parameters involved in binding of porcine pancreatic α-amylase with black bean inhibitor: role of...

Biochimie 70 (1988) 1153-1161 t~ Soci6t6 de Chimie biologique/Elsevier, Paris 1153

Parameters involved in binding of porcine pancreatic a-amylase with black bean inhibitor: role of sulfhydryl groups, chloride, calcium, solvent composition and temperature

J.R. WHITAKER*, F. FINARDI FILHO and F.M. LAJOLO

Departamento de Alimentos e Nutricdo Experimental, Faculdade de Ciencias Farmac~uticas - USP Conj. das Quimicas, Universidade de S(to Paulo, Sdo Paulo, SP, Brazil

(Received 6-11-1987, accepted after revision 18-1-1988)

Summary - - The amylase inhibitor of black (kidney) beans (Phaseolus vulgaris; MW 53 000) forms a 1:1 stoichiometric complex with porcine pancreatic a-amylase (MW 52 000) at pH 5.40. The single sulfhydryl group of the inhibitor and the two sulfhydryl groups of a-amylase are not involved in recognition and binding. Chloride ions, required for activity of a-amylase at both pH 5.40 and 6.90, are important for inhibitor-enzyme binding at pH 6.90 but not at pH 5.40. Calcium-free a-amylase binds with the inhibitor. An increase in the ionic strength of the solvent increases the rate of binding of the inhibitor with a-amylase; a decrease in the dielectric constant decreases the rate of binding; and decreasing the temperature increases the dissociation constant, Kd, of the complex. These data support the hypothesis that hydrophobic interaction is of primary importance in complex formation. The activation energy, Ea, for complex formation was found to be 12.4 kcal/mol at pH 5.40 and 24.2 kcal/mol at pH 6.90. In the presence of the poor substrate, p-nitrophenyl-a-D-maltoside, the Ea for complex formation was 4.1 kcal / mol at pH 6.90.

porcine pancreatic ~-amylase/sulfnydryl groups/black bean inhibitor

Introduction

There are three types of naturally-occurring inhibitors of a-amylase. These are: 1) the carbohydrate inhibitors, typified by acarbose [1]; 2) the polypeptides of various sizes produced by Streptomyces and some other microorganisms [2]; and 3) proteins of some higher plants (see [3] for discussion).

a-Amylase inhibitors from several legumes have been purified and partially characterized. These legumes include some varieties of the common bean (Phaseolus vulgaris), particularly the white kidney bean [4, 5], the red kidney bean [6-9] and the black kidney bean [3, 10-13].

The a-amylase inhibitors of white [4], red [6, 8] and black [12] kidney beans combine with porcine pancreatic a-amylase to form a tight 1:1 complex at both pH 5.5 and 6.9.The a-amylase- red kidney bean inhibitor complex retains about 5% of the original activity on p-nitrophenyl-a-D- maltoside [8]. The carbohydrate groups of the red kidney bean inhibitor do not appear to be involved in complex formation [9], although treatment with periodate inactivated the inhibitor [7]. Treatment of the black bean inhibitor with a-mannosidase removed some mannose and xylose without affecting the ability to bind the a-amylase [13]. Up to 70% of the carbohydrate residues were removed from red kidney

*Author to whom correspondence should be sent at permanent address: Department of Food Science and Technology, 1480 Chemistry Annex, University of California, Davis, CA 95616, U.S.A. This research was performed while J.R.W. was a Fuibright Senior Scholar in Brazil, lf35.

1154 J.R. Whitaker et al.

bean inhibitor enzymatically without affecting its inhibitory activity [9]. The red kidney bean inhibitor appears to combine with t~-amylase outside the active site of the enzyme, as indicated by retention by the complex of -,- 5% of the original activity on small substrates [8] and the ability to bind maltose [7, 8] and starch and Sephadex [6].

Partially or complete inactivation of porcine pancreatic t~-amylase with diethylpyrocarbonate (histidine modification), 5-5'-dithiobis-(2-nitrobenzoic acid) (cysteine modification), N-bromosuccinimide (tryptophan modification) and N- acetylimidazole (tyrosine modification) did not affect its binding to the black bean inhibitor [13]. Photooxidation of t~-amylase with rose bengal, known to modify a number of amino acid side chains, caused loss of enzyme activity and binding to inhibitor [13].

Effect of chemical modification of the red kidney bean inhibitor on its ability to bind with porcine pancreatic t~-amylase was reported by Wil- cox and Whitaker [9]. Oxidation of one tryptophan residue with N-bromosuccinimide caused 50% loss of inhibitory activity. Modification of 3 of the 5 histidine residues with diethylpyrocarbo- hate led to about 50?/0 loss of activity. Periodate treatment caused rapid loss of 2 tyrosine and 1 methionine residues without correlation with loss of inhibitory activity.

Data on the effect of pH and, to a lesser extent, temperature on the rate of binding of porcine pancreatic o~-amylase and inhibitors from white, red and black kidney bean [4, 7, 9, 12] have been reported and a detailed kinetic model for complex formation between porcine .pancreatic t~-amylase and red kidney bean inhibitor has been proposed by Wilcox and Whitaker [8].

Delineation of factors affecting recognition and complex formation between porcine pancreatic t~-amylase and common bean inhibitors is incomplete. In the present paper, data on the effect of temperature, pH, chloride ion and calcium ion concentrations, polarity of solvent and the sulfhydryl groups of the inhibitor and t~-amylase on complex formation are reported for the black bean inhibitor-porcine pancreatic a-amylase system.

Materials and methods

Materials a-Amy:ase inhibitor from black kidney beans (Pha-

seolus vulgaris), variety Rico 23, was prepared as described previously [3, 10]. It was homogeneous by disc gel electrophoresis [14].

Porcine pancreatic a-amylase (2 x crystallized; DFP treated; -,- 900 units per mg of protein), 5,5'- dithiobis-(2-nitrobenzoic acid), N-2-hydroxyethyl- piperazine-N'-2-ethanesulfonic acid (HEPES), p- nitrophenyl-a-D-maltoside and sodium dodecyl sulfate were from Sigma Chemical Co. Soluble potato starch was from Baker. Before use, the starch was reduced with NaBH4 as described by Strumeyer [15]. The 3,5-dinitrosalicylic acid was from Carlo Erba. All other compounds were reagent grade. Gla:~distilled water was used.

Methods The concentration of a-amyl.ase solutions was determined spectrophotometrically based on Alg o = 24.0 at 280 nm [16]. Inhibitor solutions were prepared by weight of lyophilized protein. Molar concentrations were calculated using a molecular weight of 53 000 for black bean inhibitor [3] and 52 000 for porcine pancreatic a-amylase [17].

Before use, the a-amylase was dialyzed against 0.02 M HEPES buffer, pH 6.90, to remove the NaCl and excess CaCl2 from the suspension supplied by Sigma. Fresh dilutions were made from the dialyzed stock solution each day.

The chloride-free enzyme was obtained by exhaustive dialysis against 0.02 M HEPES buffer, pH 6.90, as described by Levitzki and Steer [18] using ethyl- enediaminetetraacetic acid (EDTA)-treated dialysis membrane. The reduced starch substrate used to assay the chloride-free enzyme was also exhaustively dialyzed against the same buffer. The glass-distilled water had a chloride ion concentration < 1 x 10-5 M.

,"nl~'..--,.,a~,,lutt~_u,:. ~ o~-,tnlylase was prepared by incubat- ing exhaustively dialyzed a-amylase in which the chloride ion had been removed (above), at 76.2 /.~g/ml, with 10 mM EDTA at pH 5.40 (0.03 M acetate buffer) and 25.4oC for 13 min (total volume 1.5 ml), followed by successive dialyses at 0oC against 50 ml of 10 mM EDTA in 0.03 M acetate buffer, pH 5.40, for 20 min, and 3 times for 20 min each against 50 ml of the buffer only. The dialysis membrane was boiled in EDTA before use and polypropylene test tubes and beakers were washed with EDTA and then with glass-distilled water. The a-amylase was com- pletely inactive on exhaustively dialyzed starch (no CI- or Ca2÷), at least 90% active when 40 mM NaCI was added and 100% active when 1 mM calcium acetate plus 40 mM was added.

a-Amylase activity was determined in most experiments by the method of Bernfeld [19], using 1.0 ml of 1% reduced starch in 0.02 M phosphate (or HEPES) buffer, pH 6.90, containing 20 mM NaCI and 0.1 mM CaCI2. After equilibration at 37oC, 0.1 ml of a-amylase was added and the reaction incubated for exactly 5.0 min. The reaction was stopped by the addition of 2.0 ml of dinitrosalicylic acid reagent. The solution was heated for exactly 5.0 min in a boil-

Binding of black bean inhibitor with a-amylase 1155

ing water bath, cooled, diluted by adding 5.0 ml of H20 and read at 540 nm against a blank prepared in the same way, without added a-amylase.

Inhibitor activity was determined by a rate assay method. Inhibitor and a-amylase were mixed and incubated under the desired conditions. Aliquots (0.10 ml usually) were removed at intervals into 1.0 ml of starch solution (above) and the remainiag amylase activity determined as described in the previous paragraph. The rate constant for a-amylase activity loss was calculated for each reaction.

t~-Amylase activity on p-nitrophenyl-a-o-maltoside and the rate of binding of inhibitor to the enzyme at pH 6.90 were also determined according to the continuous kinetic assay method of Wilcox and Whitaker [8] in a thermostated Perkin-Elmer spec- trophotometer at 403 nm. E~ for p-nitrophenol at pH 6.90 was 9000 M -! • cm -] [8].

The sulfhydryl groups of a-amylase, of inhibitor and of the enzyme-inhibitor complex were determined using Ellman's method [20].

Results and Discussion

Purity of a-amylase and inhibitor based on stoichiometry of complex formation

Stoichiometry of complex formation between a- amylase and black bean inhibitor at pH 5.40 and 37.0°C was 0.96 ([/] / [E]) (data not shown). The enzyme concentration was held constant at 0.107/~M, while the inhibitor concentration was varied from 0.0117 to 0.287/.tM. The instability of a-amylase at p[4 ~ 4n and ~tTor -;,as . . . . . . . . .

by using rate assays and extrapolating to zero time. Therefore, the stoichiometry ofbinding of inhibitor with a-amylase is 1:1. Tanizaki and Lajolo [12] reported a value of 0.91 at pH 5.5 and 37oC for the black bean inhibitor and porcine pancreatic a-amylase, determined by single point assays, following incubation for 17 h.

Role of sulfhydryl groups of a-amylase and inhibitor in complex formation

Black bean inhibitor has one 1 / 2 cystine/mol as determined by amino acid analysis [3]. Por- cine pancreatic a-amylase has 2 cysteine residues/mol as determined by titration with 5,5'- dithiobis-(2-nitrobenzoic acid) (DTNB) [18]. Since enzymatic activity is lost slowly when a- amylase and inhibitor are incubated together (10-20 min for complete reaction at 37oC and 0.107/,~M each of enzyme and inhibitor), it is important to rule out disulfide bond formation in the process.

Titration of inhibitor (12.6/zM) at room temperature (16oC) with DTNB at pH 8.0 by the Ell- roan method gave no change in absorbance at 412 nm. Addition of sodium dodecvl sulfate (SDS) to 1% also gave no change in absorbance. Incubation of inhibitor (14.5 ttM) in 6.15 M gua- nidine containing 15 mM EDTA at pH 8.0 and 55°C for 30 min did not give any titratable sulfhydryl groups. Incubation of inhibitor (14.5/zM) in 1.15% SDS containing 1 mM EDTA at pH 8.9 and 55oC for 20 min, followed by titration with DTNB, gave 0.34 mol of - S H groups/mol of inhibitor. Inhibitor (189/zM) incubated for 2 h at 37°C with 10 mM dithiothreitol in 0.1 M phosphate buffer at pH 7.0 containing 10 mM EDTA, and passed through a Sephadex G - 2 5 column to remove the dithiothreitol, gave no titratable sulfhydryl groups until SDS was added to 0.2%. Then 0.91 mol of - S H groups / mol of inhibitor were titrated. These results confirm the amino acid data in showing that the black bean inhibitor contains one mol of cysteine/mol of inhibitor. The requirement for dithiothreitol could indicate that the - S H group is present in part as an - S - S - R as a result of previous reaction with a non-protein R - S H group as for streptococcal protease [21]. The 0.2% SDS appears to be required to denature the inhibitor and render all the masked - S H groups accessible to DTNB.

Titration of the sulfhydryl groups of a-amylase with DTNB is shown in Fig. 1. At 25.5°C ~.,a pH ~ n DITN, B a-a "":"- Ifhy , , , not react w,, , any su - i i . i . 1 l I . J l II..]l • l l J ,

dryl groups (data not shown). When 0.15 M EDTA was added to the reaction at pH 8.0 and 20oC, the sulfhydryl groups were titrated slowly to a maximum of 1.88 mol of - S H groups/mol of enzyme in about 2 h (1.40 - S H groups/mol enzyme after 40 min, Fig. 1). Prior removal of the Ca 2÷ permitted rapid titration of the - S H groups at pH 7.0, to a maximum of 1.58 mol / mol of enzyme. These results are in agree- ment with those of Levitzki and Steer [18]. The - S H groups were not titrated in the complex of Ca2+-free a-amylase-inhibitor at pH 7.00 (Fig. 1), indicating that complex formation with the inhibitor either sterically excludes DTNB from the - S H groups or, due to a conformational change upon complex formation, as proposed for the red kidney bean inhibitor-porcine pancreatic complex [7, 8], the - S H groups are no longer accessible to DTNB. The - S H groups cannot be titrated in free a-amylase, without removal of Ca 2+ (see above).

The Hg2+-derivative of a-amylase was prepar-


,,, 1.6 >- N z IJJ

._I 0

co P_.

0

-J 0.8 >- rr CI >- "l- la_ _J

oo 0 .4 'I,Ll - J 0

I I I I i I I I

A.A.,," . , ..~ . . . . -A . . . . b

' 7 o , / !

¢ 3 ; o , / ! i ,j/o

0 I0 2 0 3 0 4 0

#

MINUTES

Fig. 1. Rates of reaction of the sulfhydryl groups of porcine pancreatic a-amylase with 5,5'-dithiobis-(2-nitrobenzoic acid). The continuous spectrophotometric titration procedure of Ellman [20] was used at 412 rim. A: Ca2+-free a-amylase, at 5.48/~M in 0.15 M phosphate buffer, pH 7.00, and 25.5oC. e: Complex of Ca2+-free a-amylase and black bean inhibitor, at 5.48/zM in 0.15 M phosphate buffer, pH 7.00, and 25.5oC. o: CaE+-containing a-amylase, at 5.27 gM in 0.2 M phosphate buffer containing 0.15 M EDTA, pH 8.00, and 213ol.5.

ed by the method of Steer et al. [22]. The derivative was fully active on a starch substrate (as reported by Steer et al. [22]) and formed a complex with the black bean inhibitor at the same rate at pH 5.40 and 37oC as the unmodified a- amylase. Tanizaki et al. [13] reported that the disulfide derivative of a-amylase formed by reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) was fully active as an enzyme (as also reported by Steer et al. [22]) and that the 2-nitro-5-thio- benzoate derivative of a-amylase formed a complex with black bean inhibitor. Payan et al. [23] have shown by X-ray crystallography that the two - S H groups of porcine pancreatic a-amylase are close together in a sort of tunnel some distance from the active site.

Therefore, on the basis of the data presented, we conclude that the sulfhydryl groups of the inhibitor and of the a-amylase are not involved

in the complex formation.

Effect o f [Cl - ] on complex format ion

Chloride ion is a requirement for activity of porcine pancreatic a-amylase at pH 6.9 [18]. (Bro- mide, iodide, fluoride and nitrate ions can replace chloride, but give lower maximum activity.) Therefore, we have determined the effect of [Cl-] on the activity of a-amylase and on its rate of binding with the inhibitor at pH 5.40 and 6.90 (Fig. 2). Chloride ion appears to be required for a-amylase activity on starch at both pH 5.40 and 6.90. At pH 5.40 and 37.0oC, the dissociation constant, Kd, for chloride ion binding was determined to be 4.74 - 1.34 x 10-5 M. It was impossible to remove sufficient chloride ion to obtain an activity below 55% of the maximum activity because of the low Kd. At pH 6.90 and 37.0°C, the Kd was determined to be 1.66 +- 0.21 x 10 -4 M. This compares favorably with a Kd of 2.9 x 10 -4 M at pH 6.9 and 25oC determined by Levitzki and Steer [18]. Since the Kd was 3.5 times larger at pH 6.90 than at 5.40, sufficient chloride was removed from the system to achieve 80% loss in the maximum activity. Levitzki and Steer [18] reduced the activity to about 4% of the maximum activity.

As determined by the rate of binding of a- amylase with the inhibitor, chloride ion is required for complex formation at pH 6.90, but it is not required at pH 5.40 (Fig. 2). The Kd for chloride inn rlotormln~rl ~ . m ~,o ~ o , . . rate "-~ ..,,m_ l ~ J l l , l l . i l l l .# l i i i i JLL~ I~JLL I~ ,~L ll.,#ill li.#,L k l t . # l l i -

plex formation, was 2.75 _+ 0.55 x 10 -4 M at pH 6.90 and 37.0oC, comparable to the Kd determined from effect of [CI-] on a-amylase activity of 1.66 _ 0.21 x 10 -4 M. It appears that chloride ions may be required at pH 6.90 to bring the a- amylase into correct conformation for binding to the inhibitor, whereas this correct conformation may exist already at pH 5.40. Levitzki and Steer [18] found that CI-- caused a 240 times tighter binding (Keq = 1.2 x 10 -9 M and 5 x 10 -12 M in the absence and presence of Cl- , respectively) of Ca 2+ tO porcine pancreatic a-amylase. They pos- tulated that the Cl- ions, upon binding, caused a conformational change thereby increasing the binding constant for Ca 2+.

In preliminary studies based on one point assays, Lajolo and Finardi Filho [3] reported that NO$, Cl- , Br- , I- and SCN-, all known to activate salivary a-amylase, also affected the binding of inhibitor to the enzyme at pH 6.9 and 37°C. Lajolo and Finardi Filho [3] ascribed the effect of these anions to be on the inhibitor


I 0 0

~. 8O ,4

o >- I-- >

6 0 0

X

'~ 4 0 I..- Z W 0

I.U

2O

I i ~ i I i l I i u I / , ' /

.,.., .°'

i A o A o -

...~l. ....... ~ .~ . ................................... /

l I • • 0

/ / /

I I I I -I I n

- 5 - 4 - 5 - 2 LOG [a]

2.0

1.6

1 . 2 A

I i - ° ~

E v

0 . 8 ~

0 . 4

1.2

n.o

tO I 0

0.8 x

' v ' -

I r "

o ~

E 0 .6 T

v

o - 0 . 4 ""

0 .2

Fig. 2. Effect of chloride ion concentration on the activity of porcine pancreatic a-amylase and its rate of binding to black bean inhibitor. Chloride ions were removed from the enzyme and starch by exhaustive dialysis against 0.02 M HEPES buffer, pH 6.90, prepared with special glass-distilled water. Effect of [Cl-] on a-amylase activity was determined at 37.0oC and pH 5.40 (&) and 6.90 (e) by the method of Bernfeld [19] as described under Materials and Methods. The effect of [Cl-] on the rate of binding of a-amylase with inhibitor at 37oC and pH 5.40 (A) and 6.90 (o) was determined as described in the text. At pH 5.40, 0.03 M acetate buffer was used; at pH 6.90, 0.02 M HEPES buffer was used. The a-amylase concentration was 0.117/zM and the inhibitor 0.314/zM.

rather than on the a-amylase, since appropriate controls on the effect of these anions on a-amylase were not done. The data presented above (Fig. 2) indicate that the effect is on a-amylase.

Effect o f Ca 2+ on complex formation

Porcine pancreatic a-amylase contains 1 mol of Ca 2+ / m o l [24]. The best evidence indicates that Ca 2+ is important in maintaining the tertiary structure of the enzyme and is not part of the active site [25-27].

We removed the Ca 2+ from porcine pancreatic a-amylase as described under Materials and Methods. Evidence that Ca 2+ had been removed included: 1)a t pH 5.40, the enzyme had 2.0% of its original activity on dialyzed starch containing 10 mM E D T A , 11.0% on dialyzed starch, 36.2%

on dialyzed starch containing 10 mM E D T A and 40 mM NaCI, 91.2% on dialyzed starch containing 40 mM NaCl and its full original activity on starch containing 40 mM NaCl and 1 mM calcium acetate. At pH 6.90, essentially full activity was restored by adding 40 mM NaCl to dialyzed starch; 2) the CaE+-free enzyme was much less stable at pH 6.90 and 5.40 and 25.4°C than was the Ca2+-containing enzyme (Fig. 3). The Ca 2+- free a-amylase was somewhat more stable at pH 6.90 than at pH 5.40. A stock solution containing 1.47/zM Ca2+-free a-amylase in 0.03 M acetate buffer, pH 5.40, stored at 4oC retained 79.3%, 53.4% and 21.7% of the original activity after 21.5, 54.5 and 96 h, respectively, while Ca2+-a - amylase was stable under these conditions.

The Ca2+-free a-amylase bound the inhibitor at both pH 5.40 and 6.90 at 25.4°C (Fig. 3). The


I - LL UJ --I

>- I - ra >__ I - 0 < ._1 < Z

t r 0 P- Z LU 0 r r W 11-

I001~. ~r ~ ~ ~ ~ ]I -o v ~ ~ , c , ~ I A "

" . . . . -t I Q5 ~ T::J. ~. | , [ j

,o_ . \

5 0 - \ " ' " . . A . - "~... - e O " o • X%

-- "% el'I• X -

- "~... ~ -

.o...

I I I l I 1 1 1 1

2 4 6 8 I0 20 40 I00 120

20

I0

0

M I N U T E S

I r ] . . . . .

60 80

M I N U T E S

Fig. 3. Effect of calcium ions on the rate of binding of porcine pancreatic a-amylase with black bean inhibitor. The Ca 2+ was removed from a-amylase as described in the text. The a-amylase concentration was 0.117 taM; the inhibitor concentration was 0.314 taM except as noted in B. A. Performed in 0.03 M acetate buffer, pH 5.40, and 25.4oC. e: stability of Ca2+-a-amylase; a,: stability of Ca2+-free a-amylase; A: rate of binding of inhibitor with Ca2+-free a-amylase; o: rate of binding of inhibitor with Ca2+-a-amylase. B. Performed in 0.03 M phosphate buffer, pH 6.90, and 25.4oC. e: stability of Ca~+-a-amylase; A. stability of CaZ+-free a-amy!as•;., n. . . . . . . . . . . rata nf c.nrnhln~tlc~n...v...,...,...., . , , '-'f .,,.u,,,.,,~'-h:r': . . . . . . ,,,,,,,:"- Ca2+-free a-amylase; []: rate of combination of 0.162 ~M inhibitor with 0.107 taM CaZ+-a-amylase; A, x: rate of combination of 3.14/~M inhit, itor with 0.115 #M CaZ+-free a-amylase. The rate of binding of inhibitor with a-amylase was determined as described under Materials and methods.

initial rates of binding were similar for the Ca 2+- free a-amylase and the Ca2+-containing a-amylase. The longer the incubation, the more diver- gent were the rates of binding of inhibitor with the CaZ+-free a-amylase compared to the Ca 2÷- containing a-amylase. A large part, if not all, of this difference can be explained by the instability of the Ca2+-free enzyme. (Denatured enzyme also did not bind to red kidney bean inhibitor [7].) Because of the length of incubation required for binding at pH 6.90, resulting in a major loss of enzyme activity, the experiment was repeated with a 10 × higher concentration of inhibitor so that the rate of binding would be increased and the contribution of the instability of the enzyme less of a problem in interpreting the data (Fig. 3).

From these data, Ca2+-free a-amylase was found to have at least 91% of the activity of Ca2+-containing a-amylase. Native Ca2+-free a- amylase formed a complex with the inhibitor at essentially the same rate as the Ca2+-containing enzyme.

Effect of ionic strength, dielectric constant and temperature on complex formation

Sodium sulfate was chosen to increase the ionic strength of the solution, since the sulfate ion has no effect on a-amylase activity, in contrast to the chloride inn for example. The rate of binding of a-amylase with inhibitor increased as the ionic strength increased at both pH 6.90 and 5.40 (Table I). At 1.55 ionic strength, the relative


Table I. Effect of sodium sulfate concentration on the rate of binding of porcine pancreatic a-amylase with black bean inhibitor.

[Na2So4] pH 6.90 a pH 5.40 b

(M) ionic relative ionic relative strength c rate of strength c rate of

binding binding

0 0.052 1.9 0.030 1.0 0.15 0.50 2.1 0.48 1.9 0.30 0.95 2.5 0.93 2.8 0.50 1.55 3.2 1.53 3.7

aReaction at 37.0oC contained 1.07 x 10 -7 M a-amylase and 1.61 × 10 -7 M inhibitor in 0.03 M phosphate buffer containing 40 mM NaCI plus indicated [Na2SO4], pH 6.90. Remaining a-amylase activity was determined as described in the text. has in a except performed in 0.03 M acetate buffer, pH 5.40. clncludes ionic strength of the buffer.

rates, in comparison to the rate in buffer alone, were 3.2 at pH 6.90 and 3.7 at pH 5.40. Increase in the rate of binding of a-amylase with inhibitor with an increase in ionic strength of the solution is indicative of hydrophobic bonding being of primary importance in complex formation. If ionic interaction (electrostatic bonding) were of primary importance, an increase in ionic strength would decrease the rate of complex formation.

Effect of the isopropanol concentration on the rate of binding of a-amylase with inhibitor at pH 5.40 and 25.4°C is shown in Fig. 4. a-Amylase and inhibitor separately were shown to be stable under the conditions used. The rate of complex formation decreased as the isopropanol concentration increased. At 1.95 M isopropanol (dielectric constant D = 69.5) the rate of complex formation was 0.224 compared to 1.00 in acetate buffer alone (D = 78.2). The observed effect of the organic solvent on the rate of complex formation, in the absence of stability effects, is indi-

Fig. 4. Effect of isopropanol concentration on the rate of binding of porcine pancreatic a-amylase with black bean inhibitor. Experiments were performed in 0.03 M acetate buffer containing 0.04 M NaCI, pH 5.40, and 25.4°C with 0.165 /zM inhibitor and 0.107 /zM a-amylase in various concentrations of isopropanoi. The effect of isopropanol on pH of buffer was corrected by preparing the buffer with isopropanol and adjusting the pH to 5.40. Separate experiments showed that inhibitor and a-amylase alone were stable under these conditions. Dielectric constants (D) used were: buffer, 78.5; isopropanol, 18.3. The rate of binding of inhibitor with a-amylase was determined as described in the text.

cative of hydrophobic bonding being of primary importance in formation of the complex between a-amylase and inhibitor.

1 0 0 / D

0 0

x 60 0

o Z

n

~40 o _m

1.274 1.559

Ok.l -

2° f

1.445 I . . . . i

0 i I I i

0 0.5 1.0 1.5 2.0

I S O P R O P A N O L C O N C E N T R A T I O N (M)


Additional evidence for the primary importance of hydrophobic bonding in complex formation between a-amylase and inhibitor is given by the effect of temperature on the equilibrium concentration of free a-amylase present in a reaction containing an initial molar ratio of ([I]/[E]) of 2.00. At 30 .1-40 .2°C the enzyme activity on p-nitrophenyl-a-D-maltoside on completion of binding was constant at 4.31 --- 0.61% of the original activity (Fig. 5). As shown in these studies, addition of more inhibitor to give ( [ ,q / [E] ) of 4.0 did not further decrease the level of activity. Therefore, we conclude that this level of activity is due to activity of the complex. At temperatures lower than 30.1oC, the enzyme activity at equilibrium increased. It was 7.23, 10.5 and 21.2% of the original activity at 26.5, 22.5 and 18.0°C, respectively. When a new aliquot of inhibitor equivalent to the original was added at the end of the reaction at 18oC, the activity was reduced from 21.2 to 5%, clearly showing that 16% of the activity was due to free a-amylase. These results, indicating that the Kd of the a -amylase - inh ib i to r complex increased

TEMPERATURE (°(3)

r Ia_ 1.1.1 _1 >. 1.3 I -- _> !-- 0 < I--

z 0.9 I,kl 0 r r ILl I1,.

(..9 o " 0.5

39.3 29.8 I I I I

o ~ u - - 0 1 I

32

s O S

S #

o'- p

I

3 3 , ,T,,.,. 0 3 i , , I ~ r ~ l ^ I

2 0 . 9 I I

P o" J

J J

,0 s

I I

3 4

Fig. 5. Effect of temperature on di.ssociation of the porcine pancreatic (~-amvlasc-black bean inhibitor complex. The reactions, in I).l),~ M phosphatc buffer plus ().(14 M NaCI, pH 6.9(I, contained 11.488 gM o~-amylase, (I.974/xM inhibitor and 2.(1 mM p-nitrophcnyl-a-u-maltoside. Rate of binding wa.s followed to completion at 412 nm in a thcrmostatcd Pcrkin-Elmcr recording spectrophotometcr. The amount of activity at the end was determined from the constant rate of hydrolysis of the substrate, p-nitrophcnyl-a-D-maltoside.

as temperature decreased, are expected when hydrophobic bonding is of pr imary importance in complex formation. A rough estimate of the A Hdiss of the complex is 21.2 k c a l / m o l over the temperature range 26 .5 -18°C (from Fig. 5).

39 .3 I

TEMPERATURE (°C)

29 .8 I I

2 0 . 9 I

/ - 1 . 4 - -4

/ -I. 8 ~~'~'ll~re"'~ ° .-1

1 I O A " . , . , . . . • ' ~ I

-2.6 ', o F'°-'°--" "2" - - " ° - - - - " " ... 0

.° - 3 ° F k ' " a - - . . n o _

- 3 . 4

- 3 8

-4.2 -4.6

I z~:~.......~ l l °o j , oa ~

° e . o t

t "'.A • I

I I I I t

I

3 2 3 3 3 4 I IT(K) x 103

Fig. 6. Effect of temperature on a-amylase activity and on the rate of binding of porcine pancreatic a-amylase with black bean inhibitor. The reactions were performed in 0.03 M acetate buffer containing 0.04 M NaCI, pH 5.40, or in 0.03 M phosphate buffer containing 0.04 M NaCI, pH 6.90. o: pH 5.40, with 0.107/~M a-amylase and 0.162/xM inhibitor. Aliquots were removed from the reaction periodically and the remaining a-amylase activity determined on starch as described in the text. A: pH 6.90, conditions and procedure as described for pH 5.40. A: effect of temperature on a-amylase activity as measured with p-nitrophenyl-a-o-maltoside by the continuous kinetic assay method. The reaction conditions at pH 6.90, were: 0.488 O, dVl a-amylase and 2.0 mM p-nitrophenyl-a-D-maltoside, o: rate of binding of 0.488/zM a-amylase and 0.974 o~M inhibitor in the presence of 2.0 mM p-nitrophenyl-a-D-maltoside at pH 6.90, as followed by the continuous kinetic assay method. See legend to Fig. 5 for other details.

Binding o f black bean inhibitor with a-amylase 1161

Effect o f temperature on the rate o f complex f o r m a t i o n

References

The rate of complex formation between porcine pancreatic a-amylase and black bean inhibitor mcreases by about 20 times at pH 5.4 as compared to pH 6.9 ([12]; confirmed in present study). The order of the reaction also changes ~rom second order at pH 6.9 to first order at pH 5.4 ([12]; confirmed in this study). In searching for an answer to this latter observation, we examin- ed the effect of temperature on the rate of complex formation at pH 5.40 and 6.90. The continuous kinetic assay method of Wilcox and Whita- ker [8] using p-nitrophenyl-a-t)-maltoside was also used at pH 6.90. It could not be used at pH 5.40 because the background absorbance of the substrate was too high to monitor the reaction continuously at 340 nm (Ama~ for unionized nitrophenol) and the reaction was too rapid to use quenching methods available to us.

The effect of temperature on the rate of binding of a-amylase and inhibitor is shown in Fig. 6. From the starch assay for activity remaining, Ea for the reactions at pH 6.90 and 5.40 were determined to be 24.2 and 12.4 kca l /mol , respectively. An Ea of 4.10 k c a l / m o l was determined from the effect of temperature on the rate of binding of a-amylase and inhibitor as measured by the continuous kinetic assay method of Wilcox and Whitaker [8] using p-nitrophenyl-a- D-maltosi~: t.o ra~3nitor the rate of loss of a-amy- iase activity (Fig. O). l h e data obtained in the presence of the maltoside substrate indicated that binding occurs by a second order rate process.

The different Ea (12.4 aad 24.2 k c a l / m o l at pH 5.40 and 6.90, respectively) reported here for the porcine pancreatic a -amylase -b lack bean inhibitor complex formation, and first order rate process at pH 5.40 and second order at pH 6.90, indicate that t~ere are different rate- determining steps at the 2 pH's. The data for the effect of temperature on complex formation obtained in the presence of p-nitrophenyl-a-D- maltoside are too complex to be interpreted yet.

Acknowledgments

The authors thank Celly for assistance with the experiments and Virginia DuBowy for checking the references and typing the manuscript. JRW thanks the Fulbright Commission for a Fulbright Senior Fel- lowship, which made this collaborative research pos- sible.

1 Truscheit E., Frgmmer W., Junge B., Miiller L., Schmidt D.D. & Wingender W. (1981) Angew Chem. 20, 744-761

2 Frommer W., Junge B., Miiller L., Schmidt D. & Truscheit E. (1979) Planta Med. 35, 195-217

3 Lajolo F.M. & Finardi Fiiho F. (1985) J. Agric. Food Chem, 33, 132-138

4 Marshall J.J. & L a u d a C.M. (1975) J. Biol. Chem. 250, 8030-8037

5 Pick K.H. & WOber G. (1978) Hoppe-Seyler's Z. Physiol. Chem. 359, 1379-1384

6 Powers J.R. & Whitaker J.R. (1977) J. Food Bio- chem. 1,217-238

7 Powers J.R. & Whitaker J.R. (1977) J. Food Bio- chem. 1,239-260

8 Wilcox E.R. & Whitaker J.R. (1984) Biochemis- try 23, 1783-1791

9 Wilcox E.R. & Whitaker J.R. (1984) J. Food Biochem. 8, 189- 213

10 Lajolo F.M. & Finardi Filho F. (1984) Cienc. Tecnol. Aliment. 4, 1 - 11

11 Frels J.M. & Rupnow J.H. (1984) J. Food Bio- chem. 8, 281-301

12 Tanizaki M.M. & Lajolo F.M. (1985) J. Food Biochem. 9, 71-89

13 Tanizaki M.M., Lajolo F.M. & Finardi Filho F. (1985) J. Food Biochem. 9, 91 - 104

14 Davis B.J. (1964) Ann. N.Y. Acad. Sci. 121, 404-427

15 Strumeyer D.H. (1967) Anal. Biochem. 19, 61-71

16 l::lndi P ]L4"nr~ ~ ,J~, ld ' r~ t tc t~v~ ]k/[ / ' l Q T " ) l i~ ' , , r I

Biochem. 24, 577-582 17 Cozzone P., Pasero L., Beaupoil B. & Marchis-

Mouren G. (1970) Biochim. Biophys. Acta 207, 490-504

18 Levitzki A. & Steer M.L. (1974) Eur. J. Biochem 41,171-180

19 Bernfeld P. (1955) Methods Enzymol. 1, 149-158

20 Ellman G.L. (1958)Arch. Biochem. Biophys. 74. 443 -450

21 Ferdinand W., Stein W.H. & Moore S. (1965) J. Biol. Chem. 240, 1150-1155

22 Steer M.L., Tal N. & Levitzki A. (1974) Bio- chem. Biophys. Acta 334,389-397

23 Payan F., Haser R., Pierrot M., Frey M., Astier J.P., Abadie B., Duee E. & Buisson G. (1980) ~tctu ~.~ya,u,t,~r. at. A 1(,_A31 . ~ u , " l r l ~ J " l r~ . . l

24 Vallee B.L., Stein E.A., Sumerwell W.N. & Fis- cher E.H. (1959) J. Biol. Chem. 234, 2901-2905

25 Fischer E.H. & Stein E.A. (1960) Enzymes 4, 313 -343

26 Hsiu J., Fischer E.H. & Stein E.A. (1964) Bio- chemistry 3, 61-66

27 Stein E.A., Hsiu J. & Fischer E.H. (1964) Bio- chemistry 3, 56-61

parameters involved in binding of porcine pancreatic α-amylase with black bean inhibitor: role of...

Documents