Inhibition of apple 1-aminocyclopropane-1-carboxylic acid oxidase, by cyclopropane-1,1-dicarboxylic acid and trans-2-phenylcyclopropane-1-carboxylic acid

Article abstract of DOI:10.1016/S0031-9422(00)00315-0

Cyclopropane-1,1-dicarboxylic acid (CDA) and trans-2-phenylcyclopropane-1-carboxylic acid (PCCA) are the main representatives of a group of compounds that are structural analogues of 1-aminocyclopropane-1-carboxylic acid (ACC) and have been proved to have an inhibitory effect on the wound ethylene produced by Lycopersicum esculentum fruit discs. During the experiments, that were carried out in this work the inhibition pattern of PCCA and CDA were studied when tested on partially purified apple ACO and their K(i) values were determined. A mechanistic proposal was given, in order to explain the kinetic behaviour of the inhibitors. The common feature of these molecules is their cyclopropane ring, with different substitutes mainly at the positions C1 and C2. Two other compounds with similar structure where also tested as inhibitors, in order to clarify the relationship between structure and activity. These compounds are: 2-methyl cyclopropanecarboxylic acid (MCA), and cyclopropanecarboxylic acid (CCA). (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0031-9422(00)00315-0

Phytochemistry 55 (2000) 203±211
www.elsevier.com/locate/phytochem
Inhibition of apple 1-aminocyclopropane-1-carboxylic acid
oxidase, by cyclopropane-1,1-dicarboxylic acid and
trans-2-phenylcyclopropane-1-carboxylic acid
xVassilis Dourtoglou *, xElisabeth Koussissi
Vioryl S.A. Chemical and Agricultural Industry, Research, 36 Viltaniotis St, Ki®ssia, 145 64 Athens, Greece
Received 7 December 1999; received in revised form 19 July 2000
Abstract
Cyclopropane-1,1-dicarboxylic acid (CDA) and trans-2-phenylcyclopropane-1-carboxylic acid (PCCA) are the main representa-
tives of a group of compounds that are structural analogues of 1-aminocyclopropane-1-carboxylic acid (ACC) and have been
proved to have an inhibitory e€ect on the wound ethylene produced by Lycopersicum esculentum fruit discs. During the experi-
ments, that were carried out in this work the inhibition pattern of PCCA and CDA were studied when tested on partially puri®ed
apple ACO and their Ki values were determined. A mechanistic proposal was given, in order to explain the kinetic behaviour of the
inhibitors. The common feature of these molecules is their cyclopropane ring, with di€erent substitutes mainly at the positions C1
and C2. Two other compounds with similar structure where also tested as inhibitors, in order to clarify the relationship between
structure and activity. These compounds are: 2-methyl cyclopropanecarboxylic acid (MCA), and cyclopropanecarboxylic acid
(CCA). # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Apple fruit; Ethylene; 1-Aminocyclopropane-1-carboxylic acid oxidase (ACO) inhibitors
1. Introduction
In this paper we report the inhibition e€ect of four
compounds on partially puri®ed apple ACO, aiming at
the development of a new class of non gaseous, envir-
onmental friendly ``ethylene inhibitors'' that can be used
for cut ¯owers and for post harvest treatments of fruits
and vegetables. All the compounds tested for their e€ect
on apple ACO extract, are ACC analogues and are
proved to inhibit wound ethylene, produced by Lyco-
persicum esculentum fruit discs (Dourtoglou et al., 1999,
2000). Some of them also inhibit tomato fruit matura-
tion (Dourtoglou, unpublished results).
The generic term ``ethylene inhibitors'' is applied to a
large group of organic and inorganic chemical com-
pounds possessing a common property: the disturbance
of ethylene mediated plant regulation. They can be clas-
si®ed in three categories according to their action (Abeles
et al., 1992): the ®rst one includes those acting on ACC
synthase and therefore inhibit the conversion of S-ade-
nosyl-l-methionine (SAM) to 1-aminocyclopropane-1-
carboxylic acid (ACC) according to the pathway pro-
posed by Yang. The second category includes the com-
pounds acting on ACC-oxidase and could be called ACO
inhibitors (Adams and Yang, 1979; Yang and Ho€man,
1984; John et al., 1997; Gibson et al., 1998). Finally, the
last category includes all the compounds interfering
between ethylene and its receptor (Yang and Ho€man,
1984; Sisler et al., 1996).
2. Results and discussion
Various compounds have been tested as inhibitors of
ACO in order to understand the relationship of ACO
active site and ACC degradation. Most of these com-
pounds are l and d amino acids or l and d amino acid
derivatives such as hydroxamates and N-acetyl amino
acids. However, due to low inhibition and high Ki
values, most of these compounds were not suitable for
* Corresponding author. Tel.: +30-18074603-669; fax: +30-18074681.
E-mail address: dourt@ath.forthnet.gr (V. Dourtoglou).
0031-9422/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(00)00315-0

204
V. Dourtoglou, E. Koussissi / Phytochemistry 55 (2000) 203±211
agricultural use, although they provide a useful tool for
the study of ACC oxidation mechanism.
other dioxygenases (John et al., 1991). In addition, we
detected a low level substrate inhibition, which a€ected
the initial velocity of the reaction and was more fre-
quently apparent at ACC concentrations higher than
150 mM, even though it varied upon each enzymatic
activity (Fig. 3).
ACO from many fruits (Dilley et al., 1993; Dupille
et al., 1993), pear (Vioque and Castellano, 1998) and
avocado (Mc Garvey and Christo€ersen, 1992) has
been isolated and puri®ed. Due to low stability of the
native enzyme, a recombinant apple ACO, expressed in
Escherichia coli, was used to understand the mechan-
ism of ACC oxidation (Kadyrzhanova et al., 1997).
a-Aminoisobutyric acid (AIB) was proved to act as
a competitive inhibitor and the inhibition e€ect of
other amino acids has also been studied on the above
mentioned enzyme (Charng et al., 1997).
CDA was clearly shown to have a non-competitive
inhibition and its Ki value was determined at 100Æ20 mM
(Figs. 4 and 5). For PCCA by plotting inhibition data Ki
value was determined at 10Æ2 mM if the competition
was non-competitive or 5±6 mM if the competition was
competitive. In the double reciprocal plot of ACO in the
absence and presence of 10 and 5 mM of PCCA (Figs. 6
and 7) a slight displacement of the lines changes the
pattern of the inhibition from non-competitive to
competitive (dashed lines in Figs. 6 and 7). Another
hypothesis is to have a real intermediate type of inhibi-
tion. Unlike to strictly non-competitive inhibition, the
inhibitor has di€erent anities for the two forms of the
enzyme (free or bound with ACC) having two dissocia-
tion constants Ki. The straight lines obtained at di€erent
values of ACC (Fig.7) intersect at the points [PCCA]=
Ki and if at the intersection points (1/v)>0 the inhibi-
tion tends to be competitive. Concerning the rest of the
compounds, the ethylene produced, when CCA was
added to the assay mixture, was much higher compared
to the blank assay (data not shown). In addition, CCA,
as well as MCA, produced ethylene when reacted with
Fenton reagent.
In this work a partially puri®ed apple ACO was used
to evaluate the inhibition of CDA, PCCA, MCA, and
CCA and the results of inhibition were expressed by
comparing the enzymatic activity with and without each
inhibitor. Therefore, no further puri®cation of the
enzyme was required. The mean value for the activity of
the enzyme preparation obtained, was 200 nM of ethyl-
1
1
ene min mg of protein (Fig. 1). Meanwhile, the
apparent value of Km for ACO was determined at 25±
30 mM (Fig. 2), which is in accordance with the results
given for puri®ed apple fruit ACO (Dupille et al., 1993).
Preliminary experiments showed that the activity of the
partially puri®ed enzyme used in this work was directly
proportional to the amount of the enzyme added to the
reaction mixture over the range of activities determined
(Fig. 1). However, the reaction rate was not linear but
decreased as the reaction proceeded (data not shown). A
non-linear time course has been observed with many
Previous work on the catalytic mechanism of ACC oxi-
dation have shown that catalysis involves an octahedral
Fig. 1. E€ect of di€erent ACO concentrations, on the initial velocity of the reaction.

V. Dourtoglou, E. Koussissi / Phytochemistry 55 (2000) 203±211
205
Fig. 2. Double reciprocal plot (Lineweaver±Burk) of ACO activity vs concentration of ACC. Kmꢀ30 mM.
Fig. 3. Time course of ethylene production from apple ACO, at di€erent ACC concentrations.
ferrous iron (FeII) structure with H177, D179 and H234 of
apple ACO participating in the active center of the
enzyme and being ligated to FeII (Kadyrzhanova et al.,
1997; Barlow et al., 1997; Zhang et al., 1997; Gibson et
al., 1998) (structure I, Fig. 8). In our work, it was
necessary to explain the kinetic data obtained by the
inhibitors by using a mechanistic approach. The sug-
gested mechanism for the catalytic cycle of ACO involves

206
V. Dourtoglou, E. Koussissi / Phytochemistry 55 (2000) 203±211
Fig. 4. Double reciprocal plot (Lineweaver±Burk) of ACO activity vs concentration of ACC in the absence and presence of 62.5 mM cyclopropane-
1,1-dicarboxylic acid (CDA).
Fig. 5. E€ect of di€erent cyclopropane-1,1-dicarboxylic acid (CDA) concentrations on the activity of the partially puri®ed ACO [1/v=f (CDA)].
Two di€erent plots at two di€erent concentrations of ACC.
a ®rst step of oxidation using dioxygen and ascorbate and
replacement of dehydroascorbate by carbon dioxide or
bicarbonate at the FeII active centre (structure II, Fig. 9)
(Barlow et al., 1997; Zhang et al., 1997).
At this point, the hypothesis suggested in our work
for the following steps of the mechanistic approach,
di€ered from that of previous authors. In particular, we
propose that the carboxyl group of ACC can be ligated

V. Dourtoglou, E. Koussissi / Phytochemistry 55 (2000) 203±211
207
Fig. 6. Double reciprocal plot (Lineweaver±Burk) of ACO activity vs concentration of ACC in the absence and presence of 10 mM and 5 mM of
trans-2-phenylcyclopropane-1-carboxylic acid (PCCA). Km and K0m for non-competitive and competitive inhibition pattern.
Fig. 7. E€ect of di€erent trans-2-phenylcyclopropane-1-carboxylic acid concentrations on the activity of the partially puri®ed ACO [1/v=f(PCCA)].
Two di€erent plots at two di€erent concentrations of ACC). Ki and K0i for non-competitive and competitive inhibition pattern.
to FeIV replacing a molecule of water with subsequent
formation of FeIII (structure III, Fig. 8). At the next
step of catalysis, the amino group of ACC replaces the
hydroxyl ligand of FeIII by forming a bidantate ®ve
atom ring between FeIII and ACC (structure IV, Fig. 8).
This bidentate coordination has been suggested by
Dourtoglou et al. (1999) and recently con®rmed inde-
pendently by Rocklin et al. (1999) binding alanine

208
V. Dourtoglou, E. Koussissi / Phytochemistry 55 (2000) 203±211
Fig. 8. Proposed catalytic cycle of ACC oxidase.
speci®cally labelled with 15N and examined by using Q-
band electron nuclear double resonance.
of 2-ethyl-ACC mainly a€ects the stabilization of the
positively charged amino group of the substrate by the
bicarbonate anion attached to the iron. This hypotesis is
also supported by the observed ACO catalysis of d-
valine to isobutanal conversion (Gibson et al., 1998). It is
also supported by the fact that the enzyme exhibits a
much higher activity with d-amino acids than with their
l-enantiomers, when using the same substrate con-
centration and recombinant apple ACO (Charng et al.,
1997). The ®nal step of catalysis, according to our
hypothesis, involves two electron transfers. The ®rst
In addition ACO's stereospeci®city for (1S, 2R)-2-
ethyl-ACC (Ho€man et al., 1982) can be explained by
this structure. In this rigid ®ve atom bidantate ligation
of the above substrate to iron, the preferential transfor-
mation of 1R, 2S diastereoisomer of 2-ethyl-ACC to 1-
butene can be explained by the absence of steric hin-
drance of the ethyl group when it is at a trans position
to the carboxylic acid. The steric hindrance of the ethyl
group of (1S, 2R), (1S, 2S), (1R, 2R) diasteroisomers

V. Dourtoglou, E. Koussissi / Phytochemistry 55 (2000) 203±211
209
electron is transferred from the amino group of ACC to
FeIII resulting to the formation of a FeII complex
(structure V, Fig. 8). The second electron transfer takes
place within the ACC molecule (structures VI and VII,
Fig. 8).
As far as the inhibitors PCCA and CDA are concerned,
the kinetic data reveals a pattern of non-competitive
inhibition for CDA and still unclear for PCCA for
which and intermediate type should be proposed with
slightly di€erences on the value of Ki. It can be con-
cluded that the carboxyl group of the above inhibitors
can be ligated to the active site prior to the ACC liga-
tion, (Fig. 9, intermediate InIII for PCCA and CDA) or
replace the carboxyl group of ACC at the bidantate ®ve
atom ring (Fig. 9, intermediate InIV). The last is true
with certitude only for CDA while in PCCA the model
requires further work to be proved.
In any case, we adopt the mechanism proposed by
Zhang et al. (1997), in which the iron-ligated bicarbo-
nate may serve to orientated the amino group of ACC
towards the ferryl group e€ectively, in order to stabilize
the positively charged amino group (Fig. 9, intermediate
InIV) or (Fig. 8, intermediates IV, V, VI, VII).
In the above mentioned mechanistic hypothesis, the
carboxyl group of the inhibitor is ligated to the ferryl or
ferrous iron and the principal characteristic of its kinetic
behaviour is the addition of the acid to the same, as that
of ACC, intermediate (Fig. 9). We assume that the
inhibitor does not replace the residues H177, D179, H234
of the active site since the catalysis takes place according
to kinetic data.
Moreover the absence of an amino group and the
presence of a carboxyl group attached to a cyclopro-
pane ring within the structure of the inhibitor (PCCA) is
necessary for this type of inhibition.
Studies of Charng et al. (1997) with a-aminoisobu-
tyric acid (AIB), as well as other studies with amino
acids, reveal a competitive inhibition. As far as amino
acids are concerned, there is competition between their
amino groups and those of ACC. This leads to the con-
clusion that amino acids can be ligated to ferrous or
ferryl iron both with their amino and carboxyl groups
prior to ACC ligation and the resulted type of inhibition
is competitive.
In both cases, ACO can oxidize amino acids or AIB
and the obtained products are iso-butanal and aceton
when valine or AIB were employed as substrates (Gib-
son et al., 1998; Charng et al., 1997). Consequently, we
can conclude that amino acids are also ligated at a
bidantate ®ve-atom ring. This explains the stereo-
selectivity of the enzyme and the fact that they are
competitive inhibitors replacing both the amino and the
carboxylic group of ACC in the active centre.
The octahedral iron at the active site allow us to pro-
ceed to another hypothesis, that explains the steroselec-
tivity of ACO, when d-amino acids are assayed as
substrates. Two of three residues (H177, D179) of ACO
are placed on the same median plane, while the oxygen
of the carbon dioxide and the other residue (H234
)
occupy, respectively, the two opposite summits of the
octahedral bi-pyramidal structure. Amino and car-
boxylic groups of amino acids occupy the other two
sites of the median plane.
This con®guration also explains why the ethyl sub-
stituent of the pro-R methylene which is at a trans
position to the carboxyl group of (1R, 2S)-AEC (1-
amino-2-ethyl-cyclopropane-1-carboxylic acid) causes
very little or no interference with the enzyme when the
above substrate was assayed (Charng et al., 1977).
Compared to PCCA, CDA is a much more e€ective
inhibitor. This is probably due to its double carboxyl
function. Compounds such as MCA, which is also a
substrate analogue, demonstrate varying behaviour
depending on their ®nal concentration. Inhibition was
observed at a MCA concentration of 20 mM, 15 mM
corresponds to the control assay, and 10 mM induces a
higher ethylene production according to Dourtoglou et
al. (1999).
Similarly, a smaller molecule such as CCA can form a
loose connection with the active site and consequently
cannot act as an inhibitor. Thus, the fact that more
ethylene was produced when this molecule is used,
than that produced at the blank assay, may be due to a
partial breakdown of the molecule.
Fig. 9. Proposed catalytic cycle of ACC oxidase in the presence of
PCCA and CDA.

210
V. Dourtoglou, E. Koussissi / Phytochemistry 55 (2000) 203±211
3. Experimental
syringe of 100 ml was used to take samples from the vial
headspace.
3.1. Plant material
Ethylene was measured using the Varian 3800 gas
chromatograph, equipped with a capillary RT-Q-PLOT
column (RESTEC) (0.53 mm i.d., 15 m, 0.5 mm) with a
Helium ¯ow rate of 8 ml/min at 50ꢁC. Column tem-
perature was stable at 50ꢁC (isotherm), and the detector
was maintained at 150ꢁC. The GC was also equipped
with an autosampler (Varian 8200) which, by using a
special gas tight syringe, would withdrawn 100 ml samples
from the headspace of the vials through the septum and
injects them directly to the GC.
The initial velocity of the reaction was calculated by
measuring the produced ethylene both after 10 and
20 min of incubation. Initial velocity (v) was expressed
as the slope of the line joining the points corresponding
at ethylene production at 10 and 20 min using the curve
®tting facility of the Excel program (Microsoft) and the
data of each experiment. Each ethylene production value
at the corresponding time point represents the mean value
of the measurements of three relevant experiments. An
error of 5% was attributed to all the values of each curve
(Y-axis error bars). The error attributed to these values
accounts for all the possible sources of error. Experi-
mental data with error exceeding 5% was rejected. As it
was mentioned above, for some experiments the initial
velocity was measured at 10 and 20 min time points using
the same enzyme preparation for both the blank and the
inhibitor. The activity of the puri®ed enzyme was not the
same and di€ers from one experiment to the other.
When studying the e€ect of the substrate on the
enzyme and the reaction with the inhibitors varying
concentrations of ACC were used, whereas the con-
centrations of NaHCO3, FeSO4 and sodium ascorbate
remained the same.
``Golden delicious'' apples were purchased from the
local market and stored at 4ꢁC.
3.2. Chemicals
Potential inhibitors were purchased from ACROS
and FLUKA. The compound trans-2-phenylcyclopro-
pane carboxylic acid (PCCA) was also synthesized in
VIORYL S.A. Dithiothreitol (DTT) and polyvinyl-
pyrolidone (PVP) were purchased from SIGMA.
3.3. ACC oxidase extraction and partial puri®cation
Unless otherwise stated, all operations were carried
out at 4ꢁC. One hundred grammes of apple (cut in four
to ®ve pieces) were turned into thin powder in a ther-
momixer that contained liquid nitrogen, and a spoonful
of sea sand. The powder was homogenized in two
volumes (v/w) of the extraction bu€er A (0.1 M Tris±
HCl (pH 7.4), 10% (w/v) glycerol, 30 mM sodium
ascorbate, 5 mM DTT, 1% (w/v) PVP 40000). The
slurry was centrifuged at 13000 rpm for 60 min and the
supernatant was adjusted to 30% saturation with
(NH4)2SO4. After removal of precipitated material by
centrifugation (13000 rpm, 60 min), the supernatant was
brought to 90% saturation with (NH4)2SO4 and cen-
trifuged for 60 min at the same conditions. Precipitated
protein was collected by centrifugation (13000 rpm,
60 min). The resulting pellet was thawed and resus-
pended in 5 ml of bu€er B (20 mM Tris±HCl (pH 7.4),
glycerol 10% (w/v), sodium ascorbate 3 mM, DTT
1 mM), containing 1M (NH4)2SO4. Two millilitres of
the dissolved pellet were desalted by passage through a
Sephadex-G25 column (33.5 cm long, radius: 0.75 cm)
equilibrated with bu€er B. Finally, 30 fractions of
3.5 ml were collected. The fractions used for the deter-
mination of the inhibitory e€ect, were those with the
highest activity, which in general was found in fractions
6±10.
Proteins were determined according to the Bradford
method (1976) using the Bio-Rad (GmbH Laboratories,
Munich, Germany) reagent and bovine serum albumin
as a standard.
Acknowledgements
3.4. Assay for enzymatic activity and protein
determination
We are grateful to Zoe Karvouni for her valuable
comments and critical examination of the manuscript.
This work is part of a research program under the title
``Controlled ripening and increased storage life in fruits
and vegetables through ethylene control'' (FAIR CT
95-0225), supported by the funds of EU.
Enzymatic activity was measured as follows: 100 ml of
enzyme extract was added in a 2 ml vial, equipped with a
silicon septum cap, which contained the reaction mix-
ture. The reaction mixture, contained: 10 mM NaHCO3,
200 mM FeSO4, 0,25 mM ACC, and was brought to
900 ml with bu€er B. The enzymatic extract was always
added last and the vial was immediately closed. ACO
activity was assayed by measuring the ethylene pro-
duced after 10 min of incubation at 30ꢁC. A gas tight
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