or turnover number of wild-type PAL divided by kcatof the respective PAL mutant It is a measure
of how many times the mutant is less active comparedto the wild-type enzyme Pure wild-type PAL showed a kcat of 13.5 s)1anda Kmof 0.12 mMin agreement with previously reportedvalues [51] Comparison of the Kmvalues of wild-type PAL andthe various mutants revealed, that except mutants L138H andL138H/Q488E all other mutants showeda slightly higher affinity for their substrates with
Kmvalues varying between 0.019 and0.057 mM Mutation
in L138 in the PAL sequence, which is the counterpart of H83 in the HAL sequence resultedin a 14-folddecrease in activity anda decreasedaffinity forL-phenylalanine The measured Kmvalue is about 13 mMandis therefore about
100 times higher than that of wild-type PAL In the double mutant L138H/Q488E, the Kmvalue is increasedfurther to
55 mMandthe enzyme is about 145 times less active than the wild-type enzyme The PAL residues L138 and Q488 andthe unsimilar counterparts H83 andE414 in the HAL amino-acidsequence may be important for the substrate specificity of the homologous enzymes PAL andHAL Our expectation that the L138H/Q488E mutant of PAL shows activity with L-histidine was not fulfilled Although this mutant showeda similar K value forL-histidine as
Trang 5wild-Fig 2 Amino-acid sequence alignment of HAL from P putida [22], HAL from Homo sapiens [23] and PAL from P crispum [24] performed with
MALIGN (HUSAR) Active site amino-acidresidues of HAL andrespective residues in PAL are markedwith colour (A) Secondary structure of the PAL model (line: Mod) aligned with the secondary structure predictions of PHDSEC [36,37] (lines: PHD andSUB) and3 D - PSS PSI - PRED [38,39] (line: sspPP) The less reliable 1–63 and532–716 fragments are in grey, the more reliable 64–531 fragment is in blue (B).
Table 1 Kinetic constants of wild-type PAL and PAL mutants PAL activity was measuredby monitoring the formation of E-cinnamate at 290 nm
in the presence of purifiedenzyme The enzyme was preincubatedat 30 C in 0.1 M Tris/HCl (pH 8.8) The reaction was startedby addition of a
20-m M L -phenylalanine solution The L -phenylalanine concentrations were variedfrom 0.01 to 5 m M The kinetic constants K m (m M ) and V max (UÆmg)1or lmolÆmin)1Æmg)1) were determined using a double reciprocal plot [17] Turnover numbers or k cat values were determined with the relative molecular mass 311 313 for the tetrameric PAL Determination of protein concentration was carriedout according to Warburg & Christian [18], Murphy & Kies [20] andGroves et al [21].
K m (m M )
k cat (s)1)
k cat /K m (m M )1 Æs)1)
Factor (k cat wtPAL /k cat mutPAL )
wt PAL 0.12 ± 0.004 13.5 ± 0.1 112.5
S203A PAL 0.019 ± 0.001 0.031 ± 0.0001 1.63 435
R354A PAL 0.057 ± 0.003 0.104 ± 0.005 1.82 130
Y351F PAL 0.024 ± 0.004 0.057 ± 0.001 2.38 235
N260A PAL 0.033 ± 0.003 0.005 ± 0.001 0.152 2,700
Q348A PAL 0.03 ± 0.01 0.0057 ± 0.0004 0.19 2,370
F400A PAL 0.027 ± 0.005 0.039 ± 0.001 1.44 345
Q488A PAL 0.033 ± 0.002 0.022 ± 0.002 0.667 615
Q488E PAL 0.057 ± 0.006 2.1 ± 0.04 36.8 6
L138H PAL 13.5 ± 0.6 0.99 ± 0.02 0.073 14
L138H/Q488E PAL 55 ± 4.9 0.093 ± 0.004 0.0017 145
Trang 6type HAL, its kcat value was about 8000 times lower.
Construction of the PAL mutants Q488A andQ488E leads
to a 615-foldandsixfoldlower activity comparedto that of
the wild-type enzyme, respectively Q488 is the PAL
counterpart of E414 in the HAL amino-acidsequence,
but it seems to play a different role in the catalysis by PAL
Distances obtainedin models (Fig 5A,C: Q488 NH1 to N1
of the MIO is 3.3 A˚) suggest that the Q488 may interact
with the MIO ring Its replacement by an acidic residue in
mutant Q488E has no dramatic effect on the catalytic
activity of this mutant, whereas replacement by a nonpolar
residue in PAL mutant Q488A leads to a more severe
decrease The effects are not as dramatic as in the
mutagenesis of residue E414 of HAL HAL mutant
E414A showeda more than 20 900-foldlower activity
comparedto the wild-type enzyme [50] Therefore it was assumedthat E414 acts as a base in HAL catalysis Mutagenesis in residue S203 of the PAL gene led to an enzyme variant with a kcatof 0.031 s)1 The mutant S203A
is therefore 435 times less active than the wild-type enzyme This is an 10 times higher activity that was previously reportedfor this active site mutant [52] The counterpart of residue Y280 at the active site of HAL is Y351 in the amino-acidsequence of PAL PAL mutant Y351F showeda by factor 235 reduced activity compared to that of the wild-type enzyme PAL mutants N260A andQ348A showeda 2700- and2370-folddiminishedactivity, respectively This indicates that both residues may play important roles in substrate binding or catalysis PAL mutant F400A shows a less dramatic effect; this mutagenesis resulted in an enzyme
Fig 3 Comparison of X-ray structure of HAL [7] and homology model structure of PAL Compar-ison of the schematic representation of (A) tetrameric and(B) monomeric structures of HAL (left) andPAL (right) Catalytically important residues are shown as stick models: MIO moiet-ies are shown in green, the other residues are colouredby elements: C, grey; N, blue; O, red) The less reliable 1–63 and532–716 fragments of the PAL model are coloured in grey.
Trang 7with 345 times lower activity than the wild-type enzyme.
This residue seems to interact with the r-complex
interme-diate by p-stacking andthereby contributing to its
stabil-ization [28] Amino-acidresidue R354 in the PAL sequence
is the counterpart of residue R283 in the HAL sequence
The R354A mutant showed130 times lower activity than
the wild-type enzyme, whereas the Y110F mutant of PAL
resultedin an almost inactive enzyme PAL mutant Y110F
is > 75 000 times less active than the wild-type enzyme and
is therefore the least active PAL mutant examinedso far
Y110 probably plays a very important role that can be
explainedby enzyme modelling
Homology modelling of PAL
To gain further insight into the possible role of the active site
residues a theoretical model for PAL was built by homology
modelling [28] (Figs 3 and 4) According to the model, the
active structure is a homotetramer as in the case of HAL
(Fig 3A) The catalytically important residues are located
on two distinct regions in both HAL and PAL (Fig 3B) at highly isosteric positions
The active site region of the PAL homotetramer (Fig 4) closely resembles that of the X-ray structure of HAL [7,50,53] All the residues in the PAL model occupy the expectedpositions postulatedby comparison with the HAL sequence andstructure Modelling of a zwitterionic sub-strate (Fig 5A), the r-complex forming between the substrate andMIO (Fig 5B) andthe product E-cinna-mate/ammonia (Fig 5C) into the active site of the PAL model gave further insight into the role of the amino-acid residues in catalysis (Fig 6A) These ligand-binding models confirmedthe hypothesis concerning the p-stacking role of F400 This residue may stabilize the intermediate r-complex andprevent abstraction of the proton from the ortho-position of the aromatic ring by excluding any basic group [28] The close vicinity of Y351 to the pro-S b-H of the substrate in the r-complex model (3.61 A˚, Table 2)
indi-Fig 4 The substrate-free active site modelof
PAL overlaid on the experimental structure of
HAL (A) The protein chains are coloured
differently The lighter colours and the labels in
yellow are relatedto the PAL model (B)
Com-parison of the active sites in the r-complex
intermediate containing model of PAL with the
cationic intermediate binding model of HAL
[50] The thick bonds and labels in yellow are
relatedto the PAL model The thin lines and
white labels are relatedto the HAL model.
Trang 8cates that this residue might act as the b-H abstracting base.
The residues Y110, Q348 and R354 might play a role in
binding the carboxylate moiety of the substrate, whereas
residues Y110 and N260 can interact with amine/ammonia
in the substrate andproduct, respectively
In the case of HAL it was shown that in the
substrate-free state of the enzyme the MIO prosthetic group is
not substantially polarized[50] In HAL, presumably,
activation/polarization of the MIO happens when the
substrate itself is approaching it andthe MIO carbonyl oxygen can be stabilizedby partial protonation from N1-H
of the substrate Analysis of the acidity/charge distribution
in the r-complex intermediate in the PAL reaction by semiempirical and ab initio calculations (Fig 6B) revealed that the C3-H hydrogen atom is almost as positively chargedas the pro-S b-H, andtherefore it can stabilize the negatively chargedMIO carbonyl oxygen in a similar way Interaction of the C6-H of theL-phenylalanine with
Y453-O– can have a ring preorientation effect andit can also increase the nucleophilic character of the phenyl moiety of the substrate
It was observedthat the b-H abstraction in the PAL reaction does not seem to be rate determining for good substrates [54] The attack by MIO is partially rate determining; another rate-limiting step seems to be product release The kinetic significance of these two steps is substrate-dependent [28] Inspection of the whole PAL tetrameric model reveals that residue Y110, whose mutation
to F showed the most dramatic decreasing effect on the reaction rate, is locatedat the edge of a channel (see also Fig 4A) through which the substrate can enter into or the product can be released from the active site Residue Y110
in PAL (andalso in HAL its counterpart Y53) seems to play twofoldrole (Fig 7) The first role of it may be a protonation state conversion: ammonium ions (outside from active site in aqueous solution) , ammonia/amine (Fig 7A,F) Secondly, it may serve as anchoring/orienting
Fig 6 Modelfor the ammonia elimination from the r-complex inter-mediate of the PAL reaction (A) and the charge distribution in the
L -Phe–MIO r-complex intermediate calculated by PM3 method (B).
Fig 5 Calculated models for the zwitterionic L -phenylalanine binding
(A), for the r-complex intermediate (B), and for the E-cinnamate/
ammonia binding (C) state of the PAL active site.
Trang 9group for the carboxylate of the substrate/product during
the elimination/addition step in its protonated form through
hydrogen bonding (Fig 7D) Presumably, the enzymic base
exists in the substrate-free state of the enzyme as its
conjugatedacid(Fig 7A,F), from which the amino group
of the substrate can abstract a proton during entering into
the active site (Fig 7C) Interaction of the aromatic ring of
the substrate with the MIO group forms the cationic
intermediate in which the pro-S b-H is acidified and thus the
elimination step can take place (Fig 7D) After elimination
the unsaturatedproduct can be releasedfirst (Fig 7E),
followedby leaving the ammonia as ammonium ion
(Fig 7F)
Conclusions for the mechanism of action
of phenylalanine ammonia-lyase
About 30 years ago Hanson & Havir [1,6] proposeda
mechanism for the PAL reaction starting with the addition
of the a-amino group of the substrate to the prosthetic
electrophile at that time believed to be dehydroalanine
Because this mechanism cannot explain how the nonacidic b-H of the substrate can be abstractedby the enzymic base [54], about 6 years ago another mechanism was suggested involving an electrophilic attack at the phenyl ring by the prosthetic group [52] The new mechanism was supported
by the easy reaction of alternative substrates, especially 3-hydroxyphenylalanine (m-tyrosine), which facilitatedthe electrophilic attack Furthermore, the S203A mutant of PAL lacking the electrophilic MIO group reactedeasily with 4-nitrophenylalanine, in which the b-H is more acidic [52] In the older literature there are also data that seem to support the Hanson mechanism Peterkofsky foundthat HAL catalyses the incorporation of [2-14C]urocanate into
L-histidine but not of15NH4+[55] Unfortunately, in his experiments the concentration of NH4+ was too low (10–60 mM); on the basis of these andother previous experiments [56,57], he assumedthat the HAL (as well as the PAL) reaction was irreversible Later, it was shown that
at higher NH4+concentrations (up to 6M) both ammonia-lyase reactions can be completely reversed[28,58] Never-theless, Peterkofsky’s experiments support the existence of
an enzyme–NH4+intermediate and the idea that ammonia
is releasedafter the other product (urocanate or cinnamate) This does not confirm, however, that ammonia is covalently bonded to the prosthetic electrophile (MIO) as the Hanson mechanism requires Because the mutant ammonia-lyases lacking MIO (S143A for HAL andS203A for PAL) still catalyze the reaction (about 103times more slowly with the natural substrates, but much faster for their nitro-deriva-tives [52,59]), it is clear that in these cases covalent binding
of NH4+cannot occur Modelling the active sites contain-ing the product (cinnamate: Fig 5C and urocanate: Fig 2C
in [50]) suggest that the product may be released first, and before leaving thus blocks the narrow channel for the release
of ammonia/NH4+(Fig 7) Therefore, an ionic bonding of ammonia/NH4+ is a reasonable assumption In another paper purportedly supporting the Hanson mechanism,15N and2H-isotope effects on the PAL reaction are described [54] The ammonia produced in the PAL reaction was convertedin two steps into N2 whose 15N-content was determined by mass spectrometry The latter was 1% less than the one expectedfrom the natural abundance of15N (0.369%) Even if these small effects are real, it is not clear in which step they occurred Because the PAL reaction is not irreversible andthe Peterkofsky experiment showedthat
Fig 7 Proposed mode of entering the substrate (A, B and C), reaction
(D) and the release of the products (E,F) for the PAL and HAL
reac-tions Y denotes Y110 and Y53, N denotes N260 and N195, B denotes
Y251 andE414 for PAL andHAL, respectively.
Table 2 Distances in models of PAL’s active site Selecteddistances (A˚) in models for the zwitterionic L -phenylalanine binding (A), for the r-complex intermediate containing (B), and for the E-cinnamate/ammonia binding (C) state of the PAL active site are listed.
Trang 10there is a fast reverse reaction from the enzyme-NH4+
intermediate [55], discrimination of 15N can occur in the
later steps
We consider the results presented here and in our
previous papers [50–52] andconclude that the mechanism
involving the attack of MIO at the aromatic portion of the
substrates is more consistent with the experimental data and
modelling studies than the alternative mechanism proposed
in some older studies [1,54,55]
A C K N O W L E D G E M E N T S
We thank Prof G E Schulz andM Baed eker (University of
Freiburg) for providing us with the new PAL expression system and
also Dr M Stieger (Hoffmann-La Roche, Basel, Switzerland) for
vector pREP4-GroESL carrying the HSP-60 system D R thanks
the LandBad en-Wu¨rttemberg for a scholarship for graduate
students This work was supported by the Deutsche
Forschungsg-emeinschaft and the Fonds der Chemischen Industrie Financial
support by the Hungarian OTKA (T-033112) is also gratefully
acknowledged.
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