- Email: [email protected]
[48] Kinetic studies of immobilized α-chymotrypsin in aprotic solvents
[48]
537
IMMOBILIZED ot-CHYMOTRYPSIN IN APROTIC SOLVENTS
[48] K i n e t i c S t u d i e s o f I m m o b i l i z e d a - C h y m o t r y p s i n Aprotic Solvents
in
By MYRON L. BENDER
Preparation and Characterization of Immobilized a-Chymotrypsin This chapter deals primarily with immobilized chymotrypsin (EC 3.4.21.1). But in addition some work has also been done on other immobilized proteolytic enzymes such as immobilized trypsin (EC 3.4.21.4) and immobilized plasmin (EC 3.4.21.7). Immobilization Procedure
We have developed a method in which a proteolytic enzyme can be immobilized on a glass surface without any diminution of its catalytic activity (with respect to the solution enzyme). This was accomplished by covalently attaching the enzyme at a sufficient distance from the surface so that there is no interaction whatsoever between the enzyme and the surface to which it is attached. Previously there have been several reports that the activity of enzymes is diminished upon attachment of the enzyme to a surface, t,2 The present synthesis of immobilized a-chymotrypsin utilizes porous glass (Corning Glass Co., Ithaca, NY) as a solid support. To the used alkylamine support (1 g dry weight) in chloroform (25-50 ml) containing 5% triethylamine (v/v) p-nitrobenzoyl chloride (1 g) was added, and the mixture was refluxed for 4 hr to produce an amide. 3 This step was followed by reduction of the nitro groups with sodium dithionite (5% aqueous solution) to produce amino groups sufficiently far from the glass surface so that when we attach the enzyme to it, the enzyme is far removed from the glass. The attachment occurs by initial diazotization of the amine,3 followed by condensation of the diazonium salt to a phenolic group of a tyrosine of the enzyme or an imidazole group of a histidine of the enzyme through an azo link 4 (about I00 mg of enzyme were added per g of dry support in the coupling step at pH 8-93). In order to destroy any 1 M. D. Trevan, "Immobilized Enzymes." Wiley, New York, 1980. -~O. R. Zaborsky, "Immobilized Enzymes." CRC Press, Cleveland, Ohio, 1973. H. H. Weetal, this series, Vol. 44, p. 134. 4 E. W. Gelewitz, W. L. Riedeman, and 1. M. Klotz, Arch. Biochem. Biophys. 53, 411 (1954).
METHODS 1N ENZYMOLOGY, VOL. 135
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
538
APPLICATION OF IMMOBILIZED ENZYMES/CELLS
[48]
remaining diazonium groups the coupled product was treated with excess p-methoxyphenol solution? (There are only one tyrosine and two histidines in a-chymotrypsin). 6 This four-step synthesis is somewhat longer than the classical synthesis of an enzyme attached to a surface involving the cyanogen bromide "activation" of the hydroxyl group of a s u p p o r t . 7 But it produces an immobilized a-chymotrypsin which has identical catalytic properties to soluble a-chymotrypsin. It is also possible to obtain immobilized a-chymotrypsin commercially from The Worthington Biochemical Corp. (Freehold, N J). They sell an achymotrypsin bound to Sepharose produced in the more conventional manner by activation of one of the hydroxyl groups of Sepharose followed by condensation with the enzyme.
Kinetics of Immobilized a-Chymotrypsin Activity Measurements. The steady-state rates of N-acetyl-L-tyrosine ethyl ester hydrolysis catalyzed by soluble and immobilized a-chymotrypsin were determined using a Radiometer Automatic titrator II at 25°. 8 The kinetics of immobilized enzyme systems were determined spectrally in a similar fashion to those of soluble enzyme systems. 5 A thermostatted Cary 219 spectrophotometer was used. The only difference between soluble and immobilized enzyme systems is that in the latter a small circular magnet (9.5 mm diameter × 8.0 mm high) that would fit into the cuvette, operated by an external magnetic stirrer, was used. This resulted in a spectrophotometric trace that contained more noise than in the soluble enzyme system, but the noise level resulted in rate constants good to -+2% that were adequate. Active Site Determination. The concentration of immobilized a-chymotrypsin (bound to glass or Sepharose) was determined as follows. 8 Sepharose-bound a-chymotrypsin (1.4 ml) was added to 20 ml of pH 8.3, 0.1 M Tris buffer. Then 0.2 rnl of 0.01 M p-nitrophenyl-p '-guanidinobenzoate hydrochloride (NPGB) in dimethylformamide was added to the above solution, and the reaction mixture was kept well stirred. Aliquots (2 ml) were withdrawn at appropriate time intervals and were filtered through Millipore filters. The clear filtrate was then measured at 401.5 rim. The absorbances, after correcting for spontaneous burst ofp-nitrophenos K. Tanizawa and M. L. Bender, J. Biol. Chem. 249, 2130 (1974). 6 R. E. Dickerson and I. Geis, "The Structure and Action of Proteins." Harper & Row, New York, 1969. 7 j. Por~lth, R. Ax6n, and S. Ernbach, Nature (London) 215, 1491 (1967). 8 M. L. Bender, A. B. Cottingham, L. K. Sun, and K. Tanizawa, in "Protein CrossLinking, Part A " (E. M. Friedman, ed.). Plenum, New York, 1977,
[48]
539
IMMOBILIZED Ot-CHYMOTRYPSININ APROTIC SOLVENTS
late, w e r e t a k e n as a m e a s u r e for the active site c o n c e n t r a t i o n o f S e p h a r o s e - b o u n d a - c h y m o t r y p s i n . T h e active site c o n c e n t r a t i o n thus obtained f o r the c o m m e r c i a l l y available g l a s s - b o u n d a - c h y m o t r y p s i n w a s 1.0 × 10 -4 M. T h e identical p r o p e r t i e s o f soluble and immobilized a - c h y m o t r y p s i n are best s h o w n in Fig. 1 w h i c h p o r t r a y s a kcat v e r s u s p H profile for b o t h r e a c t i o n s o f soluble a - c h y m o t r y p s i n and glass-immobilized a - c h y m o t r y p sin. 8 T w o c o n c l u s i o n s c a n be d r a w n f r o m Fig. 1. O n e is that the absolute kc~t ( d e p e n d e n t o n the d e t e r m i n a t i o n o f the absolute c o n c e n t r a t i o n o f the
160
120
It,d, v 0
280
40
I
6.0
t
I
I
7.0
1
8.0
t
9.0
oH FIG. 1. The k¢,t of soluble and immobilized a-chymotrypsin-catalyzed hydrolysis of Nacetyl-L-tyrosine ethyl ester at 25°. (©) Native a-chymotrypsin (soluble) in 9.1% (v/v) acetonitrile buffer, k=t(um)= 157.03 +- 4.10 sec-t; pKa = 6.96 +--0.02. (Q) a-Chymotrypsin covalently bound to glass (immobilized) in 9.1% (v/v) acetonitrile buffer, kc~tt~i,~)= 157.17 + 5.68 sec-~; p K , = 7.09 + 0.02. From Bender et al, ~
540
APPLICATION
OF IMMOBILIZED
[481
ENZYMES/CELLS
:>5
20
X E
"
I0
O
0
f 6.0
0
0
0
I
7.O
0
0
I 8.0
0
0
9.0
pH
FIG. 2. The K~, of soluble and immobilized a-chymotrypsin-catalyzed hydrolysis of Nacetyl-L-tyrosine ethyl ester at 25°. (©) Soluble a-chymotrypsin in 9.1% (v/v) acetonitrile buffer. Km~appj= 1.47-1.81 x 10-3 M. (0) a-Chymotrypsin covalently bound to glass (immobilized) in 9.1% (v/v) acetonitrile buffer. Km(app)= 3.46-21.72 x 10-3 M. From Bender e t al. 8 e n z y m e by titration o f the active sites o f the immobilized enzyme) is identical to that o f the soluble enzyme. The other is that the p H dependence including the inflection point (the pKa) of the immobilized e n z y m e is identical to that o f the soluble enzyme. The Km(app)vs p H profiles for soluble and immobilized a-chymotrypsin are v e r y different, h o w e v e r (see Fig. 2). This is probably due to the fact that when the reaction is fast (as it is at high pH), the rate-determining step m a y be the diffusion of the product out of the glass beads. At lower pH, the reaction is slower, and depletion of products as they are formed is more efficient with respect to other steps, therefore Km(app)for immobilized a - c h y m o t r y p s i n approaches the value for soluble a-chymotrypsin. Mechanistic Studies of Immobilized a-Chymotrypsin in Aprotic Solvents The uses o f immobilized enzymes are manifold. Some are commercial and others are academic. It is beyond the scope of this chapter to discuss the commercial uses but two of the academic uses which have been highly important will be discussed. The first is the use of immobilized a-chymotrypsin in kinetic studies o f the e n z y m e in dipolar aprotic organic solvents
[48]
IMMOBILIZED ot-CHYMOTRYPSININ APROTICSOLVENTS • Asp 102
• His 5 7
.
"~7~i'<~,,a')..rd \R'
• Ser 195
/J
;,<,.. -.-.
H+
541
i_
NO
_
#o
Acylation
li S°
I --~ N
o \C@
HO x'R'
c
H
Ifl
o
.•C ~N~, #0
o -H \_/~.1
-H ÷
I
~
H 0
~ H+ LO
.n,.',--c,~
//I/AJ/~
Deacylation SCHEMEI. Mechanismof action of a-chymotrypsin. which have led to a more precise description of the catalytic mechanism of a-chymotrypsin action (Scheme I). We wanted to determine at what step water was involved in the a-chymotrypsin mechanism: binding, acylation, or deacylation. To do this we had to carry out kinetic studies with small amounts of water present, s Therefore we had to use a dipolar aprotic solvent such as dioxane (which is completely miscible with water in all proportions). If we were to use such a system we would be forced to use an enzyme operative in all conditions from 0 to 100% dioxane (by volume). In small amounts of dioxane (up to 33% dioxane) a-chymotrypsin is soluble, but above 33% dioxane, a-chymotrypsin precipitates and thus is noncatalytic. Thus we were forced to use immobilized a-chymotrypsin and the use of the immobilized enzyme enabled us to determine kinetics up to 95% dioxane-5% water. Fortunately, as discussed above in detail, we knew that the kinetic properties of soluble and immobilized o~-chymotrypsin are identical when prepared by our method, so we could use either soluble or immobilized ot-chymotrypsin interchangeably knowing we were looking at the same catalytic entity. One could then ask whether dependence on the water concentration occurs during binding, in acylation, or in deacylation. The binding was probed by following the dependence of the hydrolysis of N-benzoyl-Ltyrosine p-nitroanilide on dioxane concentration in dioxane-buffer (0.05
542
APPLICATION OF IMMOBILIZED ENZYMES/CELLS
/ ;
E
x."
o
[48]
5
I
6 0
2JO
I
40
A
60
i
80
I00
Dioxane Concentration(v/v%) FIG. 3. The effect of dioxane concentration on binding using the immobilized a-chymotrypsin-catalyzed hydrolysis of N-benzoyl-L-tyrosine p-nitroanilide.
M Tris, 0.02 M CaCI2, pH 8.2) mixtures at 25 °. It shows no dependence from about 20 up to 90% (Fig. 3). The dependence of the hydrolytic rates in dioxane concentration of N-benzoyl-L-tyrosine p-nitroanilide in 0.05 M Tris, 0.02 M CaCI2, pH 8.2 buffer-dioxane mixtures at 25° (where acylation, k2, is rate controlling) and N-acetyl-L-tryptophan p-nitrophenyl ester in 0.05 M acetate, pH 5.8 buffer-dioxane mixtures at 25° (where deacylation, k3, is rate controlling) were determined spectrophotometrically 9,~° (Figs. 4 and 5). One may legitimately ask if there is any effect in increasing the dioxane concentration since no effect occurs either in binding or acylation. The answer is a decided yes. The effect occurs in the deacylation step. Increasing the dioxane concentration from 0 to 95% results in a large diminution of the deacylation rate constant. The diminution amounts to over a factor of 30 or based on the bottom point in Fig. 5, it amounts to an infinite effect. A more reasonable statement is that the effect is somewhere between 30 and infinity. Thus the effect of dioxane concentration appears almost exclusively in deacylation. The effect of dioxane concentration on the deacylation rate constant can be explained by Scheme I. It indicates that only the deacylation rate should be dependent on the water concentration. The components of the transition state of deacylation must then include the acylserine ester, and imidazole of the enzyme, and a 9 M. L. Bender, G. E. Clement, C. R. Gunter, and F. J. K6zdy, J. Am. Chem. Soc. 86, 3697 (1964). lo H. F. Bundy, Anal. Biochem. 3, 431 (1962).
[48]
IMMOBILIZED Ot-CHYMOTRYPSIN IN APROTIC SOLVENTS
543
0.6
.j
0.4
'0
"7 V}
v
N 0.2
o
2'o
40
6=0
8=0
I00
Dioxane Concentration ( v / v % ) FIG. 4. The effect of dioxane concentration on acylation using the immobilized a-chymotrypsin-catalyzed hydrolysis of N-benzoyl-L-tyrosine p-nitroanilide.
30
2.0 ¢O O~
o_ x ,¢,
10
•
0 0
20 V/ V %
,.
25
='~
o
40 60 80 I00 Dioxane Concentration ,
,
5.0
7.5
Dioxane Molarity FIG. 5. The effect of dioxane concentration in deacylation using the immobilized achymotrypsin hydrolysis of p-N-acetyl-L-tryptophan p-nitrophenyl ester.
544
APPLICATION OF IMMOBILIZED ENZYMES/CELLS
[48]
TABLE I RATE CONSTANTS OF THE DENATURATION OF ot-CHYMOTRYPSIN a
pH 4.0 + 0.1 7.48 +- 0.05 8.50 x 0.05
11.0 - 0.2 11.9 --+ 0,2 12.9 -+ 0.2
Soluble a-chymotrypsin
Immobilized a-chymotrypsin
k2 ( M i sec-i)
k2 ( M -I sec f)
0.004 0.071 0.35
0b 0b 0b
kt (sec -1)
k~ (sec -I)
1.43 x 10 -5 6.5 x 10 -5 0.75 × 10 -3
1.74 x 10 -6 4.0 × 10 -5 1.2 × 10 -3
a Aqueous solution (25°). o The upper limit of kz is smaller than 0.001 M -~ sec -I.
molecule of water. A water molecule and imidazole are involved in the rate-determining proton transfer reaction. The first-order dependence on the nucleophile water is expected by analogy to the kinetics of methanolysis reported previously. 9 As shown in Fig. 5, it is hard to show that the reaction is also dependent on secondary effects of the aqueous organic solvent such as that on the structure of the solvent. Taking into account microscopic reversibility in respect to acylation and deacylation, the assumption that the solvent polarity affects only the deacylation step can be ruled out. Denaturation Studies of Soluble and Immobilized a-Chymotrypsin The other use we have found for immobilized a-chymotrypsin is in denaturation. The denaturation of soluble a-chymotrypsin was shown to be second order at neutrality (autolysis) and first order (with respect to enzyme) at high pH. n A cannibalistic denaturation at neutrality (involving two enzyme molecules) and a hydroxide ion reaction at high pH (involving one enzyme molecule) were proposed to explain these kinetics in a previous article. 11 These results allow one to predict that a-chymotrypsin on a solid support should show no cannibalistic denaturation, because this ~ H . - L . W u , A. Wastell, and M. L. B e n d e r , Proc. Natl. Acad. Sci. U.S.A. 78, 4116 (1981).
[48]
IMMOBILIZED ot-CHYMOTRYPSIN IN APROTIC SOLVENTS
I\
¢, II
I
/,'
~1\
/,'
'~
~1 \ ~1 \ ~_
/
~
Soluble
I
/i
I•
I t
\~n.me/,' \ / , ' En.zyme
/
¢, ~
I I
/ ' support
4
6
8
10
pH
12
545
|
14
FIG. 6. The denaturation of soluble chymotrypsin and glass-immobilized c~-chymotrypsin.
reaction is second order in enzyme, one molecule of enzyme being an active site and the other being an amino acid such as tryptophan or tyrosine. As a consequence, immobilized a-chymotrypsin should be very stable at neutral pH values, and it is (Table I and Fig. 6). At high pH, denaturation of the immobilized enzyme will be very close to that of the soluble enzyme. The experiments described in Table I were carried out as follows, t~Chymotrypsin (3.2-0.05 mM) was incubated in appropriate buffers at 25° and small aliquots (5-50/xl) of the enzyme solution were diluted into 6 mi
/t
GLASS SUPPORT I
t
I
l l l ~ l l l
I
'~
I I
T
OHOHFIG. 7. Schematic representation of denaturation of immobilized c~-chymotrypsin by OH- and by another enzyme molecule at neutrality.
546
APPLICATION OF IMMOBILIZED ENZYMES/CELLS
[49]
of 0.1 M sodium acetate buffer at pH 5.8 and incubated at 25 ° for another 20 min. The amount of enzyme in the solution was determined with Nacetyl-L-tryptophan p-nitrophenyl ester. ~2 The amount of active enzyme in the stock solution of a-chymotrypsin was titrated with n-trans-cinnamoylimidazole. 13 The reason that hydroxide ion can denature but that another molecule of chymotrypsin cannot denature when the a-chymotrypsin is immobilized 14 is schematically shown in Fig. 7 which both proves our idea about cannibalistic denaturation and provides a mechanism for the stability of the immobilized enzyme at neutrality. Although trypsin notoriously denatures more rapidly than a-chymotrypsin, the rates of denaturation of immobilized trypsin 15 and a-chymotrypsin are very similar. This is due to the fact that the rapid denaturation of trypsin is due to cannibalistic denaturation which is removed by immobilization. Acknowledgment The author gratefully acknowledges the financial assistance of NIH Grant GM-20853 in this research. 12 F. J. K6zdy, G. E. Clement, and M. L. Bender, J. Am. Chem. Soc. 86, 3690 (1964). 13G. R. Schonbaum, B. Zerner, and M. L. Bender, J. Biol. Chem. 236, 2930 (1961). ~4H.-L. Wu, D. A. Lace, and M. L. Bender, Proc. Natl. Acad. Sci. U.S.A. 78, 4118 (1981). 15 H.-L. Wu, C. Kundrot, and M. L. Bender, Biochem. Biophys. Res. Commun. 107, 742 (1982).
[49] S p e c t r o s c o p i c M e t h o d s for C h a r a c t e r i z a t i o n o f Immobilized Alcohol Dehydrogenase B y H E L G A SCHNEIDER-BERNLOHR, H E L M U T DIETRICH, a n d MICHAEL ZEPPEZAUER
Horse liver alcohol dehydrogenase (LADH, EC 1.1.1.1) is of great interest for both analytical and synthetic applications, because of its broad substrate specificity and in some cases high enantiomeric or prochiral stereospecificity. ~,2 The soluble form belongs to the best characterized metalloenzymes, x4 Due to the high stability and favorable optical i A. Wiseman, Top. Enzyme Ferment. Biotechnol. 5, 337 (1981). 2 j. B. Jones and J. F. Beck, Tech. Chem. (N.Y.) 10, Part !, 107 (1976). 3 M. Zeppezauer, I. Andersson, H. Dietrich, M. Gerber, G. Schneider, and H. SchneiderBernl6hr, J. Mol. Catal. 23, 377 (1984).
METHODS IN ENZYMOLOGY, VOL. 135
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
537
IMMOBILIZED ot-CHYMOTRYPSIN IN APROTIC SOLVENTS
[48] K i n e t i c S t u d i e s o f I m m o b i l i z e d a - C h y m o t r y p s i n Aprotic Solvents
in
By MYRON L. BENDER
Preparation and Characterization of Immobilized a-Chymotrypsin This chapter deals primarily with immobilized chymotrypsin (EC 3.4.21.1). But in addition some work has also been done on other immobilized proteolytic enzymes such as immobilized trypsin (EC 3.4.21.4) and immobilized plasmin (EC 3.4.21.7). Immobilization Procedure
We have developed a method in which a proteolytic enzyme can be immobilized on a glass surface without any diminution of its catalytic activity (with respect to the solution enzyme). This was accomplished by covalently attaching the enzyme at a sufficient distance from the surface so that there is no interaction whatsoever between the enzyme and the surface to which it is attached. Previously there have been several reports that the activity of enzymes is diminished upon attachment of the enzyme to a surface, t,2 The present synthesis of immobilized a-chymotrypsin utilizes porous glass (Corning Glass Co., Ithaca, NY) as a solid support. To the used alkylamine support (1 g dry weight) in chloroform (25-50 ml) containing 5% triethylamine (v/v) p-nitrobenzoyl chloride (1 g) was added, and the mixture was refluxed for 4 hr to produce an amide. 3 This step was followed by reduction of the nitro groups with sodium dithionite (5% aqueous solution) to produce amino groups sufficiently far from the glass surface so that when we attach the enzyme to it, the enzyme is far removed from the glass. The attachment occurs by initial diazotization of the amine,3 followed by condensation of the diazonium salt to a phenolic group of a tyrosine of the enzyme or an imidazole group of a histidine of the enzyme through an azo link 4 (about I00 mg of enzyme were added per g of dry support in the coupling step at pH 8-93). In order to destroy any 1 M. D. Trevan, "Immobilized Enzymes." Wiley, New York, 1980. -~O. R. Zaborsky, "Immobilized Enzymes." CRC Press, Cleveland, Ohio, 1973. H. H. Weetal, this series, Vol. 44, p. 134. 4 E. W. Gelewitz, W. L. Riedeman, and 1. M. Klotz, Arch. Biochem. Biophys. 53, 411 (1954).
METHODS 1N ENZYMOLOGY, VOL. 135
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
538
APPLICATION OF IMMOBILIZED ENZYMES/CELLS
[48]
remaining diazonium groups the coupled product was treated with excess p-methoxyphenol solution? (There are only one tyrosine and two histidines in a-chymotrypsin). 6 This four-step synthesis is somewhat longer than the classical synthesis of an enzyme attached to a surface involving the cyanogen bromide "activation" of the hydroxyl group of a s u p p o r t . 7 But it produces an immobilized a-chymotrypsin which has identical catalytic properties to soluble a-chymotrypsin. It is also possible to obtain immobilized a-chymotrypsin commercially from The Worthington Biochemical Corp. (Freehold, N J). They sell an achymotrypsin bound to Sepharose produced in the more conventional manner by activation of one of the hydroxyl groups of Sepharose followed by condensation with the enzyme.
Kinetics of Immobilized a-Chymotrypsin Activity Measurements. The steady-state rates of N-acetyl-L-tyrosine ethyl ester hydrolysis catalyzed by soluble and immobilized a-chymotrypsin were determined using a Radiometer Automatic titrator II at 25°. 8 The kinetics of immobilized enzyme systems were determined spectrally in a similar fashion to those of soluble enzyme systems. 5 A thermostatted Cary 219 spectrophotometer was used. The only difference between soluble and immobilized enzyme systems is that in the latter a small circular magnet (9.5 mm diameter × 8.0 mm high) that would fit into the cuvette, operated by an external magnetic stirrer, was used. This resulted in a spectrophotometric trace that contained more noise than in the soluble enzyme system, but the noise level resulted in rate constants good to -+2% that were adequate. Active Site Determination. The concentration of immobilized a-chymotrypsin (bound to glass or Sepharose) was determined as follows. 8 Sepharose-bound a-chymotrypsin (1.4 ml) was added to 20 ml of pH 8.3, 0.1 M Tris buffer. Then 0.2 rnl of 0.01 M p-nitrophenyl-p '-guanidinobenzoate hydrochloride (NPGB) in dimethylformamide was added to the above solution, and the reaction mixture was kept well stirred. Aliquots (2 ml) were withdrawn at appropriate time intervals and were filtered through Millipore filters. The clear filtrate was then measured at 401.5 rim. The absorbances, after correcting for spontaneous burst ofp-nitrophenos K. Tanizawa and M. L. Bender, J. Biol. Chem. 249, 2130 (1974). 6 R. E. Dickerson and I. Geis, "The Structure and Action of Proteins." Harper & Row, New York, 1969. 7 j. Por~lth, R. Ax6n, and S. Ernbach, Nature (London) 215, 1491 (1967). 8 M. L. Bender, A. B. Cottingham, L. K. Sun, and K. Tanizawa, in "Protein CrossLinking, Part A " (E. M. Friedman, ed.). Plenum, New York, 1977,
[48]
539
IMMOBILIZED Ot-CHYMOTRYPSININ APROTIC SOLVENTS
late, w e r e t a k e n as a m e a s u r e for the active site c o n c e n t r a t i o n o f S e p h a r o s e - b o u n d a - c h y m o t r y p s i n . T h e active site c o n c e n t r a t i o n thus obtained f o r the c o m m e r c i a l l y available g l a s s - b o u n d a - c h y m o t r y p s i n w a s 1.0 × 10 -4 M. T h e identical p r o p e r t i e s o f soluble and immobilized a - c h y m o t r y p s i n are best s h o w n in Fig. 1 w h i c h p o r t r a y s a kcat v e r s u s p H profile for b o t h r e a c t i o n s o f soluble a - c h y m o t r y p s i n and glass-immobilized a - c h y m o t r y p sin. 8 T w o c o n c l u s i o n s c a n be d r a w n f r o m Fig. 1. O n e is that the absolute kc~t ( d e p e n d e n t o n the d e t e r m i n a t i o n o f the absolute c o n c e n t r a t i o n o f the
160
120
It,d, v 0
280
40
I
6.0
t
I
I
7.0
1
8.0
t
9.0
oH FIG. 1. The k¢,t of soluble and immobilized a-chymotrypsin-catalyzed hydrolysis of Nacetyl-L-tyrosine ethyl ester at 25°. (©) Native a-chymotrypsin (soluble) in 9.1% (v/v) acetonitrile buffer, k=t(um)= 157.03 +- 4.10 sec-t; pKa = 6.96 +--0.02. (Q) a-Chymotrypsin covalently bound to glass (immobilized) in 9.1% (v/v) acetonitrile buffer, kc~tt~i,~)= 157.17 + 5.68 sec-~; p K , = 7.09 + 0.02. From Bender et al, ~
540
APPLICATION
OF IMMOBILIZED
[481
ENZYMES/CELLS
:>5
20
X E
"
I0
O
0
f 6.0
0
0
0
I
7.O
0
0
I 8.0
0
0
9.0
pH
FIG. 2. The K~, of soluble and immobilized a-chymotrypsin-catalyzed hydrolysis of Nacetyl-L-tyrosine ethyl ester at 25°. (©) Soluble a-chymotrypsin in 9.1% (v/v) acetonitrile buffer. Km~appj= 1.47-1.81 x 10-3 M. (0) a-Chymotrypsin covalently bound to glass (immobilized) in 9.1% (v/v) acetonitrile buffer. Km(app)= 3.46-21.72 x 10-3 M. From Bender e t al. 8 e n z y m e by titration o f the active sites o f the immobilized enzyme) is identical to that o f the soluble enzyme. The other is that the p H dependence including the inflection point (the pKa) of the immobilized e n z y m e is identical to that o f the soluble enzyme. The Km(app)vs p H profiles for soluble and immobilized a-chymotrypsin are v e r y different, h o w e v e r (see Fig. 2). This is probably due to the fact that when the reaction is fast (as it is at high pH), the rate-determining step m a y be the diffusion of the product out of the glass beads. At lower pH, the reaction is slower, and depletion of products as they are formed is more efficient with respect to other steps, therefore Km(app)for immobilized a - c h y m o t r y p s i n approaches the value for soluble a-chymotrypsin. Mechanistic Studies of Immobilized a-Chymotrypsin in Aprotic Solvents The uses o f immobilized enzymes are manifold. Some are commercial and others are academic. It is beyond the scope of this chapter to discuss the commercial uses but two of the academic uses which have been highly important will be discussed. The first is the use of immobilized a-chymotrypsin in kinetic studies o f the e n z y m e in dipolar aprotic organic solvents
[48]
IMMOBILIZED ot-CHYMOTRYPSININ APROTICSOLVENTS • Asp 102
• His 5 7
.
"~7~i'<~,,a')..rd \R'
• Ser 195
/J
;,<,.. -.-.
H+
541
i_
NO
_
#o
Acylation
li S°
I --~ N
o \C@
HO x'R'
c
H
Ifl
o
.•C ~N~, #0
o -H \_/~.1
-H ÷
I
~
H 0
~ H+ LO
.n,.',--c,~
//I/AJ/~
Deacylation SCHEMEI. Mechanismof action of a-chymotrypsin. which have led to a more precise description of the catalytic mechanism of a-chymotrypsin action (Scheme I). We wanted to determine at what step water was involved in the a-chymotrypsin mechanism: binding, acylation, or deacylation. To do this we had to carry out kinetic studies with small amounts of water present, s Therefore we had to use a dipolar aprotic solvent such as dioxane (which is completely miscible with water in all proportions). If we were to use such a system we would be forced to use an enzyme operative in all conditions from 0 to 100% dioxane (by volume). In small amounts of dioxane (up to 33% dioxane) a-chymotrypsin is soluble, but above 33% dioxane, a-chymotrypsin precipitates and thus is noncatalytic. Thus we were forced to use immobilized a-chymotrypsin and the use of the immobilized enzyme enabled us to determine kinetics up to 95% dioxane-5% water. Fortunately, as discussed above in detail, we knew that the kinetic properties of soluble and immobilized o~-chymotrypsin are identical when prepared by our method, so we could use either soluble or immobilized ot-chymotrypsin interchangeably knowing we were looking at the same catalytic entity. One could then ask whether dependence on the water concentration occurs during binding, in acylation, or in deacylation. The binding was probed by following the dependence of the hydrolysis of N-benzoyl-Ltyrosine p-nitroanilide on dioxane concentration in dioxane-buffer (0.05
542
APPLICATION OF IMMOBILIZED ENZYMES/CELLS
/ ;
E
x."
o
[48]
5
I
6 0
2JO
I
40
A
60
i
80
I00
Dioxane Concentration(v/v%) FIG. 3. The effect of dioxane concentration on binding using the immobilized a-chymotrypsin-catalyzed hydrolysis of N-benzoyl-L-tyrosine p-nitroanilide.
M Tris, 0.02 M CaCI2, pH 8.2) mixtures at 25 °. It shows no dependence from about 20 up to 90% (Fig. 3). The dependence of the hydrolytic rates in dioxane concentration of N-benzoyl-L-tyrosine p-nitroanilide in 0.05 M Tris, 0.02 M CaCI2, pH 8.2 buffer-dioxane mixtures at 25° (where acylation, k2, is rate controlling) and N-acetyl-L-tryptophan p-nitrophenyl ester in 0.05 M acetate, pH 5.8 buffer-dioxane mixtures at 25° (where deacylation, k3, is rate controlling) were determined spectrophotometrically 9,~° (Figs. 4 and 5). One may legitimately ask if there is any effect in increasing the dioxane concentration since no effect occurs either in binding or acylation. The answer is a decided yes. The effect occurs in the deacylation step. Increasing the dioxane concentration from 0 to 95% results in a large diminution of the deacylation rate constant. The diminution amounts to over a factor of 30 or based on the bottom point in Fig. 5, it amounts to an infinite effect. A more reasonable statement is that the effect is somewhere between 30 and infinity. Thus the effect of dioxane concentration appears almost exclusively in deacylation. The effect of dioxane concentration on the deacylation rate constant can be explained by Scheme I. It indicates that only the deacylation rate should be dependent on the water concentration. The components of the transition state of deacylation must then include the acylserine ester, and imidazole of the enzyme, and a 9 M. L. Bender, G. E. Clement, C. R. Gunter, and F. J. K6zdy, J. Am. Chem. Soc. 86, 3697 (1964). lo H. F. Bundy, Anal. Biochem. 3, 431 (1962).
[48]
IMMOBILIZED Ot-CHYMOTRYPSIN IN APROTIC SOLVENTS
543
0.6
.j
0.4
'0
"7 V}
v
N 0.2
o
2'o
40
6=0
8=0
I00
Dioxane Concentration ( v / v % ) FIG. 4. The effect of dioxane concentration on acylation using the immobilized a-chymotrypsin-catalyzed hydrolysis of N-benzoyl-L-tyrosine p-nitroanilide.
30
2.0 ¢O O~
o_ x ,¢,
10
•
0 0
20 V/ V %
,.
25
='~
o
40 60 80 I00 Dioxane Concentration ,
,
5.0
7.5
Dioxane Molarity FIG. 5. The effect of dioxane concentration in deacylation using the immobilized achymotrypsin hydrolysis of p-N-acetyl-L-tryptophan p-nitrophenyl ester.
544
APPLICATION OF IMMOBILIZED ENZYMES/CELLS
[48]
TABLE I RATE CONSTANTS OF THE DENATURATION OF ot-CHYMOTRYPSIN a
pH 4.0 + 0.1 7.48 +- 0.05 8.50 x 0.05
11.0 - 0.2 11.9 --+ 0,2 12.9 -+ 0.2
Soluble a-chymotrypsin
Immobilized a-chymotrypsin
k2 ( M i sec-i)
k2 ( M -I sec f)
0.004 0.071 0.35
0b 0b 0b
kt (sec -1)
k~ (sec -I)
1.43 x 10 -5 6.5 x 10 -5 0.75 × 10 -3
1.74 x 10 -6 4.0 × 10 -5 1.2 × 10 -3
a Aqueous solution (25°). o The upper limit of kz is smaller than 0.001 M -~ sec -I.
molecule of water. A water molecule and imidazole are involved in the rate-determining proton transfer reaction. The first-order dependence on the nucleophile water is expected by analogy to the kinetics of methanolysis reported previously. 9 As shown in Fig. 5, it is hard to show that the reaction is also dependent on secondary effects of the aqueous organic solvent such as that on the structure of the solvent. Taking into account microscopic reversibility in respect to acylation and deacylation, the assumption that the solvent polarity affects only the deacylation step can be ruled out. Denaturation Studies of Soluble and Immobilized a-Chymotrypsin The other use we have found for immobilized a-chymotrypsin is in denaturation. The denaturation of soluble a-chymotrypsin was shown to be second order at neutrality (autolysis) and first order (with respect to enzyme) at high pH. n A cannibalistic denaturation at neutrality (involving two enzyme molecules) and a hydroxide ion reaction at high pH (involving one enzyme molecule) were proposed to explain these kinetics in a previous article. 11 These results allow one to predict that a-chymotrypsin on a solid support should show no cannibalistic denaturation, because this ~ H . - L . W u , A. Wastell, and M. L. B e n d e r , Proc. Natl. Acad. Sci. U.S.A. 78, 4116 (1981).
[48]
IMMOBILIZED ot-CHYMOTRYPSIN IN APROTIC SOLVENTS
I\
¢, II
I
/,'
~1\
/,'
'~
~1 \ ~1 \ ~_
/
~
Soluble
I
/i
I•
I t
\~n.me/,' \ / , ' En.zyme
/
¢, ~
I I
/ ' support
4
6
8
10
pH
12
545
|
14
FIG. 6. The denaturation of soluble chymotrypsin and glass-immobilized c~-chymotrypsin.
reaction is second order in enzyme, one molecule of enzyme being an active site and the other being an amino acid such as tryptophan or tyrosine. As a consequence, immobilized a-chymotrypsin should be very stable at neutral pH values, and it is (Table I and Fig. 6). At high pH, denaturation of the immobilized enzyme will be very close to that of the soluble enzyme. The experiments described in Table I were carried out as follows, t~Chymotrypsin (3.2-0.05 mM) was incubated in appropriate buffers at 25° and small aliquots (5-50/xl) of the enzyme solution were diluted into 6 mi
/t
GLASS SUPPORT I
t
I
l l l ~ l l l
I
'~
I I
T
OHOHFIG. 7. Schematic representation of denaturation of immobilized c~-chymotrypsin by OH- and by another enzyme molecule at neutrality.
546
APPLICATION OF IMMOBILIZED ENZYMES/CELLS
[49]
of 0.1 M sodium acetate buffer at pH 5.8 and incubated at 25 ° for another 20 min. The amount of enzyme in the solution was determined with Nacetyl-L-tryptophan p-nitrophenyl ester. ~2 The amount of active enzyme in the stock solution of a-chymotrypsin was titrated with n-trans-cinnamoylimidazole. 13 The reason that hydroxide ion can denature but that another molecule of chymotrypsin cannot denature when the a-chymotrypsin is immobilized 14 is schematically shown in Fig. 7 which both proves our idea about cannibalistic denaturation and provides a mechanism for the stability of the immobilized enzyme at neutrality. Although trypsin notoriously denatures more rapidly than a-chymotrypsin, the rates of denaturation of immobilized trypsin 15 and a-chymotrypsin are very similar. This is due to the fact that the rapid denaturation of trypsin is due to cannibalistic denaturation which is removed by immobilization. Acknowledgment The author gratefully acknowledges the financial assistance of NIH Grant GM-20853 in this research. 12 F. J. K6zdy, G. E. Clement, and M. L. Bender, J. Am. Chem. Soc. 86, 3690 (1964). 13G. R. Schonbaum, B. Zerner, and M. L. Bender, J. Biol. Chem. 236, 2930 (1961). ~4H.-L. Wu, D. A. Lace, and M. L. Bender, Proc. Natl. Acad. Sci. U.S.A. 78, 4118 (1981). 15 H.-L. Wu, C. Kundrot, and M. L. Bender, Biochem. Biophys. Res. Commun. 107, 742 (1982).
[49] S p e c t r o s c o p i c M e t h o d s for C h a r a c t e r i z a t i o n o f Immobilized Alcohol Dehydrogenase B y H E L G A SCHNEIDER-BERNLOHR, H E L M U T DIETRICH, a n d MICHAEL ZEPPEZAUER
Horse liver alcohol dehydrogenase (LADH, EC 1.1.1.1) is of great interest for both analytical and synthetic applications, because of its broad substrate specificity and in some cases high enantiomeric or prochiral stereospecificity. ~,2 The soluble form belongs to the best characterized metalloenzymes, x4 Due to the high stability and favorable optical i A. Wiseman, Top. Enzyme Ferment. Biotechnol. 5, 337 (1981). 2 j. B. Jones and J. F. Beck, Tech. Chem. (N.Y.) 10, Part !, 107 (1976). 3 M. Zeppezauer, I. Andersson, H. Dietrich, M. Gerber, G. Schneider, and H. SchneiderBernl6hr, J. Mol. Catal. 23, 377 (1984).
METHODS IN ENZYMOLOGY, VOL. 135
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.