Vol. 178, No. 3, 1991 August 15, 1991

SUBSTRATE

BIOCHEMICAL

EFFECTS

ON THE ENZYMATIC ACTIVITY IN REVERSE MICELLES

Qingcheng

lnstitut

Received

fur Polymere,

May

14,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1705-1112

OF a-CHYMOTRYPSIN

MAO and Peter WALDE’*

ETH-Zentrum, Universitatstrasse Switzerland

6, CH-8092

Zurich,

1991

SummarX Six different substrates have been used for measuring the activity of ochymotrypsin in reverse micelles formed by sodium bis(Fethylhexyl) sulfosuccinate (AOT) in isooctane. The substrates were glutaryl-Phe p-nitroanilde, succinyl-Phe p-nitroanilide, acetylPhe p-nitroanilide, succinyl-Ala-Ala-Phe p-nitroanilide, succinyl-Ala-Ala-Pro-Phe p-nitroanilide and acetyl-Trp methyl ester. It has been shown that the dependence of the kinetic constants (k,, and K,) on the water content of the system, on w, (= [H,O]/[AOT]), is different for the different substrates. This indicates that activity-w, profiles for achymotrypsin in reverse micelles not only reflect an intrinsic feature of the enzyme alone. For the p-nitroanilides it was found that the lower k, (and the higher K,) in aqueous solution, the higher k, as well as K, in reverse micelles. “Superactivity” of a-chymotrypsin could only be found with the ester substrate and with relatively “poor” p-nitroanilides. The presence of a negative charge in the substrate molecule is not a prerequesite for achymotrypsin to show “superactivity”. 0 1991 Academic Press, 1°C.

Since the very beginning that the catalytic

of enzymology

activity of the solubilized

i.e. on w,, the molar ratio of water activity on w, has been studied variety of enzymes, theories

have been put forward micelles

whether

[l-3].

for example

which

it has been

recognized

with the overall water

This dependence

of the enzymatic

surfactant

by Luisi and Magid

content,

systems

and a

[4] or Martinek

et al.

that the activity vs. w, curve is bell-shaped

and

deal with the catalytic

behavior

of enzymes

in

to enzymes

micelles

is the question

[6-l I]. is relevant

the activity-w,

with respect

profile is an intrinsic

depends

on the substrate.

micelles

by using a-chymotrypsin

depend

on the substrate

‘I’ To whom

varies

systems

over the years with different

as summarized

reverse

micellar

enzymes

to surfactant

[5]. In many cases it has been reported

A point which

in reverse

correspondence

In the following,

feature

in reverse

of the enzyme

we will address

alone or whether

this question

it also

with AOT reverse

(EC 3.4.21 .l). We will show that the activity-w,, profiles

used. should

be addressed.

Abbreviations: AOT, sodium bis(2-ethylhexyl) sulfosuccinate; w,=[H,OY[AOT] (molar concentrations); DMSO, dimethyl sulfoxide; AC, acetyl; Sue, succinyl; Gr, glutaryl; NH-Np, pnitroanilide; K,,, or K,,, the Michaelis constant expressed with respect to the total Solution or with respect to the ‘aqueous volume of the reverse micelle solution.

1105

0006-291x/91 $1.50 Copyright 0 1991 by Aca&mic Press, Inc. All rights of reproduction in any form reserved.

Vol.

178,

BIOCHEMICAL

No. 3, 1991

MATERIALS

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

AND METHODS

Reaqents. Sue-Ala-Ala-Pro-Phe-NH-Np, Sue-Ala-Ala-Phe-NH-Np, Gr-Phe-NH-Np, Ac-PheNH-Np, Sue-Phe-NH-Np and Ac-Trp-Me were purchased from Bachem, Switzerland and were used as obtained. For all other chemicals used in this study, see [12]. Reverse Micellar Solutions. 50 mM AOT/isooctane reverse micelles were prepared as described elsewhere [12]. Aaueous Stock Solutions. All aqueous solutions were prepared in 0.1 M Tris/HCI buffer, pH 8.0. The concentration of enzyme and substrate stock solutions were determined spectrophotometrically as described earlier [12]. Spontaneous hydrolysis of the substrates could be neglected. Enzyme stock solutions were always freshly prepared and both enzyme and substrate solutions were kept on ice during the course of the experiments. In the case of Gr-Phe-NH-Np, Sue-Phe-NH-Np, Ac-Phe-NH-Np and Ac-Trp-Me the stock solutions were prepared with the help of dimethylsulfoxide (DMSO) due to their low solubilities in water. The overall DMSO concentration in these cases was 28 mM. Enzvme assay. All enzyme activity measurements were carried out spectrophotometrically at 25 “C with an Uvikon 810 spectrophotometer from Kontron (Switzerland). For the pnitroanilde substrates, the hydrolytic activity of o-chymotrypsin was measured by following the accumulation of p-nitroaniline at 410 nm in water and at 360 nm in AOT reverse micelles, the molar extinction coefficients (E) are 8800 M-‘cm-’ (in water) and 11400 M-‘cm ’ (w, = 8) 10100 M-‘cm-’ (w, = 14), 10000 M-‘cm-’ (w, = 20), and 9900 M”cm-’ (w, = 30), respectively. In the case of Ac-Trp-Me, the reaction was followed at 300 nm with de of 240 M-‘cm-’ (in water) and 360 M-‘cm-’ (w, = 8) 310 M-‘cm-’ (w, = 14), 240 M-‘cm-’ (w, = 20), and 220 M-‘cm-’ (w, = 30), respectively. Two different experimental procedures were used to measure the enzymatic activity in reverse micelles. A) “Micelle-mix”: A typical kinetic experiment with Suc-Ala-Ala-Pro-PheNH-Np, Sue-Ala-Ala-Phe-NH-Np or Ac-Trp-Me was carried out in the following way: a 1 ml quartz cuvette (with a pathlength of 1 cm) was filled with a micellar solution of substrate and buffer to a final volume of 900 ul. The enzymatic reaction was started by addition of 100 j.rl of a reverse micellar solution containing solubilized a-chymotrypsin. For each set of experiments at one particular w, value, both micellar solutions had the same w,. The formation of product was followed immediately afterwards. B) “Injection”: For the “poor” substrates, however, Gr-Phe-NH-Np, Sue-Phe-NH-Np and Ac-Phe-NH-Np, the typical injection technique was used for kinetic measurement according to the following procedure (w,=lO): to 10 ml 50 mM AOT/isooctane solution 80 f~l substrate stock solution was first added. (In all these cases the overall DMSO concentration was 28 mM). After complete substrate solubilization, 10 ul enzyme stock solution was then injected and, after good mixing, the reaction rate was measured in the transparent solution. All measurements were made with less than 0.2 mM substrate overall. Treatment of the kinetic data. The product accumulation curves were analyzed for initial velocities. Michaelis-Menten kinetics and its derived Lineweaver-Burk plots were then applied for the determination of kca and K, as a function of w,. Each determination involved duplicates at five different initial substrate concentrations. The reported values for k,, and K, are mean values whith their standard deviations as obtained from the linear regression analysis of the double reciprocal Lineweaver-Burk plots. Stopped flow measurements. Stopped flow measurements were carried out as described elsewhere [13].

RESULTS

AND DISCUSSION

The activity of a-chymotrypsin mM AOTIisooctane Gr-Phe-NH-Np,

reverse

has been measured micelles

Sue-Phe-NH-Np,

and one ester Ac-Trp-Me.

Ac-Trp-Me,

all substrates

chymotrypsin

group

terminus.

Gr-Phe-NH-Np

p-nitroanilide

or alkaline

pH negatively

In AOT reverse

[3,14],

micelles,

Sue-Ala-Ala-Pro-Phe-NH-Np 1106

solution

and in 50

substrates,

namely

Sue-Ala-Ala-Phe-NH-Np,

Note, that with the exception

are at neutral

at the amino

against

at 25 “C in aqueous

five different

Ac-Phe-NH-Np,

Phe-NH-Np carboxylic

against

Suc-Ala-Ala-Proof Ac-Phe-NH-Np

charged,

and

due to the free

the activity of o[14] and Ac-Trp-Me

Vol.

BIOCHEMICAL

178, No. 3, 1991

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

m Kinetic constants for a-chvmotrvosin Substrates

in aoueous solution for different substrates

kat(s-‘)

Km(M)

k,,/K,(M-‘s-l)

2.41f0.06x10’ 2.46x1@

4.5*0.2x10” 6.5~10.~

53.6k4.0 37.8

1.60&0.10x10-2 1.10x10-’

9.lf0.9xlO” 7.2~10.”

17.6&3 15.3

2.2+0.3x10”

48.2&l 1

A) p-Nitroanilides: Gr-Phe-NH-No 0.05 M TrislHCI, pH 8.0”’ 0.05 M Tris/HCI. DH 8.0b’ Sue-Phe-NH-No’ ’ 0.05 M Tris/HCI, pH 8.0”’ 0.05 M TrislHCI. oH 7.6b’ Ac-Phe-NH-No ’ 0.05 M Tris/HCI, pH 8.0”) Sue-Ala-Ala-Phe-NH-No 0.05 M Tris/HCI, oH 8.0”’ Sue-Ala-Ala-Pro-*he-NH-No O.lM TrisA-lCI.lOmM CaCI,, pH 8.0”’ O.lM Tris/HCI,lOmM CaCI,, pH 7.8d’ B) Ester: Ac-Tro-Me 0.1 M Tris/HCI, pH 8.0”’ 0.1 M phosphate, pH 6.98”’

13.4f3.0

8.38+2.90x10J

36.0+_5.0 45.0

32.9k2.9 27.7kO.5

3.0+-l .0x1 o-5 4.3x1 o-5

1.20+-0.50~10~ 1.05x1 o6

1.0+0.1x10” 0.95x1 o-4

3.29+0.61x105 2.91+_.05x105

a) results of this work b), c), d) and e) from Reference [22], [12], [23] and [20] respectively.

[2] has already conditions. In water, highest

been studied

A comparative the catalytic

to some extent

in the literature

study as presented

efficiency

of a-chymotrypsin

for Sue-Ala-Ala-Pro-Phe-NH-Np,

within

Ac-Trp-Me

other experimental

the group

of substrates

other compounds

are relatively

of k,, and K, on w, in the case of the six substrates is shown

substrates

the substrate

used (Fig. la). Within the w, range

studied,

“superactive”

if measured

comparison

with “poor”

with literature

experimental shaped

conditions

NH-Np

Gr-Phe-NH-Np

with already

have been obtained

seems to be obvious

with increasing

In contrast, w,, while

for the “poor”

K,,O, generally

a-chymotrypsin

data obtained

within

on

is only (higher

k,

In those cases where

to mention

of K,, another

k,, vs. w, profile depends

in reverse

a

and Sue-Ala-Ala-Pro-Phe-NH-Np,

published

our

under slightly

different

that in all cases no bell

the w, range considered. difference

between

“poor”

and “good”

p-

(Fig. 1b). In the case of Suc-Ala-Ala-Pro-Phe-

(Fig. 1b.E) and Sue-Ala-Ala-Phe-NH-Np

as w, increases.

1.

and o-chymotrypsin

or with Ac-Trp-Me

system).

[14]. It is also worthwhile

k,, vs. w, curves substrates

aqueous

is possible,

If we now look at the w,-behaviour nitroanilde

p-nitroanilides

to a comparable

results are in good agreement

the

in Fig. 1. determined

with respect

while

(high K, and low k,,), see Table

As a first result, it is clear that the experimentally

micelles

used is

and Sue-Ala-Ala-Phe-NH-NP,

The dependence

in 50 mM AOT/isooctane

“poor”

under

here has never been done before.

(Fig. lb,D), p-nitroanilde

remains

K,,,

substrates

almost constant

as well as K,,w increase K,,,

clearly

(Fig. lb,A-C).

decreases If one

compares our data for Gr-Phe-NH-Np and 50 mM AOT/isooctane (Fig. 1b.A) with literature data for the same substrate and 100 mM AOTlheptane [I 41, then both calculated K,,, 1107

Vol. 178, No. 3, 1991

BIOCHEMICAL

AND BIOPHYSICAL

10

RESEARCH COMMUNICATIONS

20 wo

30

Fia.. a-Chymotrypsin catalyzed hydrolysis of Gr-Phe-NH-Np (A), Sue-Phe-NH-Np (B), AcPhe-NH-Np (C), Sue-Ala-Ala-Phe-NH-Np (D), Sue-Ala-Ala-Pro-Phe-NH-Np (E), and Ac-TrpMe (F) in 50 mM AOT/isooctane reverse micelles at 25 “C. W, dependency of (a) k, and (b) K,,, ( 0 ) or K,,w ( A ). The dashed lines correspond to the values obtained in water. The closed triangles in Fig. la correspond to the values obtained in 100mM AOT reverse micelles by Fletcher et al. [14]. The data for Sue-Ala-Ala-Pro-Phe-NH-Np are replotted from the work of Bru & Walde [12]. 1108

Vol.

178, No. 3, 1991

values

BIOCHEMICAL

are approximately

the same (50-130

differ by a factor of 2 approximately. decrease

as w, increases

enzyme-substrate From the present

water

comparative

six different

substrates,

characteristic

property

obvious

concerning

the substrate

of substrate

KS, the dissociation

layer of the micelles

that the enzyme

alone.

The shape

used.

However,

effect, it is necessary

detail at the experimental

procedure

Trp-Me,

the “micelle

in AOT

this possibility are independent

(b) A second

difference

For the “good” used, while necessary

rule out a possible

our data by looking

dependence

since k,, is relatively

has an influence

in more

applied

high (Table

on the enzyme

I),

activity,

the determined

in the different

concentration

Sue-Phe-NH-Np concentrations

between

(0.53

measurements.

of 2-200

nM has been

and Ac-Phe-NH-Np,

effect, we have checked concentration

and Ac-

used (data not shown).

concentration

enzyme

of activity on enzyme

to be

and Methods”).

at w, = 8. However,

on the procedure

an overall a-chymotrypsin

concentration

with

final conclusions

Sue-Ala-Ala-Phe-NH-Np

with Gr-Phe-NH-Np

in the case of Gr-Phe-NH-Np, to work with overall

micelles are not a

rather seems

was used (see “Materials

methodology

is the enzyme

substrates,

reverse

before drawing

to analyze

technique

Since it could well be that the mixing we have checked

[15,16].

of these profiles

had to be applied,

the injection

k,, and K, values

of the

which we have used in each case.

mix” technique

in the other cases,

constant

activity-w,, profiles

(a) First of all, in the case of Sue-Ala-Ala-Pro-Phe-NH-Np, while

therefore

both K,,O, and K,,,

kinetic study of a-chymotrypsin

of the enzyme

by the nature

on wJ; K,,Ov values

that K, can vary with w, due to partitioning

pool and surfactant

it seems

influenced

mM, depending

it is understandable

between

RESEARCH COMMUNICATIONS

In the case of Ac-Trp-Me,

(Fig. 1 b,F). Since K, contains

complex,

Of the substrate

AND BIOPHYSICAL

it was

1 and 4 uM. In order to

at different

w, values

PM, data not shown).

the

In all cases,

the dependence was linear. In addition, we have estimated for Sue-Ala-Ala-Pro-Phe-NH-W (one of the “good” substrates), k,, at w, = 8 with an overall a-chymotrypsin concentration of 1 PM in a stopped

flow apparatus.

The value

= 7 [12] or 1.8 s-’ at w, = 8 (Table chymotrypsin. not depend

Again,

in the present (c) DMSO

However,

of enzyme

with 6-34 nM a-

from measurements

for a-chymotrypsin

used (at least within

has been used as cosolvent

in AOT

reverse

the concentration

only in those cases where

Sue-Phe-NH-Np,

in all these cases,

Ac-Phe-NH-Np

a-chymotrypsin

as seen from Table

activity against

2 - where

Sue-Ala-Ala-Pro-Phe-NH-Np

a direct consequence of the presence K,, the effect is relatively moderate. (d) In a next step, we have looked discussed

with 0.9 s-’ at w, micelles range

do

applied

study).

was low (Gr-Phe-NH-Np, enough,

2), as obtained

the kinetic parameters

on the amount

of 0.7 s’ is comparable

that high k,

values

of the high salt (counterion)

results

of DMSO.

in AOT reverse

[17] have then shown

Although

aqueous

indeed

micelles

experienced that k,

DMSO

micelles.

on the enzyme that “superactivity”

increases

and Parrott

iS

k,, as well as [17] have recently

at low w, could simply be the result by the enzyme

in the micelle’s

for Gr-Phe-NH-Np/a-chymotrypsin

as the NaCl concentration 1109

in reverse

- it is unlikely

solubility

Interestingly

on the effect of DMSO

at the salt effect. Fletcher

concentration increases

“superactivity”

are reported

pool. The authors solution

shows

the substrate

and Ac-Trp-Me).

is increased.

Water

in an

Comparable

Vol.

BIOCHEMICAL

178, No. 3, 1991

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table 2 Effect of dimethvl sulfoxide (DMSO) on the activitv of a-chvmotrvosin. Substrate: Sue-Ala-Ala-Pro-Phe-NH-Np in aqueous solution (A) and in 50 mM AOTlisooctane reverse micelles (B). (A) in 0.1 M Tris/HCI, pH 8.0 [DMSO] (% v/v)

k,(s-‘1

K,(M)

37.4k0.8 41.4k0.8 47.1~1~2.2

3.21~0.05x10-5 521~0.26~10"

0-M

0

0

0.2 5.0

28 705

(6) in 50mM AOT/isooctane [DMSO] (% v/v)

reverse micelles, w, = 8 (0.1 M Tris/HCI, pH 8.0) kc&‘)

0-M

0

0

0.2

3.13ti.09x10-5

28

k,(M)

1.8OrtO.18

0.76?1O.llxlO-’

2.1mO.36

2.271kO.46xlO-'

results have been found in the case of Sue-Ala-Ala-Pro-Phe-NH-Np: Tris/HCI)

in the presence

absence

of salt. For Gr-Phe-NH-Np,

observations,

of 1 M NaCl or 2 M NaCl is 1.5 - 1.6 times

we therefore

the increase

conclude,

it has been discussed

due to a high local substrate between

negatively

indicate

charged

that the charge

activity in (charged) negatively NH-Np).

charged

micelles;

of cx-chymotrypsin

charged

substrates

changed

on going from bulk water

to reverse

[13]. It could also be that possible

of o-chymotrypsin

in reverse

the case of the p-nitroanildes chymotrypsin hydrophobic

can namely substrate

micelles

nitroanilde

substrates,

with respect

affinity for the AOT interface experimental

as already

k,

reverse mechanism

discussed

are

in the case of

binding

behavior

This may be of significance

p-nitroanilde

substrates

of IXring in the

(and K,). Such non-productive

the smaller the (artificial) substrate is, which values in water in the case of the “good” p-

to the “bad”

p-nitroanilides.

(see [I91 and the molar extinction

Part), one can imagine

(e.g.

used and on w,.

mode, with the p-nitroanilide

1181, lowering

binding is expected to be more significant, would partly explain the relatively high k,

individual

in the non-productive

solution,

(e.g. Ac-Phe-

“superactivity”

on the substrate

may be of importance.

used. In aqueous pocket

showed

the enzyme

was found for

steps of the enzyme

micelles,

changes

bind in a non-productive

binding

[l I]. Our data

substrates

rates between

role [9] and depend

On the other hand, it may well be that the individual trypsin

surfactant

as well as for uncharged

did not). The exchange

may well play an important

charged

may be

repulsions

is not the basic factor that determines

“superactivity”

not all negatively

Sue-Ala-Ala-Pro-Phe-NH-Np micelles

in the case of cr-chymotrypsin may arise from electrostatic

and negatively

of the substrate

at low

salt effect. which

(e.g. Gr-Phe-NH-Np)

Furthermore,

than in the

is by a factor of 2.6 [17]. From these

that “superactivity”

concentration substrate

reverse

higher

that the low k,, for Sue-Ala-Ala-Pro-Phe-NH-Np

w, can not simply be the result of a lacking Recently,

k,, at pH 8.0 (0.1 M

that non-productive 1110

binding

Since p-nitroaniline coefficients of amino

shows

in the acid p-nitroanildes

in

Vol.

178,

No.

3, 1991

is decreased indeed

in AOT

observed

Trp-Me,

BIOCHEMICAL

reverse

micelles,

for Gr-Phe-NH-Np,

the situation

the p-nitroanilides:

is different.

leading

BIOPHYSICAL

In water,

COMMUNICATIONS

in k,, (and K,). This is

and Ac-Phe-NH-Np

there are two main differences

substrate

step is different.

RESEARCH

to an increase

Sue-Phe-NH-Np

(1) Nonproductive

(2) the rate determining

AND

binding

is expected

For Ac-Trp-Me

(Fig. 1). For

AC-

with respect

to

to be unimportant,

deacylation

and

is rate determining

[20], K, being a function

of K, as well as k, and b, the acylation

and the deacylation

constants:

The possibility

step is changed

K, = K&/b).

case of ester substrates

that the rate determining

in reverse

micelles

observation

which

is now studied

by stopped

rate

in the

flow

spectrophotometry. Let us mention

another

enzymologists.

Recent

vs. w, profiles

(as obtained

not necessarily chymotrypsin,

investigations

represent

expected

- along

In conclusion, profiles Therefore, enzyme

thermodynamic

the present

we conclude alone

the effect of adding

comparative

for reverse

ruaosa

have shown

situations

that v,,

apparently

do

[21]. In the case of CL-

buffer solution

as substrates.

miCelIar

during

Changes

the reaction were

with

however

- as

(data not shown). study has shown

in AOT reverse that these profiles

but rather depend

from Candida

interest

with olive oil as substrate)

equilibrium

and Gr-Phe-NH-Np

the v,, vs. w, profiles

for cc-chymotrypsin

of a lipase

from measurements

we have studied

Sue-Ala-Ala-Pro-Phe-NH-Np

may be of general

micelles

that the shape

are sensitive

do not only reflect

on the substrate

of activity-w,

to the substrate

intrinsic

features

applied.

Of the

used.

ACKNOWLEDGMENTS The fruitful discussions with Pier Luigi Luisi, Marco LeSer are gratefully acknowledged.

Maestro,

Heribert

Watzke

and Martin

REFERENCES

111

El 171

PI PI

v41

[151

1161

Martinek, K., Levashov, A.V., Klyachko, N.L. & Berezin, I.V. (1977) Dokl. Akad. Nauk SSSR 236, 920-923. Menger, F. & Yamada, K. (1979) J. Am. Chem. Sot. 101, 6731-6734. Barbaric, S. & Luisi, P.L. (1981) J. Am. Chem. Sot. 103, 4239-4244. Luisi, P.L. & Magid, L. (1986) Crit. Rev. Biochem. 20, 409-474. Martinek, K., Levashov, A.V., Klyachko, N., Khmelnitski, Y.L. & Berezin, I.V. (1986) Eur. J. Biochem. 155, 453-468. Kabanov, A.V., Levashov, A.V., Klyachko, N.L., Namyotkin, S.N. & Pshezhetsky, A.N. (1988) J. Theor. Biol. 133, 327-343. Bru, R., Sanchez-Ferrer, A. & Garcia-Carmona, F. (1989) Biochem. J. 259, 355-361. Bianucci, M., Maestro, M. & Walde, P. (1990) Chem. Phys. 141, 273-283. Verhaert, R.M.D., Hilhorst, R., Vermue, M., Schaafsma, T.J. & Veeger, C. (1990) Eur. J. Biochem. 187, 59-72. Oldfield, C. (1990) Biochem. J. 272, 15-22. Ruckenstein, E. & Karpe, P. (1990) Biotechnol. Lett. 12, 241-246. Bru, R. & Walde, P. (1991) Eur. J. Biochem., in press. Walde, P., Peng, O., Fadnavis, N. W., Battistel, E. & Luisi, P. L. (1988) Eur. J. Biochem. 173, 401-409. Fletcher, P.D.I., Freedman, R.B., Mead, J., Oldfield, C. & Robinson, B.H. (1984) Colloids Surf. 10, 193-203. Bru, R., Sanchez-Ferrer, A. & Garcia-Carmona, F. (1990) Biochem. J. 268, 679-684. Leodidis, E.B. & Hatton, T.A. (1990) J. Phvs. Chem. 94, 641 I-6420. 1111

Vol.

178,

[17] [18] [19] [20] [21] [22] [23]

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Fletcher, P. D. I. & Parrott, D. (1988) J. Chem. Sot. Faradav Trans. 1. 1144. Fastrez, J. & Fersht, A. (1973) Biochemistrv 12, 1067-1074. Fletcher, P.D.I. (1986) J. Chem. Sot., Faradav Trans. 1 82, 2651-2664. Zerner, B., Bond, R.P.M. & Bender, M.L. (1964) J. Am. Chem. Sot. 86, Han, D., Walde, P. & Luisi, P.L. (1990) Biocatalvsis 4, 153-1. Erlanger, B.F., Edel, F. & Cooper, A.D. (1966) Arch. Biochem. BioDhvs. 210. DelMar, E.G., Largman, C., Brodrick, J.W. & Geokas, M.C. (1979) Anal. 99, 3-320.

1112

84(4), 1131-

3674-3679. 115, 206Biochem.