J. Biol. Macromol. 3(1), 23-29 (2003)

The Rhizopus niveus glucoamylase-catalyzed reaction for
substrate maltose in the absence or presence of acetonitrile
Katsuo Takahashi and Masatake Ohnishi

Research Field of Cellular Macromolecules, Science Department of Biological Function,
Graduate School of Agriculture, The Prefecture University of Kyoto(Shimogamo, Sakyo-ward, Kyoto City, Kyoto 606-8522,Japan)
Received January 14, 2003, Received February 3, 2003

In the presence or absence of acetonitrile CH 3 CN, the molecular mechanism on the

Rhizopus niveus glucoamylse (GA)-catalyzed reaction was studied by the steady-statekinetics for a substrate maltose (G2).

At high concentration of G2, the GA-catalyzed

reaction was observed not to obey the Michaelis kinetics and was reasonably explainedwith a mechanism of the substrate-inhibition involving the ternary complex ESS.
Moreover, CH 3 CN was found to effect on (decrease in) the dissociation constants Ks(for ES) and Ks' (for ESS).

No transglucosylation was confirmed for the G2 reaction.

These findings will support the hydrophobic-driven mechanism proposed in a previous
study.

Key words:

Rhizopus niveus , glucoamylase, substrate-inhibition, hydrophobic-driven mechanism,
acetonitrile,

Glucoamylase(GA) catalyzes the hydrolytic

niveus 1,2). It is concluded that its active

reaction for the non-reducing-end glucosidic

site is constructed with 7 subsites in totalbond of substrate α-glucans including

and the catalytic site is located at between

starch and malto-oligosaccharides. Basedsubsites 1 and 21-12). Based on the subsite

on the steady-state kinetic observations

structure of GA, the ligand binding to thewith a series of linear substrate malto-

enzyme is explained by the two-stepoligosaccharides Gn (degree of polymeriz-mechanism as follows: at the first step,

ation n = 2〜7), the subsite theory has been

which is the fast association process, a

successsfully applied to GA from Rhizopus

ligand is bound to subsite 2, then moves toMechanism of the GA-catalyzed reaction

24

subsite 1 at the successive second step,solvents for precolumn labeling were used ofwhich is slow isomerization process. Thus

the highest grade in the commerciallythe isomerizaiton process refers to theavailable reagents.

productive binding, in which subsite 1 should

Steady-state kinetics on the GA-catalyzed

be occupied by the ligand3-12).

hydrolysis for G2.

On the other hand, enzymes catalyze

The time course of the GA-catalyzed

sometimes an interesting reaction under thereaction for G2 was observed to evaluate thepresence of an organic solvent. It must be

initial velocity v in an 0.025M acetate

an excellent idea to employ a non-alcoholic

buffer at pH 4.5 and 5.0 ℃. The amount ofsolvent CH3CN, which would not be concerneda product glucose released by the catalyzed

to the solvolysis. Because CH3CN has no OH

reaction was measured using a commerciallygroup in the molecule.

available glucose-assay kit, Glucose C-II

In this paper, the steady-state kinetics

Test Wako.

for a substrate maltose G2 in the presence or

High-performance liquid chromatography.

absence of CH3CN was carried out to invest-

The High-performance liquid chromatography

igate the effect of CH3CN on the GA-catalyzed(HPLC) apparatus employed was composed of areaction.

Shimadzu Liquid Chromatograph LC-4A, a
Hitachi 833 data processor, a Jasco UVIDECEXPERIMENTAL

100-Ⅱ UV detection spectrophotometer, and a
Cosmosil Packed Column 5C18-AR-II(4.6-mm I.D.

Materials.

x 150-mm length). Elution was carried out

The enzyme sample GA from Rhizopus niveus

with a mixture of 2.5mM ammonium dihydrogen-(1,4-α-D-glucan glucohydolase, EC 3.2.1.3)

phosphate and 20% acetonitrile at flow rate

was purchased from Toyobo Co., Osaka, and1.0ml/min and column temperature was 40 ℃.

used without further purification. It was

Preparation of the precolumn labeling withconfirmed to be pure using an SDS-PAGE

1-phenyl-3-methylpyrazolone (PMP) was

technique. Concentration of the enzyme wascarried out by the same procedures described

determined spectrophotometrically with theelsewhere13).

absorption unit A1% 280nm of 16.3 cm-1 and the

Examination of the CH3CN-induced change inmolecular weight of 58,00010). G2, CH3CN andconformation of GA.

other chemicals were purchased from Nacalai

For examination of CH3CN-induced change inTesque Inc., Kyoto, which were used without

conformation, UV-difference absorption-

further purification. 1-Phenyl- 3-methyl-

spectra of GA were observed using a Shimadzu

pyrazolone (PMP) was purchased from Kisida

UV-1600 spectrophotometer under the presence

Chemicals Co., and the other reagents andor absence of CH3CN in an 0.025M acetate

Takahashi and Ohnishi

25

0.09

(a)

0.07

60

0.06

50

1/v (1/mM/min)v (mM/min)

0.08

0.05
0.04
0.03
0.02
0.01

40
30
20
10
0

0
0

50

100

0

150

1

[S]o (mM)

Fig.1. The v-[S]o plots of the GA-

CH3CN 20%, closed triangles; CH3CN 40%.
Lines were obtained theoretically using

1/v (1/mM/min)closed circles; CH3CN 0%, closed squares;

60
50
40
30
20
10
0
0

50

100

150

[S]o (mM)

Eqs.(1) and (2) with the values summarizedin Table 1.

3(b)catalyzed reaction for G2[E]o, 3μM, at pH 4.5 and 5℃;

2

1/[S]o (1/mM)

Fig.2. The linear plots of the GA-catalyzed
reaction for G2.
Conditions were the same as describedbuffer, pH 4.5 at 5.0 ℃. Changes in thein Fig.1.

(circles, CH3CN 0% )molar difference absorption of the enzyme at
292nm (Δε292) were evaluated in the absenceor presence of 40% and 20% CH3CN.
At higher concentration of G2, v does not
RESULTS AND DISCUSSION

increase but decreases with [S]0. Thus forthis case, a possible mechanism, substrate

GA-catalyzed reaction for a substrate G2 ininhibition, was suggested as follows;

the presence or absence of CH3CN.
In the presence or absence of CH3CN, theE + S

examples of the results are shown in Fig. 1.,

E + P

(1)

Ks

GA-catalyzed reactions for G2 were observed
and the v- [S]0 plots were obtained as typicalES

ES + S

ESS

(2)

Ks'

of which plots are rather characteristic for

Based on the reaction scheme in Eqs. (1) andthe enzyme-catalyzed reactions.

(2), the rate equation is represented,

26

Mechanism of the GA-catalyzed reactionv = k0[E]0/(1+Ks/[S]0+[S]0/Ks')(3),

where the dissociation constants Ks and Ks'

amean as follows: Ks = [E]0[S]0 /[ES] andb

Ks' = [ES][S]0 /[ESS], respectively. Thus,
the kinetic parameters Ks, Ks' and k0 can beevaluated using a linear equation derived
from Eq. (3)14). At low concentration of G2,
the term [S]0/Ks' can be reasonably neglected
then Ks can be evaluated, whereas at highconcentration of G2, Ks' can be reasonably
estimated. Typical examples of linear plot

Retention time (min)for low G2 and for high G2 in 0% CH3CN are
illustrated in Fig. 2(a) and Fig. 2(b),respectively. Based on these plots, therate parameters were obtained as summarized

Fig.3. HPLC of the reaction mixture[E]o,3μM [S]o,50mM at pH4.5, 20℃ andin Table 1. Theoretical curves shown in Fig.

30min in the presence of CH3CN 20%

1 were obtained by Eq. (3) with the values

Elutant, 20 v/v% CH3CN in 2.5mM ammonium

listed in Table 1, according to the reaction

dihydrogenphosphate

scheme in Eqs. (1) and (2). The experimental

Products, a:G2 b:glucose

points are in almost consistent with thetheoretical curves, nevertheless in theabsence or presence of CH3CN, suggesting that

Effect of CH3CN on the mechanism of GA-

the reaction scheme is compatible with thecatalyzed reactions.
In the presence of CH3CN, the kinetic

experimental results for G2.

parameters, Ks' and k0 for G2 are not so much

Table 1. Kinetic parameters of the
GA-catalyzed reaction for G2 in the

different but almost identical to those in
the absence of CH3CN, whereas Ks is muchdifferent (see Table 1.). Thus, CH3CN ispresence and absence of CH3CN

supposed to effect on the binding of the

CH3CN

Ks

Ks'

k0

(%)

(mM)

(mM)

(sec-1)

0

1.9

270

0.51

20

5.1

310

0.35

40

7.2

590

0.35

substrate, where Ks may refer to thedissociation constant for the Michaelis
complex. However, we do not have evidence on
the Ks and Ks' processes of the substratebinding. Subsite structure of GA has been
analyzed using the steady-state kinetics

Takahashi and Ohnishi

27

with a series of malto-oligosaccharides as

Examination of the CH3CN-induced change insubstrate1-2). Subsites 2 and 3 have binding

conformation of GA.

affinities 4.9 and 1.6 kcal/mol, respec-

UV-difference absorption spectrum of thetively, and other subsites have smaller

enzyme was observed in the presence of CH3CN

affinity than these, thus G2 is mainly boundto examine the CH3CN-induced change into occupy subsites 1 and 2, and to form theconformation. The molar change in dfference

ES complex. According to the subsite theory,

aborption ⊿ε292 was evaluated to be 450 in

subsite affinity can give the Ks value1-12).

the presence of 20% CH3CN. When one

The Ks value (1.9mM) in Table 1 is almost

tryptophan residue is exposed from the insideconsistent with the value calculated with theof a protein molecule to its surface,affinities of subsites 1 and 2. When anotherΔε 292, which is called denaturation-blue

G2 molecule is bound to subsites 3 and 4 andshift, is estimated to about 1,500 4,15-16).

makes to form the ESS complex, the dissoc-

Thus, the difference absorption observed

iation constant Ks' is calculated to morehere by the addition of 20% CH3CN corresponds

than 1000mM, however, the experimental

to 0.3 residue of tryptophan exposed. In the

result was obtained to be 270mM as shown inpresence of 40% CH3CN, 2/3 parts of a

Table 1. It is suggested that another G2tryptophan residue are estimated to bemolecule is bound at different subsites fromexposed, then still not so much change insubsites 3 and 4. In this stage of theconformation can be induced by CH3CN. In theinvestigation, we cannot conclude at whatcase of 8M guanidine hydrochloride at 25 ℃,

site the second substrate is bound to form the

4.0 and more residues of tryptophan were

ESS complex.

exposed to the surface of GA molecule. Thus,

The GA-catalyzed reaction was observed byit can be concluded that little change inthe HPLC technique to confirm the hydrolytic

conformation of GA was produced by thereaction for the substrate G2. As an example

addition of CH3CN under the experimentalof the results is illustrated in Fig.3, aconditions employed.

product by transglucosylation was not foundThe kinetic parameters Ks and Ks' obtained

under the experimental conditions employed.

in the presence of CH3CN are larger than thoseThus, in the presence of CH3CN, the GA-

in its absence, whereas CH3CN little effects

catalyzed reaction for G2 will be explainedon change in the conformation of GA. Thus,by the mechanism “substrate inhibition”

CH3CN can be concluded to affect on the

represented in the reaction scheme shown withbinding process of the complex formation

Eqs.(1) and (2).

between GA and G2. In the previous studies
on the saccharide binding to an enzyme β–glucosidase, we have found that an organic

28

Mechanism of the GA-catalyzed reaction

Table 2. Molar difference absorption of

method. J. Biochem., 76, 1365-1367 (1974)

at 292nm (⊿ε292) in the presence of CH4) M. Ohnishi, H. Kegai and K. Hiromi:

at pH 4.5 and 5℃

Studies on the subsite structure ofamylases.I. Interaction of glucoamylas with

CH3CN(%)

20

40

substrate and analogues studied by⊿ε292(M-1cm-1)

440

1060

difference-spectrophotometry. J. Biochem.,
77, 695-703 (1975)
5) M. Ohnishi, T. Yamashita and K. Hiromi:
Studies on the subsite structure of amylase.
Ⅲ. Inhibition by gluconolactone of thesolvent will weaken the hydrophobichydrolysis of maltodextrin catalyzed byinteraction between a substrate and the

glucoamylase from Rhizopus niveus. J.

enzyme, and have proposed a hypothesis,

Biochem., 79, 1007-1012 (1976)“hydrophobic driven” mechanism14,17).

6) M. Ohnishi, T. Yamashita and K. Hiromi:

In this time, these experimental results

Static and kinetic studies by fluorometry on

with GA will support the hydrophobic-driven

the interaction between gluconolactone andmechanism on the productive substrateglucoamylase from Rh. niveus. J. Biochem.,binding proposed previously.

81, 99-105 (1977)
7) M. Ohnishi and K. Hiromi: Kinetic studies

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29

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Communicated by Ueno Hiroshi