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421
values of the parameters necessary for fitting equations to
the measured variables, were obtained by using graphical
procedures described by Henderson (1972) and Cha
Amrhein, N., Godeke, K.H., 1977. a-Aminooxy-b-phenylpropionic
¨
acid- a potent inhibitor of phenylalanine ammonia-lyase in vitro
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¨
Amrhein, N., Hollander, H., 1979. Inhibition of anthocyanin forma-
(1975).
tion in seedlings and flowers by the enantiomers of a-aminooxy-b-
phenylpropionic acid and their N-benzyloxycarbonyl derivatives.
Planta 144, 385–389.
4
.4. Stopped-flowkinetics
Amrhein, N., 1986. Specific inhibitors as probes into the biosynthesis
and metabolism of aromatic amino acids. In: Conn, E.E. (Ed.), The
Shikimic Acid Pathway. Rec. Adv. Phytochem., Vol. 20. Plenum
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Rapid kinetics were performed using the rapid kinet-
ics spectrometer accessory RX.1000 (Applied Photo-
physics, Leatherhead, UK).
Appert, C., Logemann, E., Hahlbrock, K., Schmid, J., Amrhein, N.,
1
994. Structural and catalytic properties of the four phenylalanine
ammonia-lyases from parsley (Petroselinum crispum Nym.). Eur. J.
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4
.5. Analysis of reaction products
Cha, S., 1975. Tight-binding inhibitors-I. Kinetic behavior. Biochem.
Pharmacol. 24, 2177–2185.
After incubation with PAL, reaction product origi-
nating from 2-aminoindan-2-carboxylic acid (AIC) was
Cha, S., 1976a. Erratum to ‘‘Cha, S., 1975. Tight-binding inhibitors-I.
Kinetic behavior. Biochem. Pharmacol. 24, 2177–2185’’. Biochem.
Pharmacol. 25, 1561.
separated on a C-18 reversed phase column (mBOND-
PAK C18, Waters, Millipore, Volketswil, CH) using a
TM
Cha, S., 1976b. Tight-binding inhibitors III. A new approach for the
determination of competition between tight-binding inhibitors and
substrates—inhibition of adenosine deaminase by coformycin. Bio-
chem. Pharmacol. 25, 2695–2702.
linear gradient from 5% acetic acid to 20% acetic acid/
0% acetonitrile in water. The HPLC-purified reaction
4
product from 2-aminoindan-2-carboxylic acid (AIC)
was separated both as the free acid and as its methy-
lester by GC on a Finnigan MAT-Magnum instrument
equipped with a 25 m column (ID=0.25 mm, film
thickness=0.25 mm, DB17, J&W Scientific, UK) using a
De-Eknamkul, W., Ellis, B.E., 1987. Tyrosine aminotransferase: the
entrypoint enzyme of the tyrosine-derived pathway in rosmarinic
acid biosynthesis. Phytochemistry 26, 1941–1946.
Hanson, K.R., Havir, E.A., 1981. Phenylalanine ammonia-lyase. In:
Stumpf, P.K., Conn, E.E. (Eds.), The Biochemistry of Plants, Vol.
ꢂ
temperature gradient from 80 to 250 C. The purified
substances were analyzed by MS. The mass spectro-
meter was operated at 70 eV.
7
. Academic Press, New York, pp. 577–625.
Hanson, K.R., 1981. Phenylalanine ammonia-lyase: mirror-image
packing of D- and L- phenylalanine and D- and L-transition state
analogs into the active site. Arch. Biochem. Biophys. 211, 575–588.
Henderson, P.J.F., 1972. A linear equation that describes the steady-
state kinetics of enzymes and subcellular particles interacting with
tightly bound inhibitors. Biochem. J. 127, 321–333.
4
.6. Determination of the catalytic-center activity
The catalytic-center activity of PAL for the alter-
¨
Hollander, H., Kiltz, H.-H., Amrhein, N., 1979. Interference of
L-a-aminooxy-b-phenylpropionic acid with phenylalanine meta-
bolism in buckwheat. Z. Naturforsch. 34C, 1162–1173.
Janas, K.M., Filipiak, A., Kowalik, J., Mastalerz, P., Knypl, J.S.,
native substrate AIC was determined by comparing the
rate of product formation with that for (S)-phenylala-
nine. The previously determined catalytic-center activity
1
985. 1-Amino-2-phenylphosphonic acid: an inhibitor of L-
ꢁ
1
ꢂ
for PAL (40 s per active site, at 40 C; Appert et al.,
994) was used to calculate the catalytic-center activity
for the alternative substrate.
phenylalanine ammonia-layse in vitro. Acta Biochem. Polon. 32,
131–143.
1
Laber, B., Kiltz, H.-H., Amrhein, N., 1986. Inhibition of phenylala-
nine ammonia-lyase in vitro and in vivo by (1-amino-2-phenylethyl)
phosphoric acid, the phosphonic analogue of phenylalanine. Z.
Naturforsch. 41C, 33–39.
Acknowledgements
Liebermann, C., Zsuffa, M., 1911. Zur Carboxylierung mehrkerniger
aromatischer Kohlenwasserstoffe. Chem. Ber. 44, 202–210.
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We are grateful to Prof. Dr. E. W. Weiler and Mr. J.
Schab (Ruhr-University Bochum, Germany) for the
¨
GC-MS analysis. We thank Prof. E. Schonbrunn (Uni-
versity of Kansas) for his helpful advice on interpreta-
tion of the kinetic data. This research was supported in
part by the Polish Ministry of Education (MEN), the
Komitet Badan Naukowych and by the Oststaaten-
Soforthilfeprogramm of the Swiss National Science
Foundation (Grant 70PP-029654).
1
05.
Morrison, J.F., Walsh, C.T., 1987. The behavior and the significance
of slow-binding enzyme inhibitors. Adv. Enzymol. Relat. Areas.
Mol. Biol. 61, 201–368.
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alanine ammonia-lyase has tyrosine ammonia-lyase activity. Plant
Physiol. 113, 175–179.
Ro
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ther, D., Merkel, D., Re
-methylidene imidazol-5-one in histidine ans phenylalanine ammo-
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