J. Biol. Macromol. 3 (2) 47-55, 2003

ArticleEffect of polyethylene glycol on binding of a transition-state analogue
glucono-1:5-lacton to glucoamylase from Rhizopus niveus *Katsuo TAKAHASHI1,** and Masatake OHNISHI1,***
1
Research Field of Cellular Macromolecules, Science Department of Biological
Functions, Graduate School of Agriculture, The Prefecture University of Kyoto.
(Shimogamo, Sakyo-ward, Kyoto City, Kyoto 606-8522, Japan)
1,**
Shigaken Pharm. Ind. Co. Ltd.
(879 Taki, Koka-cho, Koka-gun, Shiga, 520-3432, Japan)
Received May 1, 2003; Accepted June 16, 2003
In the presence of polyethylene glycol (PEG), binding of a transition-state analogue,
glucono-1:5-lactone (GLN) to glucoamylase from Rhizopus niveus was investigated by means of
the transient-phase kinetics using a stopped-flow spectrophotometer in a 0.025 acetate buffer pH
4.5 at 5.0 °C.

GLN was supposed to be bound by the two-step mechanism, a fast association

process (first step, K-1) followed with a slow isomerization process (second step, k+2 and k-2). A
transient-phase kinetic parameter, K-1 in the presence of high concentration PEG was found to be
identical with that in the absence of PEG, suggesting that PEG does not effect on the association
process of the ligand.

Moreover the parameter of k-2 was increased in the high concentration of

PEG, suggesting that EL complex was formed loosely.

Key words; Rhizopus niveus, glucoamylase, polyethylene glycol, glucono-1:5-lactone,transient-phase kinetics, viscosity.
Glucoamylase (1,4-α-D-glucan glucohydrolase, EC 3.2.1.3, abbreviated to GA)
catalyzes the release of β-D-glucose from thenon-reducing ends of starch, amylose, amylo*

Presented in part at the Annual Meeting of Japan

Society of Glycoscience, Nagano, 2000.
***

To whom correspondence should be addressed.

pectin, and related malto-oligosaccharides [1].
Thus, GA is one of the most important enzymes
for the production of glucose in the fields ofprocessing industries for starch and foodmaterials. Based on the steady-state kinetic
observations with a series ofmalto-oligosaccharides Gn (degree ofpolymerization n=2-7) as substrates, the subsite

48

Effect of PEG on the GLN-GA reaction

theory has been proposed by Hiromi, who has
adequately applied its idea to GA from
Rhizopus niveus and analyzed the subsite
structure in the enzyme active site at the firsttime in the world [2,3].
Binding reactions of the ligands (substrates,
analogues, and inhibitors) to the enzyme active
site are observed to investigate its mechanism
using the kinetic and static methods [4-14].
Especially, the transient-phase kinetics using a
stopped-flow apparatus is inevitable for
evaluation of the elementary process in thebinding reaction [4,7-13]. For study on thebinding mechanism, glucono-1:5-lactone(gluconolactone, GLN) is one of the best
ligands for enzyme mechanism, because GLN
is thought to be an analogue of thetransition-state, which is a decisive process inthe enzyme-catalyzed reaction. Thus thetransient-phase kinetics on the GLN binding is
an interesting and important project of thestudies on the GA-catalyzed reaction.
Enzyme-catalyzed reactions are usually
carried out at the dilute solution, of which
reaction vessel is under not viscousenvironment, at the most laboratories ofindustry and research institutes. However, incytosol of biological cells and in a reaction
vessel of the manufacture process for making
food and industrial materials, almost all thereactions cannot proceed under the diluteenvironment [15]. Thus, it could be very
interesting and important that enzyme-catalyzed
reactions are carried out in the unusual
conditions, for instance, in the presence of highconcentration of reactants and reagents, and areinvestigated about the environmental factor
such as viscosity. Here we were interested inpolyethylene glycol (PEG) for providing of aviscous environment around the
enzyme-catalyzed reaction. Thus, thetransient-phase kinetics on the binding reactionof GLN to GA was tried to analyze its
mechanism in the presence and absence (0〜10%) of PEG using the stopped-flow apparatus.
The steady-state kinetics on the GA-catalyzed
reactions for maltose as a substrate in thepresence and absence (0〜20%) of PEG could
be useful for investigation of its effect on the
kinetic parameters, the Michaelis constant Km
and molar activity k0.
In this paper we aimed to elucidate theeffect of PEG on what process of the ligand
binding to GA using the transient-phasekinetics.

EXPERIMENTAL
Materials
GA form Rhizopus niveus, pure grade, waspurchased from Toyobo Co., Osaka, and usedwithout further purification. Concentration of
the enzyme preparation was determined
spectrophotometrically with an absorption unit
AU1%280nm of 16.3 cm-1 and the molecularweight of 58000 [11,13]. GLN of a product ofNacalai Tesque Inc., Kyoto, was used within 10
min after dissolution to minimize its hydrolysis
into gluconic acid and conversion into gluconic

K. Takahashi and M. Ohnishi

Methods
Stopped-flow fluorescence spectrophotometry
Binding reactions were observed using a
Photal RA-451 stopped-flow apparatus, which
is a gas-pressure driven type with a 150W-D2
lamp, as the same procedures described
elsewhere [7,9,11]. Change in the
fluorescence intensity produced by the
excitation at 280nm was followed through a
Toshiba Kasei Kogyo cut-off filter (UV-31,
50% transmittance at 310 nm) from right angle
to incident beam [7,9,11]. The dead-time ofthis apparatus was determined to be 1.0 msec
under the 5 kg/cm2 nitrogen gas pressure fordriving using the method with ascorbic acid and2,6-dichlorophenol-indophenol [16-18].
Reaction curves were usually accumulated 9times by a Union Giken RA-450 data-averaging
processor to improve the signal to noise ratio.
A fixed concentration (6.1 µM) of
glucoamylase (GA) was used for the binding
reaction of GLN.
Concentration of GLN was examined
finally 0 to 80 mM in an 0.025 M acetate buffer
pH 4.5 at 5.0 ℃.
Steady-state kinetics for a maltose substrate

The GA-catalyzed hydrolysis of maltose asa substrate was observed in a 0.025 M acetate
buffer pH 4.5 at 5.0 ℃ as the same procedures
described elsewhere [14]. Amount of glucose
released from maltose by the hydrolysis
reaction was routinely measured using acommercially available glucose determination
kit, Glucose C-II Test Wako. Based on thereaction time curves and the initial velocity v,
the kinetic parameters, the Michaelis constant
Km and molar activity ko were evaluated using
an NEC PC-9801 personal computer and a
Kyoto Soft Bio-graph software [10].
Measurement of viscosity.
Viscosity of the 0-10% PEG solutions
made with an 0.025 M acetate buffer pH 4.5
was measured at 5 ℃ using a Toki Sangyo Co.

kapp(1/sec)

acid-1:4-lactone. The hydrolysis rate of GLN
in the solution at 10 min was confirmed to beless than 2% of the initial concentration underthe experimental conditions employed.
PEG-6000, maltose, and other chemicals,
guranteed grade products, were purchased fromWakenyaku Inc., Kyoto, and used without
further purification.

49

350
300
250
200
150
100
50
0

2
5
10
20
40
60
80

0

52
99
120
225
280
290
280

2
5
10
20
40
60
80

50

47
80
113
217
250
280
300

100

GLN (mM)

Fig.1. Dependence of the first-order rate
constant k app on the initial concentration of
GLN, evaluated in 0% or 3% PEG-6000
solution.

□ ; 0% PEG, ○ ; 3% PEG,

0.025M acetate buffer, pH 4.5, 5.0 ℃

50

Effect of PEG on the GLN-GA reaction

viscometer model RE100L.
High performance liquid chromatography(HPLC)A Shimadzu liquid chromatograph LC-4A
and a Hitachi 833 data processor were used for
the HPLC analysis with a Nacalai Tesque
Cosmosil Packed Column 5C18-AR-II of 4.6
mm I.D. x 150 mm length. Detection was
performed with a JASCO UVIDEC-100-IV
UV spectrophotometer. A mixture of 2.5 mM
ammonium dihydrogen phosphate and 20%
acetonitrile was used for elution of the sample atflow rate of 1.0 ml/min, 40 ℃. A precolumn
labeling with 1-phenyl-3-methylpyrazolone(PMP) was prepared with the same procedures
as described elsewhere [19].

RESULTS AND DISCUSSION
Transient-phase kinetics on the binding of
gluconolactone
Binding reaction of glucono-1:5-lactone(gluconolactone, GLN) to gluco-amylase (GA)has been observed using a stopped-flow
spectrophotometer, where only one relaxation is
confirmed to obey first-order kinetics [8,11].
Here, using the same procedures, the binding
reaction was observed in the presence of PEG
to investigate its effect on the reaction of GLN
with GA at pH 4.5 and 5.0 ℃. The apparent
first-order rate constant kapp was obtained for thebinding reactions of GLN and dependence ofkapp on the initial concentration of GLN was
analyzed as shown in Fig 1, where theF ig.2 . Tw o - step m ec ha n is m o n b in d in g a lig a nd to t he
sub s ite o f G A .
L; lig a nd . T he w ed ge ind icate s t he ca ta ly t ic s it e. A
tr yp to p ha n res id u e is e xp e cted to lo ca te at s ub s ite 1 .

K. Takahashi and M. Ohnishiconcentration of gluconolactone is represented
with GLN. Such a hyperbolic dependence ofkapp on ligand ([L]o) implies that the reaction
obeys the two-step mechanism, which
comprises of a fast bimolecular association
process followed by a slow unimolecularisomerization process as follows,
k+1

k+2
*
(EL)

E +L

EL

(1)

k-2

k-1

where EL is a tightly bound form of the
GLN-GA complex and isomerized to (EL)*,51

which is a less tightly bound form of the
GLN-GA complex, and k+1, k-1, k+2 and k-2 are
the rate constants of each elementary process asshown in Eq. (1). For this mechanism, tworelaxation times (τ) can be most expected, thenthe reciprocal of slower relaxation time (1/τ) is
given under the condition [L]o>>[E]o by thefollowing equation,
1/τ= kapp = k+2[L]o/(K-1 + [L]o) + k-2 (2),where K-1 is the dissociation constant of (EL)*defined as
K-1 = [E][L]/[(EL)*] = k-1/k+1(3).
The solid and dotted lines in Fig. 1 aretheoretical curves, which were drawn according

Table 1. Effects of PEG on the transient-phase kinetic and static parameters K-1,k+2,k-2,
and Kd for the GA-GLN binding reaction
PEG-6000K−1(ｍＭ)k＋2(sec-1)
k−2(sec-1)
Kｄ（ｍＭ）

0%
20
380
20
1.0

1.5%
25
430
20
1.1

3.0%
19
340
22
1.2

5.0%
21
370
29
1.5

10.0%
22
300
28
1.8

Table 2. Effects of PEG on the steady-state kinetic parameters Km and ko of the
GA-catalyzed hydrolysis for maltose (pH4.5, 5.0℃)

PEG-6000 (%)
0
1.5
3
5
10
20

Km (mM)
0.92
0.90
1.09
1.02
0.81
0.98

ko (min-1)
15
18
19
16
14
11

ko/Km (min-1mM-1)
17
21
19
16
17
11

52

Effect of PEG on the GLN-GA reaction

to Eq. (2) with the values thus obtained,
indicating that these lines are fairly well covered
the experimental results.
In the two-step mechanism, theoverall dissociation constant of the EL
complex Kd is represented as
Kd = [E][L]/([(EL)*]+[EL])= K-1/ [1+(k+2/k-2)](4).
Based on the subsite structure of GA, the mostpossible mechanism of the GLN binding was
illustrated in Fig. 2, where Trp residue(s) could
be located at subsite 1 [5,9,10,13]. That is tosay, GLN transiently attaches to subsite 2 andimmediately relocates to subsite 1 accompany
by change in the fluorescence intensity of the
Trp residue.
The kinetic parameters, K-1, k+2, k-2, and Kd
for the binding reaction were evaluated in thepresence and absence of PEG as the results are
summarized in Table 1. The dissociation
constant of the (EL)* complex, K-1 is almost
identical and is not dependent on PEG in thepresence of PEG, whereas k-2 and Kd areincreased in high concentration of PEG. Thus,
it will be concluded that PEG brings little effecton the first bimolecular association process but
in the high concentration of PEG, EL complex isloosely formed.
Steady state kinetics on the GA-catalyzed
reactions for maltose
Reaction time course of the GA-catalyzedhydrolysis for a substrate maltose was observedin the presence and absence of PEG at pH 4.5
and 5.0 ℃, then the initial velocity v was

obtained on the basis of these reaction
time-curves. Dependency of v onconcentration of the substrate maltose s (the s-vplot) gives the steady-state kinetic parameters,
the Michaelis constant Km and molar activity ko ,by according to the Michaelis-Menten kinetics
and the non-liner least-squares method [11].
The experimental results are summarized inTable 2, indicating that the kinetic parameters in
the presence of PEG (1.5 - 10%) are almost
identical with those in the absence of PEG,
especially the ko/Km value is quite identical.
Based on the subsite theory, ko/Km refers to the
productive binding of substrates [2,14]. Thus,
these kinetic results suggest that PEG (1.5 10%) does not effect on the productive bindingof substrates but PEG (20%) effects on. It isagreement with the result showing in Table 1, of
which k-2 and Kd are increased in highconcentration of PEG.
Viscosity of the polyethylene glycol solutions
In the biological cells, especially in cytosol
viscosity is one of the most important factors,
which affect on the various biological functions,and the degree of viscosity (usually 3〜5 cP in
cytosol) is higher than that in water (1.5 cP at
5.0 ℃) [15]. Viscosity of the PEG solutions
was determined at 5.0 ℃ as the results areillustrated in Fig. 3, clearly indicating that a 5%
PEG solution gives around 8cP under theexperimental conditions employed. In therelation with viscosity, we may conclude thatthe binding mechanism of ligands found in thepresence of PEG (1.5-10%) is almost the same

K. Takahashi and M. Ohnishi

53

with that in cytosol. Viscosity effects not somuch on the association process of the substratebinding under the experimental conditions
emplyed.

HPLC analysis of the GA-catalyzed reaction
Many carbohydrolases catalyzetransglucosylation in addition to the hydrolysis.
Here high concentration of PEG was employed
for the experiments, therefore the GA-catalyzed
reaction has to be confirmed fortransglucosylation.
Because PEG has many OH groups in its
molecule, thus it is very possible that OH
becomes an acceptor instead of water(hydrolysis) for the enzyme-catalyzed reaction.
The HPLC procedures were employed toobserve the existence of transglucosylation
reaction. A typical example of the results is
illustrated in Fig. 4, where a substrate maltose
was examined in the presence of 10% PEG at
pH 4.5 and 5.0 ℃. The substrate maltose and

60
0
1.25
2.5
5
7.5
10
15
20

50

cp

40
30
20
10

2.38
3.59
6.09
8.07
11
13.08
22.8
43.86

0
0

5

10

15

20

PEG-6000(%)

Fig.3. The viscosity of PEG-6000
solutions.

25

a

b

Fig.4. HPLC of the reaction mixture. [E]o;
3 µM, [S]o; 50 mM, pH 4.5, 5.0 ℃ and 60
min in the presence of 10% PEG. Elutant,
20v/v% CH3CN in 2.5 mM ammonium
dihydrogenphosphate,.Flow rate, 1 ml/min.
Saccharides: a; maltose, b; glucose

a product glucose can be clearly seen, however,other products cannot be confirmed in the
HPLC chromatogram, indicating that there is
not a transfer product in the GA-catalyzed
reaction mixture. Thus, it can be recognizedthat no transglucosylation was occurred at the
GA-catalyzed reaction in the presence of PEG.
In this study, PEG was used for a viscous
system of the enzyme-catalyzed reaction and it
was shown that viscosity effected not so muchon the association process of the substratebinding in the presence of PEG 1.5 - 10%, ofwhich viscosity is almost the same with that incytosol. However, in cytosol, many factors
could be concerned to the enzyme-catalyzed
reactions. Thus, It should be very important to54

Effect of PEG on the GLN-GA reaction

investigate the enzyme-catalyzed reactions in
unusual conditions such as high viscosity.
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