An active site homology model of phenylalanine ammonia-lyase
from
Petroselinum crispum
Dagmar Ro¨ ther
1
,La
´
szlo
´
Poppe
2
, Gaby Morlock
1
, Sandra Viergutz
1
and Ja
´
nos Re
´
tey
1
1
Institute for Organic Chemistry, University of Karlsruhe, Germany;
2
Institute 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 solved by X-ray crystal-
lography. Based on amino-acid sequence alignment of the
two enzymes, mutagenesis was performed on amino-acid

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 related enzyme 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 believed that
a dehydroalanine acts as electrophilic prosthetic group at
theactivesiteofbothHALandPAL[4–6].Recentlythe
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 proposed that this MIO
group is generated by 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 and by a homology model of PAL.

P. crispum is consistent with the SWISS-PROT database (P24481)
record but not with the numbering used in previous PAL papers.
(Received 15 February 2002, accepted 8 May 2002)
Eur. J. Biochem. 269, 3065–3075 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02984.x
5¢-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 verified by 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
and PAL was purified as described previously [10].
SDS/PAGE and Western blot analysis
SDS/PAGE was carried out according to Laemmli [13]
using 10% polyacrylamide gels. The gels were stained with
Coomassie Brillant Blue R250. Western Blot analyses were
performed following a previously described method using
nitrocellulose blotting filters [14,15]. Wild-type PAL and
mutants were detected with rabbit polyclonal antibodies
raised against PAL from P. crispum (the antibody was a
generous gift of N. Amrhein, Eidgeno

tions were varied from 0.01 to 5 m
M
. Kinetic constants (K
m
,
V
max
) were determined using a double reciprocal plot [17].
The isolated enzymes were electrophoretically pure as
verified by staining with Coomassie Brillant Blue R250
and therefore it was possible to measure the turnover
numbers (k
cat
) with the relative molecular mass of 311.313
for the tetrameric PAL. Determination of protein concen-
tration was carried out according to Warburg & Christian
[18,19], Murphy & Kies [20] and Groves et al. [21]. BSA was
used as reference protein for the measurements.
Sequence comparison
Amino-acid sequences of HAL from P. putida [22], HAL
from Homo sapiens [23] and PAL from P. crispum [24] were
extracted from the SWISS-PROT database. Sequence
alignment was carried out using the computer program
MALIGN
(HUSAR, DKFZ Heidelberg).
MALIGN
is a
HUSAR adaptation of the program
MAP
[25].

(
REDHAT
6.2). A switched smoothing
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 performed by using default settings of the
program packages. These optimized structures were
compared by
GROMOS
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.
3066 D. Ro
¨
ther et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Ramachandran plot analyses using the
SWISS
-
PDBVIEWER
3.6 package [27]. For further modelling, the PAL 64–531
monomer optimized by MM+ force-field of
HYPERCHEM

terminal fragment (532–716) of the PAL sequence (Swiss-
Prot: P24481). Although several reasonable hits were
found when the 532–716 fragment was submitted to
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]
showed no 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 and the 91–275 fragment of
chain B in the aspartase structure (PDB accession no.
1JSW) [40,41]. Because aspartase is also an ammonia-
lyase and its structure shows substantial structural
similarity to that of the template HAL (long parallel
helices and a 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 [27] package. No significant
clashes were found in the contact regions between the
unique chains. The aspherical nature of the active homo-
tetramer form of PAL is supported by 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 found in the 64–531 and the 532–716
fragments (29 and 14, 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. bond lengths 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 found for I141:
)3.68, H309: )2.93, R52: )2.80, F
621
: )2.73, E291: )2.51,
P699: )2.50] and
PROCHECK
V3.5 [45,46] through EMBL
the Biotech Validation Suite, Heidelberg [47]. Although

Conformational analysis of phenylalanine in its zwitter-
ionic state by PM3 calculations of
PC SPARTAN PRO
[48]
package was performed [28], and the lowest energy confor-
mation was used as 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 C
2
position of the phenyl ring of
L
-Phe should
be close enough to the methylene of the MIO to perform the
nucleophilic addition to the C ¼ C double bond; and (b) the
NH
3
+
and the pro-S b-H should be antiperiplanar [28].
Several, slightly different arrangements satisfied these
requirements. These starting structures containing the
zwitterionic
L
-
PHE
substrate were optimized by MM+
Ó FEBS 2002 Active-site model of phenylalanine ammonia-lyase (Eur. J. Biochem. 269) 3067
method of the

active sites containing the cationic intermediate state of
the substrate for PAL and HAL [50] were aligned
similarly (Fig. 4B).
Calculation of the charge distribution in the
L
-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.
RESULTS AND DISCUSSION
Amino-acid sequence comparison and site-directed
mutagenesis
Amino-acid sequence comparison of HAL and PAL from
different organisms showed a sequence identity of about 40
and 20% when comparing different sequences of HAL
among one another and comparing sequences of HAL and
PAL, respectively [3].
Recently, the X-ray structure of HAL from P. putida was
solved by Schwede et al., who discovered a new electrophilic
prosthetic group at the active site, namely MIO (Fig. 1) [7].

The plasmids containing the mutated genes were trans-
formed in cells of E. coli BL21(DE3) that were previously
transformed with the chaperonin-expressing plasmid
pREP4-groESL [10]. After expression and purification of
wild-type PAL and the various PAL mutants SDS/PAGE
and Western-Blot analysis of crude extracts were performed
to check expression levels and monomeric size of the enzyme
variants. In all cases similar quantities of recombinant
enzymes were produced showing the same monomeric size.
Western-Blot analysis revealed that all enzyme variants
were detected with anti-PAL antibodies. After purification
of wild-type PAL and the different PAL mutants yields
between 5 and 30 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 measured at substrate concentrations
varying from 0.01 to 5 m
ML
-phenylalanine. Table 1 shows
the kinetic constants of wild-type PAL and the constructed
and measured PAL mutants. The factor k
cat wtPAL
/k
cat
mutPAL
is the k
cat
or turnover number of wild-type PAL
divided by k

m
value is about 13 m
M
and is therefore about
100 times higher than that of wild-type PAL. In the double
mutant L138H/Q488E, the K
m
value is increased further to
55 m
M
and the enzyme is about 145 times less active than
the wild-type enzyme. The PAL residues L138 and Q488
and the unsimilar counterparts H83 and E414 in the HAL
amino-acid sequence may be important for the substrate
specificity of the homologous enzymes PAL and HAL. Our
expectation that the L138H/Q488E mutant of PAL shows
activity with
L
-histidine was not fulfilled. Although this
mutant showed a similar K
m
value for
L
-histidine as wild-
3068 D. Ro
¨
ther et al. (Eur. J. Biochem. 269) Ó FEBS 2002
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-acid residues of HAL and respective residues in PAL are marked with colour (A). Secondary structure of the

Æmg
)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 carried out according to Warburg & Christian
[18], Murphy & Kies [20] and Groves et al. [21].
K
m
(m
M
)
k
cat
(s
)1
)
k
cat
/K
m
(m
M
)1
Æs
)1
)
Factor
(k
cat wtPAL

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 showed a more than 20 900-fold lower activity
compared to the wild-type enzyme [50]. Therefore it was
assumed that E414 acts as a base in HAL catalysis.
Mutagenesis in residue S203 of the PAL gene led to an
enzyme variant with a k
cat
of 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
reported for this active site mutant [52]. The counterpart of
residue Y280 at the active site of HAL is Y351 in the amino-
acid sequence of PAL. PAL mutant Y351F showed a by
factor 235 reduced activity compared to that of the wild-
type enzyme. PAL mutants N260A and Q348A showed a
2700- and 2370-fold diminished activity, 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) and PAL (right). Catalytically important
residues are shown as stick models: MIO moiet-

expected positions postulated by comparison with the HAL
sequence and structure. Modelling of a zwitterionic sub-
strate (Fig. 5A), the r-complex forming between the
substrate and MIO (Fig. 5B) and the 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
confirmed the hypothesis concerning the p-stacking role of
F400. This residue may stabilize the intermediate r-complex
and prevent 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 model of
PAL overlaid on the experimental structure of
HAL. (A) The protein chains are coloured
differently. The lighter colours and the labels in
yellow are related to 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
related to the PAL model. The thin lines and
white labels are related to the HAL model.
Ó FEBS 2002 Active-site model of phenylalanine ammonia-lyase (Eur. J. Biochem. 269) 3071
cates 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

reaction rate, is located at 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 (and also in HAL its counterpart Y53) seems to play
twofold role (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. Model for 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.
3072 D. Ro
¨
ther et al. (Eur. J. Biochem. 269) Ó FEBS 2002
group 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
conjugated acid (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

[55]. Unfortunately, in his
experiments the concentration of NH
4
+
was too low
(10–60 m
M
); on the basis of these and other previous
experiments [56,57], he assumed that the HAL (as well as
the PAL) reaction was irreversible. Later, it was shown that
at higher NH
4
+
concentrations (up to 6
M
) both ammonia-
lyase reactions can be completely reversed [28,58]. Never-
theless, Peterkofsky’s experiments support the existence of
an enzyme–NH
4
+
intermediate and the idea that ammonia
is released after 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 and S203A for PAL) still
catalyze the reaction (about 10
3
times more slowly with the

than the one expected from the natural abundance of
15
N
(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 and the Peterkofsky experiment showed that
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 and E414 for PAL and HAL, respectively.
Table 2. Distances in models of PAL’s active site. Selected distances (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.
Atomic pairs Model 5A Model 5B Model 5C
S203
C3
–Phe
C2¢
4.18 1.54 4.65
S203
O1
–Phe
C3¢
3.78 3.05 3.73
Y435
O4¢
–Phe

O1
3.01 2.90 3.10
Q348
N4
–Phe
O1
3.39 3.93 3.97
R354
NH1
–Phe
O¢1
3.36 3.26 4.54
R354
NH2
–Phe
O¢1
3.93 3.36 5.03
Ó FEBS 2002 Active-site model of phenylalanine ammonia-lyase (Eur. J. Biochem. 269) 3073
there is a fast reverse reaction from the enzyme-NH
4
+
intermediate [55], discrimination of
15
N can occur in the
later steps.
We consider the results presented here and in our
previous papers [50–52] and conclude 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

6. Hanson, K.R. & Havir, E.A. (1970)
L
-Phenylalanine ammonia-
lyase. IV. Evidence that the prosthetic group contains a
dehydroalanyl residue and mechanism of action. Arch. Biochem.
Biophys. 141, 1–17.
7. Schwede, T.F., Re
´
tey, J. & Schulz, G.E. (1999) Crystal structure of
histidine ammonia-lyase revealing a novel polypeptide modifica-
tion as the catalytic electrophile. Biochemistry 38, 5355–5361.
8. Ormo
¨
,M.,Cubitt,A.B.,Kallio,K.,Gross,L.A.,Tsien,R.Y.&
Remington, S.J. (1996) Crystal structure of the Aequorea victoria
green fluorescent protein. Science 273, 1392–1395.
9. Ro
¨
ther, D., Merkel, D. & Re
´
tey, J. (2000) Spectroscopic evidence
for a 4-methylidene imidazol-5-one in histidine and phenylalanine
ammonia-lyases. Angew. Chem. 112, 2592–2594. Angew. Chem.
Int. Eds 39, 2462–2464.
10. Baedeker, M. & Schulz, G.E. (1999) Overexpression of a designed
2.2 kb gene of eukaryotic phenylalanine ammonia-lyase in
Escherichia coli. FEBS Lett. 457, 57–60.
11. Braman, J., Papworth, C. & Greener, A. (1996) Site-directed
mutagenesis using double-stranded plasmid DNA templates.
Methods Mol. Biol. 57, 31–44.

without nucleic acid interference. Anal. Biochem. 22, 195–210.
22. Consevage, M.W. & Phillips, A.T. (1990) Sequence analysis of the
hutH gene encoding histidine ammonia-lyase in Pseudomonas
putida. J. Bacteriol. 172, 2224–2229.
23. Suchi, M., Harada, N., Wada, Y. & Takagi, Y. (1993) Molecular
cloning of a cDNA encoding human histidase. Biochim. Biophys.
Acta 1216, 293–295.
24. Lois, R., Dietrich, A., Hahlbrock, K. & Schulz, W. (1989) A
phenylalanine ammonia-lyase gene from parsley: structure, reg-
ulation and identification of elicitor and light responsive cis-acting
elements. EMBO J. 8, 1641–1648.
25. Huang, X. (1994) On global sequence alignment. Comput. Appl.
Biosci. 10, 227–235.
26. Peitsch, M.C. (1996) ProMod and Swiss-Model: Internet-based
tools for automated comparative protein modelling. Biochem. Soc.
Trans. 24, 274–279.
27. Peitsch, M.C. & Guex, N. (1997) Swiss-Model and the Swiss-
PDBviewer: an environment for comparative protein modelling.
Electrophoresis 18, 2714–2723.
28.Gloge,A.,Zon,J.,K
}
oova
´
ri, A
´
., Poppe, L. & Re
´
tey, J. (2000)
Phenylalanine ammonia-lyase: the use of its broad substrate spe-
cificity for mechanistic investigations and biocatalysis. Synthesis of

36. Rost, B., Sander, C. & Schneider, R. (1994) PHD – an Automatic
Mail Server for Protein Secondary Structure Prediction.CABIOS,
10, 53–60.
37. Rost, B. & Sander, C. (1994) Combining evolutionary information
and neural networks to predict protein secondary structure.
Proteins 19, 55–72.
38. Fischer, D., Barret, C., Bryson, K., Elofsson, A., Godzik, A.,
Jones, D., Karplus, K.J., Kelley, L.A., Maccallum, R.M.,
Pawowski, K., Rost, B., Rychlewski, L. & Sternberg, M.J. (1999)
CAFASP-1: Critical assessment of fully automated structure
prediction methods. Prot. Struct. Funct. Genet. Suppl. 3, 209–217.
39. Kelley, L.A., Maccallum, R. & Sternberg, M.J.E. (1999)
Recognition of remote protein homologies using three-dimen-
sional information to generate a position specific scoring matrix in
the program 3D-PSSM, RECOMB 99. In Proceedings of the Third
Annual Conference on Computational Molecular Biology (Istrail,
S,. Pevzner, P., Waterman, M., eds), pp. 218–225. The Associ-
ation for Computing Machinery, New York.
40. Shi, W., Kidd, R., Giorgianni, F., Schindler, J.F., Viola, R.E. &
Farber. G.K. (1993) Crystallization and preliminary X-ray studies
of
L
-aspartase from Escherichia coli. J. Mol. Biol. 234, 1248–1256.
41. Shi, W., Dunbar, J., Jayasekera, M.M., Viola, R.E. & Farber,
G.K. (1997) The structure of
L
-aspartate ammonia-lyase from
Escherichia coli. Biochemistry 36, 9136–9144.
42. Havir, E.A. & Hanson, K.R. (1968)
L

´
tey, J. (1997) Identification of
essential amino acids in phenylalanine ammonia-lyase by site-
directed mutagenesis. Biochemistry 36, 10867–10871.
52. Schuster, B. & Re
´
tey, J. (1995) The mechanism of action of phe-
nylalanine ammonia-lyase: the role of prosthetic dehydroalanine.
Proc. Natl Acad. Sci. USA 92, 8433–8437.
53. Baedeker, M. & Schulz, G.E. (2002) Autocatalytic peptide cycli-
zation during chain folding of histidine ammonia-lyase. Structure
10, 61–67.
54. Hermes, J.D., Weiss, P.M. & Cleland, W.W. (1985) Use of
nitrogen-15 and deuterium isotope effects to determine the
chemical mechanism of phenylalanine ammonia-lyase. Biochem-
istry 24, 2959–2967.
55. Peterkofsky, A. (1962) Mechanism of action of histidase. Ami-
noenzyme formation and partial reactions. J. Biol. Chem. 237,
787–795.
56. Mehler, A.H. & Tabor, H. (1953) Deamination of histidine to
form urocanic acid in liver. J. Biol. Chem. 201, 775–784.
57. Yoshioka, Y. (1955) Histidine deaminase isolated from guinea-pig
liver. Osaka Daigaku Igaku Zassi 7, 377–387.
58. Williams, V.R. & Hiroms, J.M. (1967) Reversibility of the Ôirre-
versibleÕ histidine ammonia-lyase reaction. Biochim. Biophys. Acta
139, 214–216.
59. Langer, M., Pauling, A. & Re
´
tey, J. (1995) The Role of
Dehydroalanine in the Catalysis by Histidine Ammonia-Lyase.