Probing osmotic effects on invertase with l-(-)-sucrose

Article abstract of DOI:10.1039/b810158b

l-(-)-Sucrose was efficiently synthesized using intramolecular aglycon delivery and used to elucidate osmotic effects on the activity of invertase, which catalyzes the hydrolysis of d-(+)-sucrose. The osmotic effect imposed by l-sucrose was responsible fo

Full text of DOI:10.1039/b810158b

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Probing osmotic effects on invertase with L-(−)-sucrose†‡
Seung-kee Seo and Alexander Wei*
Received 16th June 2008, Accepted 16th June 2008
First published as an Advance Article on the web 25th July 2008
DOI: 10.1039/b810158b
L-(−)-Sucrose was efficiently synthesized using intramolecular aglycon delivery and used to elucidate
osmotic effects on the activity of invertase, which catalyzes the hydrolysis of D-(+)-sucrose. The osmotic
effect imposed by L-sucrose was responsible for more than 30% of the activity loss ascribed otherwise to
“substrate inhibition.”
Introduction
The diverse roles of carbohydrates in nature can be divided
into two broad categories: those defined by molecule-specific
recognition such as metabolism and protein-based signaling path-
ways, and those derived from physicochemical properties such as
biomechanical stability and colligative effects. Although the latter
is often thought to be nonspecific, molecular fine structure can
Fig. 1 Models of sucrose-induced inhibition of invertase. (a) Catalytic
have a strong influence on the solution properties of sugars. This
makes it challenging to differentiate recognition processes from
nonspecific effects in the biological functions of carbohydrates.
Here we deconvolute solute effects from substrate recognition
by evaluating the activity of a sugar-processing enzyme in the
presence of a mirror-image carbohydrate. This approach is pred-
icated on the condition that such molecules are not recognized
as substrates, but are otherwise physicochemically identical to the
natural enantiomer. In this study we examine the inhibitory effects
of sucrose on yeast invertase (b-fructofuranosidase; E.C. 3.2.1.26),
and its association with the phenomenon known as substrate
inhibition.1,2 By using L-(−)-sucrose (1) as the dominant solute,
we show that osmotic effects contribute significantly toward the
apparent substrate inhibition of invertase. In addition, we find the
hydrolysis of sucrose by invertase (dark square = active site; dots = H2O).
(b) Substrate inhibition with active site blocked by second molecule S [cf.
eqn (1)]. (c) Lower conformational mobility due to reduction in water
activity, based on the preferential exclusion principle.
(1)
where v, kcat, [E0], and KM are the velocity, forward rate constant,
total enzyme concentration, and Michaelis constant respectively,
and KꢀS is the dissociation constant of the inactivated complex.
However, the substrate inhibition model does not actually fit
well with the kinetics of invertase activity (Fig. 2). The relevance
of substrate inhibition is further challenged by a recent crystal
structure of an invertase–sucrose complex, which suggests a well-
defined binding domain in the active site for the fructofuranoside
ring, but no comparable points of contact for the accompanying
a-glucopyranoside.7 This, coupled with glucose’s poor ability to act
as a competitive inhibitor, indicates that the active site of invertase
does not encourage the occupancy of a second sucrose molecule
by glucopyranose recognition.
k
cat and KM values of invertase to be sensitive to osmolytes such as
L-sucrose, even at relatively low concentrations.
The invertase family has a long and cherished history in
enzymology and biotechnology, having served as the classic model
of Michaelis–Menten kinetics at low substrate concentrations,3
and also as a confectionery agent for increasing sweetness and
forestalling sucrose crystallization by the in situ generation of
fructose.4 The decrease in invertase activity with increasing sucrose
concentration is well known, and has been ascribed to the
simultaneous occupancy of two sucrose molecules in the active
site (Fig. 1b).2,5 This popular model for substrate inhibition can
be expressed as a modified form of the Michaelis–Menten equation
[eqn (1)]:1,6
Department of Chemistry, Purdue University, 560 Oval Drive, West
Lafayette, IN 47907-2084, USA. E-mail: alexwei@purdue.edu; Fax: +1
765 4940239; Tel: +1 765 4945257
† The IUPAC designation of D- and L-sugars is specifically valid for
monosaccharides. However, extension of this designation toward common
disaccharides is both convenient and in widespread practice.
‡ Electronic supplementary information (ESI) available: Experimental
procedures and NMR characterization related to the synthesis ofL-sucrose,
and invertase purification and kinetics. See DOI: 10.1039/b810158b
Fig. 2 Substrate inhibition of yeast invertase by D-(+)-sucrose. Exper-
imental data (•) compared with Michaelis–Menten kinetics (—; KM
=
7.76 mM, Vmax= kcat[E0] = 0.13 lmol min−1) and least-squares fit according
to eqn (1) (—; KꢀS = 436 45 mM).
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Colligative effects, which are often nonlinear with respect to
solute fraction,8,9 may be responsible for the discrepancies in
enzyme kinetics derived from eqn (1). In particular, sucrose
is an effective osmolyte and can reduce water activity to a
greater extent than most neutral solutes.10,11 The role of osmotic
effects in substrate inhibition is likely driven by the preferential
exclusion principle, introduced by Lee and Timasheff (Fig. 1c).12
The preferential exclusion of sucrose from protein surfaces is
thought to provide some protection against thermal denaturation
by increasing the surface tension of the macromolecular cavity and
reinforcing the hydration shell, forcing the protein to adopt a more
compact conformation.13 The same effect may also reduce the
conformational mobility of enzymes, with a subsequent decrease
in kcat. In the case of invertase, experimental verification of an
osmotic effect by sucrose is complicated by its dual role as osmolyte
and substrate. We address this problem by using biochemically
inert L-sucrose as a surrogate osmolyte.
Fig. 3 (a) Osmotic effects on invertase activity, using L-sucrose (•),
D-trehalose (ꢀ), and lactose (ꢁ). Invertase activity as a function of pure
D-sucrose (ؠ) is shown for comparison. Total amount of osmolyte includes
the initial concentration of D-sucrose ([S0] = 60 mM). (b) Lineweaver–Burk
concentrations (10–60 mM). Trehalose concentrations: 0 mM (•D);-s4u0cmroMse
plot illustrating the osmotic effect (using trehalose) at low
(ؠ); 140 mM (ꢁ); 240 mM (ꢁ); 340 mM (ꢂ); 440 mM (♦).
The nonspecific osmotic effect of L-sucrose on invertase was es-
tablished in two ways. First, a parallel study with the disaccharide
D-trehalose, a constitutional isomer of sucrose and a well known
osmolyte, revealed a very similar reduction in the catalytic rate
of invertase, whereas the weak osmolyte D-lactose had nearly no
effect on enzyme activity (Fig. 3a). A Lineweaver–Burk analysis of
invertase activity as a function of trehalose concentration reveals
a modest decrease in KM and Vmax reminiscent of uncompetitive
inhibition (Fig. 3b),6,18 but the large inhibition constant (Ki =
1.6 0.2 M) implies that the enzyme–osmolyte interactions are
nonspecific.
Second, competitive inhibition assays with D-fructose, L-
fructose, and glycerol were run to address the role of chiral
recognition in substrate inhibition. The loss of fructofuranosidase
activity in the presence of D-fructose (Ki = 7 mM) was 47% at
100 mM, whereas inhibition by glycerol and L-fructose at 100 mM
were only 5% and 2% respectively, establishing that competitive
inhibition is chirospecific.18 By comparison, the decelerating effect
of L-sucrose at 100 mM was 17%, indicating that chiral recognition
is not relevant in osmotic pressure effects.
Results and discussion
An expedient synthesis of L-sucrose 1 was developed based on
intramolecular aglycone delivery (IAD),14–16 using the oxidative
coupling of L-glucoside 2b and L-fructofuranosyl donor 3 to
form mixed acetals (Scheme 1). This strategy has been used
in the synthesis of other mirror-image disaccharides such as L-
trehalose,17 as well as the coupling of b-D-fructofuranosides to
p-methoxybenzyl (PMB) ethers.16 However, PMB a-L-glucoside
2a could not be coupled with 3, possibly due to the short lifetime
of the intermediate quinone methide. The coupling efficiency was
improved by replacing 2a with 3,4,5-trimethoxybenzyl L-glucoside
2b, whose lower oxidation potential enabled the stereoselective
formation of L-sucrose derivative 4 in 41% yield, followed by
hydrogenation to afford 1 in sufficient amounts for our studies.18
Evaluation of other crowding agents revealed a general reduc-
tion in invertase activity with increasing solute concentration.19–21
Interestingly, both small achiral polyols (glycerol, mw 92) and
macromolecular solutes such as dextran (40 kd) and polyethylene
glycol (PEG; 5 kd) had similar decelerating effects as the disac-
charide osmolytes, but with variable impact on KM and catalytic
efficiency kcat/KM (Fig. 4).18 Dextran and PEG enhanced the
catalytic efficiency of invertase with increasing solute fraction,
in accord with a recent study on enzyme kinetics under crowded
conditions.20 In contrast, a slight decrease in kcat/KM was observed
for D-trehalose and glycerol. We presume L-sucrose to have the
same effect on catalytic efficiency as the other osmolytes, although
our limited supply precluded a parallel study.
Scheme 1 Synthesis of L-(−)-sucrose 1 using the IAD strategy. DDQ =
2,3-dichloro-5,6-dicyanoquinone; DMTST = dimethyl(methylthio) sulfo-
nium triflate; DTBMP = 2,6-di-tert-butyl-4-methylpyridine.
L-Sucrose was tested as a substrate against purified yeast
invertase, and found to be inert to enzymatic hydrolysis.18 The
osmotic effect of sucrose on invertase was then evaluated by
adding increasing amounts of L-sucrose to solutions of invertase
with 60 mM D-sucrose, the optimal concentration for enzymatic
activity (Fig. 3a). The relative decrease in activity at the highest
D/L-sucrose concentrations used (0.5 M) was more than 1/3 of the
activity loss ascribed otherwise to canonical substrate inhibition.
These experiments indicate that crowding agents decelerate
invertase kinetics by limiting conformational mobility.19 In the case
of small, neutral osmolytes such as sucrose, preferential exclusion
of solute increases the surface tension of the enzyme’s hydration
cavity, raising the activation barrier between conformational
states. In the case of macromolecular solutes, a greater frequency of
repulsive steric interactions drives proteins toward more compact
conformations.22 Experimental evidence for crowding-induced
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Fig. 4 (a) Influence of crowding agents on invertase activity as a function
of solute fraction: trehalose (•); glycerol (ꢃ); 40 kd dextran (ꢀ); 5 kd
PEG (ꢁ). [S0]= 60 mM or 2 wt%; invertase activity as a function of pure
D-sucrose (ؠ) is shown for comparison. (b) Influence of macromolecular
crowding agents versus osmolytes on catalytic efficiency (kcat/KM). Lines
are drawn to guide the eye.
Fig. 6 Substrate inhibition by D-sucrose corrected for osmotic effects (•),
and least-squares fit according to eqn (1) (—) using KM and kcat as functions
of log[S] (cf. Fig. 5). Original data (ؠ) is shown for comparison.
crowding effects on invertase activity by other osmolytes and
macromolecular solutes. The strong osmotic effect on invertase
may have implications on its native functions in plant physiol-
ogy: recent developments suggest possible osmoregulatory roles
associated with root and stem elongation.26–28 It is remarkable
that relatively low levels of osmolytes can cause kcat and KM
values to deviate significantly from those measured under ideal
solution conditions. Such environmental effects should be taken
into account when measuring enzyme kinetics with high solute
concentrations or in physiologically relevant environments.
changes in protein conformation has been reported in both
cases.20,23 Therefore, despite their different mechanisms, both
osmotic pressure and entropic steric repulsion can modulate
enzyme activity by reducing its effective free volume.24
The association and rate constants for invertase are also sensi-
tive to osmotic pressure. Kinetic analyses are typically performed
under the assumption of ideal solution conditions, with constant
K
M and kcat. However, these rate constants cannot be extrapolated
toward studies involving osmolytes. In our case, we find that KM
and kcat values are affected by sugar concentrations as low as
100 mM, well below the levels typically associated with nonlinear
colligative effects. We find that KM and kcat are best described as
log functions with respect to osmolyte concentration (see Fig. 5).18
Experimental
Synthesis of L-sucrose
Substituted a-benzyl L-glucosides 1a–e were prepared from com-
mercial sources of L-glucose or from L-glucal as previously
described.17,29 L-Fructose was prepared in gram quantities from
L-sorbose using a protocol reported by Zhao and Shi,30 and
converted into a-thioethyl derivative 2 by adapting previously
reported methods developed with the natural enantiomer.16
˚
A mixture of DDQ (330 mg, 1.44 mmol) and 4 A molecular
◦
sieves in CH2Cl2 (4 mL) at 0 C was treated with a solution of 2
(400 mg, 0.80 mmol) and 1e (760 mg, 1.12 mmol) in CH2Cl2 (4 mL).
The mixture was warmed to RT and stirred for 6 h, then quenched
with an aqueous solution of 0.7% ascorbic acid, 1.3% citric acid,
and 0.9% NaOH (12 mL). The reaction mixture, which formed
a yellow suspension upon standing, was diluted with EtOAc (50
mL) and filtered through Celite prior to aqueous workup. The
crude acetal was concentrated to a brown oil, dried by azeotropic
distillation with toluene and placed under high vacuum for 1 h,
then dissolved in anhydrous CH2Cl2 (10 mL) and transferred to a
Fig. 5 Semilogarithmic plots of kcat (left) and KM (right) versus total sugar
concentration, up to 0.5 M ([S0] = 10–60 mM). kcat(S) = 4.539–0.590 log[S]
(in L min−1); KM(S) = 9.318–0.913 log[S] (in mM).
The issue of substrate inhibition was revisited after subtracting
the osmotic effects of added L-sucrose (cf. Fig. 3a) from the activity
profile of invertase with D-sucrose. Applying kcat and KM as log[S]
functions (cf. Fig. 5) produced a good least-squares fit of the
corrected data to eqn (1) with a KꢀS of 1.03 0.10 M (Fig. 6).
With osmotic effects taken into account, the substrate inhibition
constant is more than double the earlier value. The revised analysis
reveals that true substrate inhibition has considerably less influence
on invertase activity than previously thought.25
˚
flask containing DTBMP (920 mg, 4.4 mmol) and 4 A molecular
sieves (200 mg). The mixture was stirred for 15 min at RT, treated
with DMTST (88 mg, 4.0 mmol), then stirred for 16 h. The mixture
was diluted with EtOAc (50 mL) and filtered through Celite prior
to aqueous workup and silica gel chromatography (10% EtOAc in
hexanes), which yielded heptabenzyl L-sucrose 3 as a light yellow
oil (520 mg, 41%).
Conclusions
Our study demonstrates that the physicochemical influence of
substrates on enzymes may be distinguished from activities in-
volving enantioselective recognition by introducing mirror-image
molecules as co-solutes. In the case of invertase, the application
of L-sucrose enabled the separation of osmotic effects from true
substrate inhibition, and provided a benchmark for evaluating
Heptabenzyl derivative 3 (600 mg, 0.6 mmol) was dissolved in
MeOH (25 mL) and treated with Pd(OH)2 on carbon (250 mg),
then stirred under an atmosphere of H2 for 24 h. The reaction
mixture was filtered through a pad of prewashed Celite and
concentrated to yield L-sucrose 4 as a waxy solid (200 mg, 98%).
IR (neat) 3312, 1605, 1420, 1054; dH (500 MHz; CD3OD) 5.36
3364 | Org. Biomol. Chem., 2008, 6, 3362–3365
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(d, 1H, J = 4 Hz), 4.07 (d, 1H, J = 9.3 Hz), 4.08–3.99 (m, 1H),
3.80–3.64 (m, 7H), 3.57 (d, 1H, J = 3.3 Hz), 3.41–3.33 (m, 4H);
dC (125 MHz; 10% D2O in CD3OD) 103.84, 92.18, 82.20, 77.62,
74.18, 72.88, 71.69, 69.76, 62.41, 61.99, 60.64; HRMS(ESI): calcd
for C12H22O11Na 365.1060 [M + Na]+, found 365.1062; [a]D20 = −67
(c = 1, H2O).
6 K. J. Laidler and P. S. Bunting, The Chemical Kinetics of Enzyme Action,
Clarendon Press, Oxford, 2nd edn, 1973.
7 J. Ma´trai, W. Lammens, A. Jonckheer, K. L. Roy, A. Rabijns, W. V. d.
Ende and M. D. Maeyer, Proteins: Struct., Funct., Bioinf., 2008, 71,
552.
8 A. P. Minton, J. Biol. Chem., 2001, 276, 10577.
9 M. J. Blandmer, J. B. F. N. Engberts, P. T. Gleesonc and J. C. R. Reis,
Chem. Soc. Rev., 2005, 34, 440.
10 G. Scatchard, W. J. Hamer and S. E. Wood, J. Am. Chem. Soc., 1938,
60, 3061.
Invertase kinetics with L-sucrose and other crowding agents
11 R. H. Stokes and R. A. Robinson, J. Phys. Chem., 1966, 70, 2126.
12 (a) J. C. Lee and S. N. Timasheff, J. Biol. Chem., 1981, 256, 7193;
(b) S. N. Timasheff, Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 9721.
13 B. S. Kendrick, B. S. Chang, T. Arakawa, B. Peterson, T. W. Randolph,
M. C. Manning and J. F. Carpenter, Proc. Natl. Acad. Sci. U. S. A.,
1997, 94, 11917.
14 (a) Y. Ito, H. Ando, H. Wada, T. Kawai, Y. Ohnish and Y. Nakahara,
Tetrahedron, 2001, 57, 4123; (b) A. J. Fairbanks, Synlett, 2003, 6,
1945.
15 (a) M. R. Pratt, C. D. Leigh and C. R. Bertozzi, Org. Lett., 2003,
5, 3185; (b) M. Gelin, V. Ferrie`res, M. Lefeuvre and D. Plusquellec,
Eur. J. Org. Chem., 2003, 1285.
16 (a) C. Krog-Jensen and S. Oscarson, J. Org. Chem., 1998, 63, 1780; (b) S.
Oscarson, in Top. Curr. Chem., ed. H. Driguez and J. Thiem, Springer
Verlag, Berlin, 1997, vol. 186, p. 171.
Activity measurements were performed at optimum substrate
concentrations ([S0] = 60 mM) in the presence of crowding agents,
using a microscale version of the dinitrosalicylate (DNS) assay
for reducing sugars.31 kcat and KM values were obtained using low
substrate concentrations ([S0] = 10–60 mM) at constant solute
fraction, based on the combined wt% of D-sucrose and crowding
agent. In a typical assay, a 40 lL aqueous solution containing
D-sucrose (120 mM) and L-sucrose (0–880 mM) was diluted in a
test tube with 30 lL of 0.05 M sodium acetate (pH 4.7) buffer and
heated to 55 ◦C. 10 lL of a yeast invertase stock solution (0.3 lM)
was added and the reaction mixture was incubated for 10 min at
55 ◦C, then quenched by the addition of DNS reagent (1000 lL)
and heated to near reflux for 5 min. The solution was quickly
cooled to RT and diluted twofold, followed by an absorbance
measurement at 540 nm.
17 S.-K. Seo, M. L. McClintock and A. Wei, ChemBioChem, 2006, 7,
1959.
18 See Supporting Information for more details‡.
19 T. G. A. Lonhienne and D. J. Winzor, Biochemistry, 2001, 40, 9618.
20 M. Jiang and Z. Guo, J. Am. Chem. Soc., 2007, 129, 730.
21 N. Asaad and J. B. F. N. Engberts, J. Am. Chem. Soc., 2003, 125, 6874.
22 D. D. L. Minh, C.-e. Chang, J. Trylska, V. Tozzini and J. A. McCammon,
J. Am. Chem. Soc., 2006, 128, 6006.
Acknowledgements
23 V. A. Parsegian, R. P. Rand and D. C. Rau, in Methods in Enzymology,
ed. M. L. Johnson and G. K. Ackers, Academic Press, San Diego, 1995,
vol. 259, p. 43.
24 We note that osmotic pressure has the opposite effect to hydrostatic
pressure, which increases the activity of invertase and other enzymes.
This effect has been attributed to a negative volume of activation. See:
H. Eyring, F. H. Johnson and R. L. Gensler, J. Phys. Chem., 1946, 50,
453.
We gratefully acknowledge the National Institutes of Health (GM-
06982) for financial support, and Mr Emmanuel Johnson and Prof.
Kavita Shah (Purdue Univ.) for their generous assistance with
enzyme purification.
Notes and references
25 Least-squares analyses based on other substrate inhibition models did
1 (a) J. B. S. Haldane, Enzymes, Longmans, London, 1930, p. 84;
(b) D. R. P. Murray, Biochem. J., 1930, 24, 1890.
2 A. D. McLaren, Enzymologia, 1963, 26, 1.
not improve the significance or quality of fit6.
26 A. Sturm and G.-Q. Tang, Trends Plant Sci., 1999, 4, 401.
27 K. Koch, Curr. Opin. Plant Biol., 2004, 7, 235.
3 L. Michaelis and M. L. Menten, Biochem. Z., 1913, 49, 333.
4 R. Torres, C. Mateo, M. Fuentes, J. Palomo, C. Ortiz, R. Ferna´ndez-
Lafuente, J. Guisan, A. Tam and M. Daminati, Biotechnol. Prog., 2002,
18, 1221.
5 L. Bowski, R. Saini, D. Y. Ryu and W. R. Vieth, Biotechnol. Bioeng.,
1971, 13, 641.
28 L. I. Sergeeva, J. J. B. Keurentjes, L. Bentsink, J. Vonk, L. H. W. van der
Plas, M. Koornneef and D. Vreugdenhil, Proc. Natl. Acad. Sci. U. S. A.,
2006, 103, 2994.
29 F. P. Boulineau and A. Wei, J. Org. Chem., 2004, 69, 3391.
30 M.-X. Zhao and Y. Shi, J. Org. Chem., 2006, 71, 5377.
31 G. L. Miller, Anal. Chem., 1959, 31, 426.
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The Royal Society of Chemistry 2008
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