J. Biol. Macromol., 3(3) 105-116 (2003)

ArticleUrea-induced change in subsite structure of glucoamylase from Rhizopus neveus:
Evaluated by steady-state kinetics with substrate malto-oligosaccharides
Saori Yasui, Mutsumi Shimada, Katsuo Takahashi and Masatake Ohnishi*

Research Field of Cellular Macromolecules, Science Department of Biological
Functions, Graduate School of Agriculture, The Prefecture University of Kyoto.
(1-5, Nakaragi-cho, Shimogamo, Sakyo-ward, Kyoto City, Kyoto 606-8522, Japan)
Received June 16, 2003, accepted September 19, 2003
Subsite structure, number of subsites and their affinities Ai for glucose residue, of
glucoamylase from Rhizopus niveus was analyzed under the presence of urea bymeans of the steady-state kinetics for the substrate malto-oligosaccharides Gn(degree of polymerization, n = 2~7). Urea was found to effect on (decrease in) the
subsite affinities A2 and A6, suggesting that hydrophobic interactions between
substrate and these subsites (2 and 6) are decreased in the presence of urea. Basedon the experimental results, tryptophan residues are supposed to locate around thetwo subsites, 2 and 6.
Keywords
steady-state kinetics, rate parameters, subsite structure, glucoamylase, urea,

Rhizopus niveus, hydrophobic effect.
Enzyme-catalyzed reactions areamylase and evaluated their subsite

usually carried out in the dilute

structures on the basis of the steady-solution system at the most laborat-state kinetics in the dilute solution

ories, in fact we have studied thesystems [1-9].

steady-state kinetics on the enzyme-

However, in the cytosol of biologicalcatalyzed hydrolysis reaction of several

cells and also in the reaction vessel ofkinds of amylase including gluco-

the manufacture process for making
materials, almost all the reactions*To whom correspondence should be addressed.

cannot proceed in the dilute solution

106

Urea-induced change in subsite structure of R. neveus glucoamylase

system. The enzyme-catalyzed

kinetics was carried out in the prese-

reactions must be investigated in the

nce and absence of urea for evaluation

unusual (not dilute) conditions to get

and characterization of the subsite

information on the structure andstructure of glucoamylase from Rhizo-

reaction, thus we are interesting inpus niveus.

effects of some chemicals for the
enzyme-catalyzed reaction under the

EXPERIMENTAL

unusual conditions. Urea is very

Materials

worthy, because urea has no OH group,

An enzyme preparation glucoam-then urea would not be an acceptor forylase from Rhizopus niveus (1,4-D-

the transfer/condensation reactions, if

glucan glucohydrolase, EC 3.2.1.3.), a

possible, under high concentration inpure (crystalline) grade, was purchased

the reaction mixture. Urea is known

from Toyobo Co., Osaka, and was used

to be one of the reagents for denatur-without further purification.

ation of proteins and inactivation ofConcentration of the enzyme

enzymes. Here, no denaturation andsolution was determined spectrophoto-no change in conformation of gluco-

metrically assuming A1% 1cm at 280 nm

amylase was confirmed to be caused

to be 16.3 and a molecular weight ofby urea under the experimental cond-

58,000. Malto-oligosaccharides Gnitions employed. Thus, urea could be(abbreviation G2-G7 represents a

effect on the enzyme function without

degree of polymerization n=2~7) werechange in conformation. Anothergiven from Nihon Shokuhin Kako Co.,

reagent, popular for protein denatur-

Ltd., Fuji City, and confirmed to be

ant, guanidine HCl has dissociating

pure by an HPLC analysis. An

ionic group (electric charge) in the

enzymatic glucose-determination kit,

molecule, whereas urea does not.

Glucose C-IΙ Test Wako, which is

Urea does not concern an electrostatic

consisted with GOD, was obtainedinteraction with the enzyme, thus urea

from Wako Pure Chemicals Co., Osaka,

is an excellent reagent for investigat-

and used after 1:3 dilution with 0.06 Mtion of effect on the enzyme function.

phosphate buffer pH 7.1. Urea was

For the basic and applied fields, it iscrystallized from water before use.

interesting and important to invest-

Other chemical reagents in guaran-

igate the effect of urea on the glucoam-teed grade were purchased fromylase-catalyzed reaction for substrate

Nakalai Tesque Inc., Kyoto, and usedmalto-oligosaccharids.

without further purification.

In this study, the steady-state

S. Yasui et al.

107

Methods
Steady-state kinetics of the glucoamylase-catalyzed reactions for maltooligosaccharides as substrates

mixed just prior to add the substrate

Five minutes after mixing urea withthe enzyme, where the final concentr-

to initiate the reaction. The reaction
mixture was subjected to measure thedifference UV-absorption spectrum of

enzyme solution (20 mM acetate buffer

ation of the enzyme and urea was

pH 4.5, 5.0ºC), the substrate solution

fixed to be 13.1 µM, and 5.4 (or 2.7) M,

containing urea in 20 mM acetate

respectively. The molar difference

buffer pH 4.5, 5.0ºC was added toabsorption at 290 nm (∆ε290) was

the enzyme-urea solution to initiate

evaluated on the time curve obtained

the enzyme-catalyzed reaction. Afterfrom the reaction between the enzyme

the start of the reaction, at 0, 2, 4, 6,

and urea by using a Shimadzu

and 8 min, an appropriate volume of

UV-1600 spectrophotometer at 5.0ºC in

the reaction mixture was taken out

an 0.02 M acetate buffer, pH 4.5 as

and was added into a 50 mM sodium

the same procedures employedhydroxide solution to stop thepreviously [11].

catalyzed reaction. The amount of

Viscosity in the mixture of the

glucose released in the catalyzed

enzyme and urea was measured using

reaction was measured spectro-

an Ostwald viscometer at 5.0ºC in

photometrically using a glucose assay-

0.02 M acetate buffer, pH 4.5.

kit, Glucose C-ΙΙ Test Wako to observe
the initial velocity.

RESULTS AND DISCUSSION

The steady-state kinetic parameters,
the Michaelis constant Km and thethe basis of those initial velocities

Steady-state kinetics on the
glucoamylase-catalyzed reaction for
malto-oligosaccharides as substrates

obtained from the catalyzed-reaction

Before beginning of the enzyme-

time-curves for malto-oligosaccharide

catalyzed reaction, the urea solution

substrates and evaluated by thewas combined with the enzyme

steady-state kinetic procedures using

solution in a final concentration of

an NEC PC and a Bio-graph software

5.4 M and after 5 min the substrate(Kyoto Soft Co.) [10].

solution in the presence of 5.4 M urea

molar activity k0, were determined on

was added to the enzyme-urea

Examination of urea-induced change
in conformation of glucoamylase
The enzyme and urea solutions were

mixture for start of the enzymecatalyzed reaction at 5.0ºC and pH 4.5.
The product glucose was measured at

108

Urea-induced change in subsite structure of R. neveus glucoamylase

Table 1. Steady-state kinetic parameters for the glucoamylasecatalyzed reaction in the presence and absence of 5.4 M urea.
Substrate
G2
G3
G4
G5
G6
G7

Km (mM)k0 (sec-1)00.93±0.06

0.68±0.03

0.73

5.4

2.70±0.07

0.43±0.03

0.16

00.31±0.11

1.50±0.28

4.8

5.4

0.38±0.01

0.47±0.01

1.2

00.49±0.03

4.01±0.29

8.2

5.4

0.83±0.14

2.30±0.112.8

00.15±0.05

4.20±0.1328

5.4

0.17±0.01

1.80±0.03

110

0.04±0.01

3.40±0.2185

5.4

0.30±0.03

2.10±0.147.0

00.03±0.003

2.90±0.15

975.4

0.27±0.051

2.30±0.20

8.5

Urea (M)k0/Km (mM-1sec-1) ∆ k0/Km
4.6
4.0
2.9
3.5
12
11

Substrate; malto-oligosaccarides Gn, at pH 4.5 and 5.0ºC,± means the standard deviation.
the appropriate time intervals tokinds of the substrate. The rate

observe the reaction time-course. The

parameters, the Michaelis constant Km

initial velocities obtained for theand the molar activity k0, were

substrate malto-oligosaccharides G2 ~evaluated using a linear plot v0/[S]0

G7 are plotted against the initial

versus [S]0 and the least squares

concentration of substrate; somemethod, where v0 and [S]0 are thetypical examples of the results are

initial velocity and the initial

illustrated in Fig. 1, where the solid

concentration of the substrate,lines are obtained theoretically using

respectively. The rate parameters

the rate parameters summarized in

evaluated for the substrates G2 ~ G7

Table 1. It is clearly shown thattogether with their standard

an addition of urea affects on thedeviations are summarized in Table 1,

enzyme-catalyzed reactions for all

where k0/Km was obtained based uponS. Yasui et al.

109

Fig. 1. Typical examples of s-v plot for the glucoamylase- catalyzed
reaction in the presence and absence of 5.4 M urea.
Substrate: G2, G4 and G6 (left hand side from up to down), G3 and G5(right hand side from up to down), Enzyme: for G2; 3.0 µM, for G3; 1.5 µM,
for G4; 1.0 µM, for G5; 0.5 µM and for G6; 0.3 µM, at pH 4.5 and 5.0ºC.
steady-state kinetics, we can reasonthese rate parameters. An addition ofably refer Km to the strength ofurea results in an increase in Km forbinding between substrate andall the substrates, especially a large

enzyme. Thus, change in free energy

increase was observed for G6 and G7,−∆G for the binding is calculated based

where is only a small difference wasupon these Km values according to an

observed on k0. For G6 and G7,∆k0/Km a difference in k0/Km between
in the presence and absence of urea, is
11 ~12 times. However, that is only 3~4 times for G3 ~ G5.
Based on the results obtained by the110equation −∆G = RTlnKm, where R and
T are gas constant and temperature,
respectively (Table 2). For substrates
G3 ~ G5, ∆∆G, a difference in −∆G
between the presence and absence of

110

Urea-induced change in subsite structure of R. neveus glucoamylase

Table 2. Binding free energy of Gn to glucoamylase.
Substrate

Urea (M)

G2

G3

G4

G5

G6

G7

−∆G (kcal/mol)∆∆G (kcal/mol)

0

3.8

0.5

5.4

3.3

0

4.5

5.4

4.3

0

4.2

5.4

3.9

0

4.9

5.4

4.8

0

5.6

5.4

4.5

0

5.7

5.4

4.5

0.2

0.3

0.1

1.1

1.2

Based on the Km values, the binding free energy −∆G was evaluated in
0 M and 5.4 M urea, at pH 4.5 and 5.0ºC. ∆∆G is the difference
between −∆G in 0 M urea and −∆G in 5.4 M urea.
urea, is small and almost identical;

rate constant kint. The rate constant∆∆G is 0.1 ~ 0.3 kcal/mol. However,kint is defined not to be dependent on

for G6 and G7, ∆∆G value is pretty

the size of substrate saccharide

large; 1.1 ~ 1.2 kcal/mol.

(degree of polymerization n) as
described elsewhere [12]. The subsite

Urea-induced change in the subsite
structure of glucoamylase
Based on the subsite theory

parameters, i, Ai, and kint, are called“subsite structure”, as summarized inTable 3. It clearly shows that thedescribed previously [1,10,12,13], thesecond subsite (i =2, thus A2) has the

kinetic parameters, the Michaelis

largest affinity and the other Ai’s areconstant Km and the molar activity k0,

much smaller than A2, nevertheless inallow us to evaluate subsite affinity Ai,

the presence of urea. The subsite

number of subsites, and the intrinsic

structures evaluated in the presence or

S. Yasui et al.

111

Table 3. Subsite structure (number of subsite, affinities of thesesubsite and kint) of glucoamylase.
Subsite (i)

1

2

3

4

5

6

7

Subsite affinity (Ai)

A1

A2

A3

A4

A5

A6

A7

5.0

1.0

0.29

0.68

0.63

-0.01

4.4

1.1

0.44

0.74

-0.20

0.11

Ai (kcal/mol)

kint (sec-1)

0
M
-0.05
Urea
5.4 M
-0.02
Urea

0 M Urea; 12.0, 5.4 M Urea; 2.9
Evaluated in the presence and absence of 5.4 M urea, kint; intrinsic
rate constant.

absence of urea are schematicallyincrease in A1 and a decrease in A2, of

illustrated with histograms for goodwhich findings are brought about bycomparison of those between presence

the presence of urea, have an import-

and absence of urea in Fig. 2.

ant meaning for the enzyme-catalyzed

It is interesting that an addition ofreaction. Based on the kinetic

urea to glucoamylase-catalyzed

parameters k0 and Km for n-mer

reaction decreases A2 and A6, whereas

substrates, a value k0/Km is given by

A1, A3, A4, A5, and A7 increase.

the sum of the affinities Ai of the i-th

Anyway, A2 and A3 are considerablysubsites occupied bycovthe binding of thelarger than A4 ~ A7, then subsites 2

substrate molecule, ∑,iand 3 carry the essential role in thebinding of substrate saccharides.

(k0/Km)nHere the catalytic site must be adequately situated at between subsites 1
and 2, thus malto-oligosaccharides arecov= (0.018) kint∑exp (∑Ai/RT)n,p
p

i

(1)

exclusively bound in the two modes:

where 0.018 arises from theone productive and one nonproductive

contribution of mixing entropy inbinding modes. In the productive

water (= 2.4 kcal/mol) and kint isbinding mode, maltose, for instance, isassumed constant irrespective of n andbound to occupy subsites 1 and 2,

j, which is the total binding mode, thenwhereas its nonproductive mode

j conceives productive p andoccupies subistes 2 and 3. Thus an

nonproductive q; j = p + q. Thus Eq.

Urea-induced change in subsite structure of R. neveus glucoamylase

112

subsite affinity (kcal/mol)

6
5
4
3

0M Urea
5.4M Urea

2
1
0
-1

1

2

3

4

5

6

7

subsite (Ai)Fig.Fig.
2.2.Subsite
glucoamylase,
evaluated
the presence
Subsite structure
structure ofof
glucoamylase,
evaluatedin theinpresence
and andabsenceof 5.4M
on of
theof thosekinetic
steady-statekinetic
absenceof 5.4urea.
M urea,Evaluatedon the basis
thebasis
steady-stateparametersparameters
the histograms,
the catalytic site;
obtained.obtained.
The catalyticRepresented
site; betweenbysubsite
1 and 2.
between subsites 1 and 2.

(1) indicates that only a productive

between the presence and absence ofbinding (p) out of the total (j) isurea (around 4 times) is almost

involved in a term k0/Km, which is

identical with that in k0/Km (3 to 4

proportional to kint as seen in Eq. (1).

times). On the other hand, theA k0/Km is evaluated with the kinetic

difference in k0/Km (= around 10 ~12)parameters Km and k0, which arefor G6 and G7 is obviously much larger

obtained experimentally for thethan that in kint (around 4 times),

substrate malto-oligosaccharides Gn,

suggesting that urea results in muchas summarized in Table 1.

more effect on the productive binding

For malto-oligosaccharides G2~G5,

of G6 and G7. Nozaki and Tanford

a difference in k0/Km between thehave confirmed that the hydrophobicpresence and absence of urea

interaction between tryptophan(k0/Km)0Murea/(k0/Km)5.4Murea = around 3

residue and water is decisively~ 4) should be reflected by kint, as seen

weakened by urea [14]. Moreover,in Table 3, where the difference in kint

using kinetic methods a tryptophan

S. Yasui et al.

113

residue is suggested to locate at

found to be very dependent on reaction

subsite 1 of the enzyme [4-6,10], thus

temperature and reaction time. ∆A290

it is possible that urea affects on

at 5.0ºC was found to be much smaller

weakening the hydrophobic interact-

than that at 45ºC. On the otherion between the substrate saccharides

hand, after an addition of urea, theand the tryptophan residue in the

difference absorption was caused

enzyme subsite.

within five minutes and after that theIn this stage of the investigation, we

difference absorption was almost

have not explained why kint decreases

constant. Thus the difference absorp-

in the presence of urea and cannottion at 290 nm was recorded for about

deny the possibility that urea has

30 min on the spectrophotometer

another effect on the enzyme-

and the reaction curves were extra-

catalyzed reaction including viscosity.

polated to time zero for evaluation of

Anyway, we would like to conclude

the ∆A290 value. Based on the differ-

here that urea brings about a goodence absorption value ∆A290, a molar

result in the glucoamylase-catalyzed

difference absorption at 290 nm,

hydrolysis of malto-oligosaccharides∆ε290 was evaluated to be around 700,

having smaller degree of polymeriz-of which value was produced by 5.4 M

ation than that of G6~G7.

urea at pH 4.5 and 5.0ºC. In case of achange in conformation, an exposure

Effect of urea on the conformation of
glucoamylase
In general, urea is one of the popularof one tryptophan residue from innerpart to surface of a protein moleculerefers to a decrease in 1,500 ~ 2,000 of

denaturation reagent for proteins and∆ε290, which corresponds to the

enzymes, then it is very essential todenaturation blue-shift [15]. Thus,

examine wheter there is or not an∆ε value of 700 corresponds to a half

urea-induced change in conformation(0.5 residue) of tryptophan exposed.

of the enzyme preparation. The

It should be concluded that only aexamination was carried out using theslight change in conformation is

UV-difference absorption spectrophot-

produced by addition of 5.4 M urea atometry in the presence and absence of

5.0ºC, pH 4.5.

urea as can be seen in the previous

The urea-induced change in confor-

paper [11]. A decrease in difference

mation of glucoamylase was also

absorption ∆A at 290 nm (−∆A290),

observed using the fluorescence differ-

which was produced by the urea-

ence-spectrophotometry, of which

induced change in conformation, wasspectra have a peak at 290 nm when114

Urea-induced change in subsite structure of R. neveus glucoamylase

excited at 280 nm as describedgiven so high viscosity, suggesting thatelsewhere [10]. The experimental

urea does not affect so mach on the

results concluded that little change inglucoamylase-catalyzed activity[16].

conformation was produced by an add-

These experimental results indicate

ition of 5.4 M urea at 5.0ºC and pH 4.5.

that urea effects on (decrease in) A2

Thus, the findings are consistent withand A6, suggesting that hydrophobic

the experimental results carried out

interactions between substrate andwith the difference absorption

the Trp residues, which are supposed

spectrophotometry as described above.

to locate around subsites 2 and 6, andsubstrate are decreased by an effect of

Viscosity of the experimental conditions employed for the glucoamylasecatalyzed reaction in the presence orabsence of urea
Viscosity would be one of the envir-

urea molecule.
Nozaki and Tanford pointed out thathydrophobic interaction between Trp
residue and water is weakened byurea [14,16]. Thus, our present

onmental factors, which are highly

findings may support theaffecting to its biological function, in“hydrophobic-driven mechanism” forfact, viscosity in cytosol of biologicalthe productive formation of thecells is generally 3~5 cP (centipoise).

substrate-enzyme complex [17].

In this experiment, urea at theconcentration of 2.7 or 5.4 M was

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