(Received for publication, March 20, 1995; and in revised form, May 24, 1995)
From the
Myrosinase (EC 3.2.3.1) is the -thioglucosidase enzyme
responsible for the hydrolysis of glucosinolates, a group of naturally
occurring plant metabolites. The enzyme catalyzes the hydrolysis of
these S-glucosides to give D-glucose and an aglycone
fragment, which then rearranges to give sulfate and an isothiocyanate.
As part of ongoing mechanistic studies on myrosinase, the ability of
the enzyme to catalyze transglycosylation reactions has been examined.
Enzyme activity and stability were both decreased in the presence of
various organic solvents, including simple alcohols, but not
sufficiently to prevent reaction taking place. However, in contrast to
most other
-glycosidases, myrosinase did not catalyze
transglycosylation reactions either with the alcohols or other suitable
glycosyl acceptors. Although a wide range of potential acceptors were
investigated, none proved to be effective. Even when appropriately
charged side chains were included in the acceptor molecule to mimic the
sulfonic acid in the glucosinolate structure, transglycosylation did
not take place. The putative enzyme-glycosyl intermediate therefore
appears to be unavailable for reaction, possibly because D-glucose is the first product released from the enzyme. The
transition state analogue, glucono-
-lactone, a potent competitive
inhibitor of
-glucosidase, was found to be a poor noncompetitive
inhibitor of myrosinase. Myrosinase is specifically activated by
ascorbic acid, and it is proposed that the inhibitor is binding at this
alternative site.
Myrosinase (EC 3.2.3.1) is the trivial name for the
-thioglucosidase enzyme responsible for the hydrolysis of
glucosinolates (I) (Fig. 1), a group of sulfur-containing
glycosides that occur in all members of the Cruciferae, including the
brassica vegetables(1) . Over 100 different examples have been
isolated and characterized, containing a variety of substituents in the
side chain R including allyl (sinigrin), benzyl, and
indoyl(1) . Enzymic hydrolysis usually occurs when cells are
damaged as a result of plant injury or food processing giving as
products
-D-glucose (II) and the aglycone fragment (III).
The aglycone is unstable and reacts further giving the isothiocyanate
(IV) by means of a Lossen-type rearrangement (Fig. 1). Some
evidence implies that the rearrangement occurs spontaneously and is not
enzyme catalyzed, but it is not conclusive(2) .
Figure 1: Metabolism of glucosinolates as catalyzed by myrosinase.
Considerable work has been carried out in this area because of the agricultural and dietary importance of the Cruciferae. Products of glucosinolate hydrolysis are responsible for the distinctive flavor and aroma characteristics of crucifers. However, they can also have deleterious effects due to their pungency and in some cases goitrogenic activity and hepatoxicity(3, 4) . This is a particular problem with oilseed rape and its food and feed derivatives. EC recommendations concerning the maximum permitted levels of glucosinolates in rapeseed have resulted in major programs to breed safer varieties. However, the chemistry of the system is still not fully understood and the mechanism of myrosinase has not been elucidated.
We were interested in
studying the chemical mechanism of myrosinase and, in particular,
comparing it with the much more widely studied -glycosidases. In
recent years, there has been a massive increase in interest in
glycosidases(5) . Inhibition of the enzymes that process
carbohydrates has potential therapeutic applications for the treatment
of cancer, AIDS, and other viral infections. Crystallographic studies,
identification of active sites by mechanism-based labeling, and
determination of transition state structure by kinetic analysis have
allowed the construction of a detailed picture of glycosidase action.
Myrosinase has been much less studied, and it is not known how the
enzymatic hydrolysis of S-glycosides fits into the established
picture of enzymatic O-glycoside hydrolysis.
Myrosinase has
been isolated from a number of plant sources including Lepidium
sativum L. (light grown cress)(6) , Sinapis
alba (white mustard)(7) , and Brassica napus (rapeseed) seeds(7) . The mustard and rapeseed (7) enzymes have molecular weights of 120,000-150,000.
The activity of the L. sativum L. enzyme (6) was tested with 29 different glycosides, and only 4 were
found to be good substrates. These were, in order of decreasing
activity, sinigrin (the natural substrate), benzylglucosinolate, p-nitrophenyl--D-glucoside (PNPG), (
)and o-nitrophenyl-
-D-glucoside.
-D-Glucose could not be replaced by any other sugar.
-Glucosides were inactive, as were unreactive
-glucosides,
such as methyl or phenyl. Interestingly, there appears to be no
discrimination between different glucosinolates, and all are hydrolyzed
at similar rates regardless of the nature of the side chain. Early work
by Ettlinger et al.(2) demonstrated that the sulfate
group was required for optimum activity as the desulfoglucosinolate did
not act as a substrate with the Sinapis enzyme. The various
isozymes have broad pH optima, from pH 5 to pH
7(6, 7) . No metal ion requirement has been found for
the enzyme. For most plant enzymes, inhibition has been observed by
-SH-directed reagents, implying that a cysteine residue is
essential for catalysis. Myrosinase is specifically activated by
ascorbic acid. The rate of hydrolysis of sinigrin, catalyzed by the
mustard enzyme, is increased more than 25-fold by 1 mM ascorbic acid(8) , although high concentrations begin to
competitively inhibit reaction. Analogues of ascorbic acid are not
effective. Hydrolysis of PNPG, by the same enzyme, however, is not
increased by ascorbic acid(9) . Spectroscopic studies implied
that there was a conformational change in the enzyme on binding
ascorbic acid, and it was proposed that this brought about the rate
enhancement.
We report the results of our initial studies on the chemical mechanism of myrosinase. These comprise examination of the activity of the enzyme in the presence of alternative glycosyl acceptors and inhibition studies.
For
incubations involving PNPG, a similar procedure was employed, except
that a larger concentration of potassium phosphate buffer, 50
mM, was employed. In this case, the absorbance due to the
product, p-nitrophenol, was monitored at 430 nm ( 7002 M
cm
). The extinction
coefficient was found to change on addition of high concentrations of
methanol, and it was necessary to determine the values. These were 4176
and 3193 M
cm
in 6.25
and 12.5 M methanol, respectively. Concentrations of PNPG from
1.0 to 30 mM were employed, and each measurement was repeated
in triplicate. The reactions were linear over the time course measured,
up to approximately 5% of the overall reaction. The kinetic data were
then analyzed as above.
It is well documented that many glycosidases are able to
catalyze transglycosylation reactions, namely the transfer of residues
from glycoside substrates to acceptor molecules other than
water(12, 13) . Synthetic organic chemists have
frequently exploited this useful property and employed
-glycosidases in the synthesis of novel glycosides(14) .
For example, when o-nitrophenyl-
-D-galactoside
(V) is hydrolyzed by
-galactosidase in the presence of the epoxy
alcohol (VI), the galactosyl moiety is transferred to the acceptor
hydroxyl group to give the new
-galactoside (VII) (15) (Fig. 2).
Figure 2:
The use of -galactosidase-catalyzed
transglycosylation in the synthesis of novel
galactosides.
The ability of myrosinase to catalyze
similar transglycosylation reactions had not been examined. Therefore,
preliminary preparative scale experiments were carried out using
myrosinase under similar conditions with PNPG as the substrate and
allyl alcohol as the glycosyl acceptor. Allyl alcohol was chosen
because it resembles the side chain of the glucosinolate, sinigrin, a
natural substrate for myrosinase, and should have some chance of
binding at the enzyme active site. However, no
allyl--D-glucoside, the product of transglycosylation,
was observed in any reaction. The same substrate was also hydrolyzed in
a 9:1 ethanol:water mixture to produce the
ethyl-
-D-glucoside. Again, no product was observed. These
early failures to observe any transglycosylation prompted us to study
the activity of myrosinase in alcohol:water mixtures in more detail and
show whether the reactions failed due to mechanistic reasons or simply
because of reduced enzyme activity.
Two myrosinase activities were
monitored. The hydrolysis of sinigrin was followed by UV spectroscopy,
monitoring the disappearance of the sinigrin absorbance at 227 nm. In
separate experiments, PNPG hydrolysis was also followed by UV
spectroscopy monitoring the appearance of the p-nitrophenol
absorbance at 430 nm. The kinetics for the hydrolysis of sinigrin were
determined under the standard assay conditions (pH 7.0, 33 mM phosphate buffer, 37 ± 0.1 °C). The K for sinigrin was found to be 0.42
± 0.05 mM, in good agreement with previous
workers(16) , and V
was also comparable (10) at (48 ± 0.2)
10
mol
dm
min
. The measurements were
then repeated in the presence of increasing amounts of methanol, from
0.2 to 12.5 M (40% by volume) (Table 1). The hydrolysis
of PNPG was then examined under similar conditions but using 50 mM phosphate buffer. In aqueous solution, a high K
of 89 ± 30 mM was obtained, as well as a V
of (0.59 ± 0.2)
10
mol dm
min
. Thus, the rate
of PNPG was much lower than that of sinigrin as expected(16) .
The experiments were then repeated in methanol:water mixtures as for
sinigrin (Table 2).
For both activities, it can be seen that
the rate of hydrolysis decreases as the amount of methanol increases.
Values of K for sinigrin remained
constant and the observed rate decrease was all due to changes in V
. Thus, binding of the substrate to the enzyme
active site was unaffected by the change in the reaction medium, while
the rate of the reaction decreased. Even with 12.5 M (40% v/v)
methanol, there was an appreciable rate of reaction with only a 50%
decrease from the rate in aqueous solution. Data for PNPG showed
similar trends. Therefore, the enzyme is not terribly sensitive to the
presence of the organic solvent, and so the chances of observing
transglycosylation were reasonable. The measurements were also repeated
in a range of other water-miscible organic solvents (ethanol, dioxan,
acetonitrile) ( Table 1and Table 2), again showing reduced,
although significant, activity.
The kinetic results were all initial rate measurements and so gave no information concerning the long term stability of myrosinase under the reaction conditions. Therefore, the stability of myrosinase was examined in the methanol:water mixtures by incubating the enzyme in buffer at 37 °C, removing aliquots at various time intervals, and measuring the myrosinase activity using the standard sinigrin assay (Fig. 3). If the enzyme proved to be very unstable, then the chances of transglycosylation reactions may be significantly reduced. However, myrosinase was in fact very stable. The half-life in the presence of 6.25 M (20%) methanol at 37 °C was 40 h but was reduced to 8 h with 12.5 M (40%) methanol. Myrosinase was generally found to be very stable. Concentrated stock solutions used for kinetic experiments, stored at 4 °C, did not lose appreciable amounts of activity even after 2-3 weeks.
Figure 3:
Stability of myrosinase in methanol:water
mixtures. Myrosinase (4 units) was incubated at 37 ± 0.1 °C
at pH 7.0 in 33 mM potassium phosphate buffer (1 ml),
containing the appropriate amount of methanol. Aliquots (30 µl)
were withdrawn at various time intervals, and the enzyme activity was
measured using the standard assay. The incubation conditions were as
follows: , no methanol;
, 6.25 M (20%) methanol;
, 12.5 M (40%) methanol.
Once the activity and stability of myrosinase in
the presence of glycosyl acceptors had been examined, it was then
necessary to determine the degree of transglycosylation that was taking
place. This required measurement of the relative amounts of D-glucose and p-nitrophenol produced on hydrolysis of
the PNPG under various conditions. A coupled enzyme assay was used to
measure D-glucose concentrations, while the p-nitrophenol was determined spectrophotometrically. In the
presence of a glycosyl acceptor, the amount of transglycosylation
product can be calculated from the difference in these two values, i.e. less glucose will be produced than p-nitrophenol. Using methanol as the glycosyl acceptor, the
transglycosylation product would be methyl--D-glucoside.
A control experiment incubating this with myrosinase under the standard
conditions resulted in no release of glucose. This demonstrated that
methyl-
-D-glucoside was not a substrate for myrosinase
and was stable under the reaction conditions. Indeed, the myrosinase
preparation used in these studies has not been found to hydrolyze any
of the alkyl O-glycosides or S-glycosides that have
thus far been tested. (
)This also implies that there are no
contaminating
-glucosidase enzymes, which would cause
complications in the interpretation of our results.
For the
transglycosylation experiments, incubations containing 20 mM PNPG were employed. To test the procedure, a reaction was run in
aqueous solution, and, as expected, equal amounts of D-glucose
and p-nitrophenol were produced. When the experiment was then
repeated in 0.2, 6.25, and 12.5 M methanol, it was observed
that equal amounts of D-glucose and p-nitrophenol
were also obtained, implying that no observable transglycosylation was
taking place. This was in agreement with the qualitative observations
of the synthetic scale experiments but was still a very surprising
result. For most of the -glycosidases, significant amounts of
transglycosylation have been observed under similar
conditions(12, 13) .
A range of other glycosyl acceptors were then investigated, all at 0.2 M concentrations. The amounts of D-glucose and p-nitrophenol produced after a 24-h incubation under standard conditions were determined in the presence of each potential glycosyl acceptor (Table 3). The larger alcohols, ethanol, propan-1-ol, and butan-1-ol, proved to be ineffective in transglycosylation reactions. As the glucosinolates are actually S-glycosides, a thiol acceptor was also tried. However, mercaptoethanol did not appear to trap out any of the glucose. The other part of the glucosinolate structure that probably provides important binding interactions is the sulfate group. This charged moiety is presumably balanced by some positively charged residue at the enzyme active site. It therefore seemed feasible that glycosyl acceptors containing a negatively charged group might have a better chance of binding at the active site and intercepting the glycosyl moiety. Three examples were chosen, which were commercially available, namely, 3-hydroxy-1-propanesulfonate (VIII), glycerol 2-phosphate (IX), and 2-mercaptoethanesulfonate (X) (Fig. 4). When PNPG was incubated with myrosinase in the presence of these compounds at 0.2 M concentration, equal amounts of glucose and p-nitrophenol were produced, again implying that no transglycosylation had taken place. Interestingly, it can also be observed that none of these compounds seems to be a particularly effective inhibitor of myrosinase, as the amounts of D-glucose and p-nitrophenol produced do not seem to be very sensitive to the presence of the glycosyl acceptor. The yields are all approximately 30% after 24 h. In the case of 12.5 M methanol, this drops to 21%, as may be expected from the effect of high methanol concentrations on the enzyme activity. The three charged glycosyl acceptors also have a significant effect, giving 22-25% yields, reflecting some degree of inhibition of the hydrolysis reaction. The detailed inhibitory properties of these compounds are currently under investigation. Preliminary results using sinigrin as the glycosyl donor, rather than PNPG, have indicated that transglycosylation does not take place with this substrate either. Crude measurements of glucose yields on completion of sinigrin hydrolysis have been unaffected by the presence of glycosyl acceptors.
Figure 4: Structures of inhibitors.
The above results
call into question the existence of a long lived glycosyl-enzyme
intermediate that can be trapped by nucleophilic species. In the case
of the -glycosidases, this intermediate has been thought to be
either a stabilized oxocarbonium ion or a covalently bound glycosyl
group, resulting from attack at the anomeric carbon by an active site
nucleophile. In the former case, the oxocarbonium ion structure (XI)
can been employed as a model for the transition state for the reaction.
Enzyme inhibitors have then been designed that mimic this structure.
One simple, readily available example is glucono-
-lactone (XII) (Fig. 4). This molecule possesses a planar sp
hybridized carbon atom at the 1-position of the sugar, which
mimics the planar structure of the transition state at that position.
As a result, glucono-
-lactone has been found to be a very tight
binding competitive inhibitor for
-glucosidase, with a K
of 0.2 ± 0.2
mM(18) . We were interested in examining the
interaction of myrosinase with this inhibitor to see if a similar type
of inhibition was observed.
Thus, the rates of hydrolysis of both
sinigrin and PNPG by myrosinase were measured in the presence of a
range of concentrations of glucono--lactone. The results are shown
in Lineweaver-Burk form in Fig. 5and Fig. 6. These show
that for both substrates, glucono-
-lactone acts as a
noncompetitive inhibitor, that is, the K
is unaffected, while V
decreases with
increasing inhibitor concentration. The variation of V
with inhibitor concentration is not simple, but it can be
estimated that K
is approximately 5
mM for each substrate. Therefore, glucono-
-lactone
appears to bind much less tightly to myrosinase than to
-glucosidase.
Figure 5:
Inhibition of myrosinase-catalyzed
sinigrin hydrolysis by glucono--lactone. Incubations were carried
out under the standard conditions containing increasing concentrations
of glucono-
-lactone:
, 0 mM;
, 1 mM;
, 5 mM;
, 10
mM.
Figure 6:
Inhibition of myrosinase-catalyzed p-nitrophenyl--D-glucoside hydrolysis by
glucono-
-lactone. Incubations were carried out under the standard
conditions containing increasing concentrations of
glucono-
-lactone:
, 0 mM;
, 5 mM;
, 10 mM;
, 20
mM.
The effect of the specific activator, L-ascorbic acid(8) , on the degree of inhibition of
myrosinase by glucono--lactone was examined (Table 4). At a
concentration of 1 mM, L-ascorbic acid exerts its
maximum effect, increasing V
for the hydrolysis
of sinigrin by a factor of 1.5 but leaving K
unchanged. When the measurements are repeated in the
presence of glucono-
-lactone, the degree of activation decreases
as the concentration of the inhibitor increases. Thus, at 1
mM, glucono-
-lactone V
increases
by a factor of 1.4, but at 10 mM, V
actually decreases by a factor of 0.8. This raises the
interesting possibility that the glucono-
-lactone is actually
binding at the activator site rather than at the substrate site, which
would fit with the noncompetitive inhibition that is observed.
Methyl--D-glucoside (XIII) was found to be a simple
competitive inhibitor of myrosinase, albeit with a rather poor K
of 120 mM. The product,
-D-glucose, was also a very poor inhibitor of the enzyme.
At a 1 mM concentration, it gave a 20% decrease in V
, but as the concentration was increased, the
rate also increased, approaching its original value.
The reaction catalyzed by myrosinase superficially resembles
the hydrolysis of glycosides catalyzed by many -glycosidase
enzymes. It is obviously different in two respects. First, the
substrate is a sulfur rather than an oxygen glycoside, and second,
there is a subsequent rearrangement of the aglycone fragment, which may
or may not be catalyzed by the enzyme. The initial results of our study
of the chemical mechanism of myrosinase, reported here, have shown that
the resemblance may indeed be only superficial, as there seem to be
some important mechanistic differences. One interesting feature is that
myrosinase is a very stable enzyme. This probably results from the role
of the glucosinolate/myrosinase system as a defensive mechanism in the
plant. In the cell, myrosinase is normally separated from the
glucosinolates, and they only come together after cell damage, such as
attack by pests or pathogens(4) . Glucosinolate hydrolysis then
releases isothiocyanates, which are noxious to pests and deter them
from further attack. Immediate response is vital, and therefore there
must always be active enzyme present in the cell. As a result,
myrosinase must be stable and long lived. This is in contrast to many
other enzymes, which are synthesized by the cell machinery when they
are required, in response to the current needs of the cell.
Transglycosylation is commonly observed with -glycosidases;
however, we have been unable to obtain evidence for this reaction
taking place with myrosinase. For example, Gopalan and co-workers (19) observed significant transglycosylation using mammalian
cytosolic
-glucosidase in the presence of alcohol acceptors. The
rate of hydrolysis of PNPG was increased 8-fold by the addition of 0.2 Mn-butyl alcohol, and the ratio of n-butyl-
-D-glucoside to D-glucose was
24:1. The proposed reaction pathway for the transglycosylation is given
in Fig. 7. In the case of the
-glucosidase, it was proposed
that for PNPG, k
was rate-limiting. So on addition
of butan-1-ol, which is more nucleophilic than water, an overall
increase in rate was observed as k
> k
.
Figure 7:
Reaction pathway for hydrolysis of
-D-glucosides in the presence of competing glycosyl
acceptors.
However, the rate of p-nitrophenyl--D-glucoside hydrolysis catalyzed
by myrosinase was not increased by the addition of alternative, and
more reactive, glycosyl acceptors, and in fact decreased. Also, there
was no decrease in the amount of D-glucose produced as a
result of transglycosylation. A wide range of potential glycosyl
acceptors were employed, none of which were successful. Even the very
nucleophilic thiols did not trap out the glycosyl moiety. It was
reasoned that the acceptors simply were unable to gain access to the
active site. The natural substrate contains a negatively charged group
in the aglycone, and so acceptors containing similar charged side
chains were employed. Again, these proved to be unsuccessful at
transglycosylation.
The ease with which transglycosylation takes
place with myrosinase is obviously much less than with
-glucosidase and
-galactosidase, despite the apparent
similarities between the reactions that they catalyze. In the case of
the
-glycosidases, reaction is thought to proceed either via an
oxocarbonium ion (XI) or a covalent enzyme-glycosyl intermediate (XIV) (Fig. 8). This is formed following expulsion of the aglycone
from the substrate. A similar pathway is feasible for myrosinase except
that there is then a subsequent rearrangement of the aglycone.
Figure 8:
Proposed chemical mechanisms for
-glucosidases.
Inhibition of myrosinase by glucono--lactone, a tight binding
competitive inhibitor of
-glucosidase that mimics the oxocarbonium
ion intermediate, was examined. However, this compound was not a
competitive inhibitor for myrosinase and instead showed noncompetitive
inhibition with a poor K
. This gives a
second example of the unusual behavior of myrosinase. One possible
reason for the observed noncompetitive inhibition is that the
glucono-
-lactone is not binding at the active site but at some
other site on the enzyme. In the case of myrosinase, there exists a
candidate for this alternative binding site. The enzyme is specifically
activated by ascorbic acid(8) , which apparently binds to the
enzyme causing a change in its conformation. Therefore, it is possible
that the glucono-
-lactone binds at the ascorbic acid site, but in
this case causes a decrease in the rate of reaction. Some preliminary
experiments (Table 4) indicated that the degree of rate
enhancement due to ascorbic acid is reduced in the presence of
glucono-
-lactone, decreasing as the inhibitor concentration
increases. It certainly appears that the glucono-
-lactone is not
acting as a transition state analogue from its mode and degree of
inhibitory activity. However, the simple glucoside,
methyl-
-D-glucoside, was a competitive inhibitor, albeit
with a poor K
.
When discussing the implications of these findings on the chemical mechanism, it must always be remembered that myrosinase could also be involved in catalysis of the rearrangement of the aglycone. The early evidence for this reaction being spontaneous and not involving the enzyme is certainly not conclusive(2) . A particularly interesting observation is that the desulfoglucosinolates are only inhibitors of myrosinase and are not hydrolyzed by myrosinase(17) . This may imply that the driving force for the reaction is actually the rearrangement step. If the enzyme was indeed required to effect this rearrangement, then it is very possible that the aglycone is not the first product released during the catalytic cycle. If the other product, D-glucose, is released first, then the ease of transglycosylation would be expected to be greatly reduced, as there would be little room for the glycosyl acceptor to bind at the active site while the aglycone was still present. Determination of the product debinding order, using product inhibition studies, would allow this possibility to be examined. Unfortunately, D-glucose gave very poor inhibition, which could not be assigned as either competitive or uncompetitive. Work is now underway to prepare the aglycone fragment, and stable analogues thereof, to assess their inhibitory properties and address this possibility in more detail.