Enzyme-Catalyzed Reactions in Unfrozen, Noncellular Systems at

3 Enzyme-Catalyzed Reactions in Unfrozen,

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Noncellular Systems at Subzero Temperatures ANTHONY L. FINK Division of Natural Sciences, University of California, Santa Cruz, CA 95064 Methods used to determine the effects of both cryosolvents and low temperatures on the catalytic and structural properties of enzymes are detailed, along with representative results. Observed effects of subzero temperatures on the structure of enzymes include increased association of oligo mers, minor temperature-induced structural changes, and in most cases no detectable effects. Examples demonstrating the very good correspondence between the kinetics observed at subzero temperatures and those for the corresponding reaction techniques, are given. Systems illustrating changes in the rate-determining step and ΔΗ with a decrease in temperature, and the potential of cryoenzymology to provide details about individual intermediates and their interconversions during catalysis, are presented.

T V J " a n y b i o c h e m i c a l processes i n v o l v e v e r y r a p i d reactions a n d transient intermediates.

F r e q u e n t l y t h e r a p i d i t y of t h e r e a c t i o n causes m a j o r

t e c h n i c a l difficulties i n a s c e r t a i n i n g t h e details of t h e events o c c u r r i n g i n t h e process.

O n e approach

t o o v e r c o m e this i n h e r e n t p r o b l e m

u t i l i z e t h e fact t h a t most c h e m i c a l reactions are t e m p e r a t u r e

is t o

dependent.

T h i s r e l a t i o n s h i p is q u a n t i t a t i v e l y d e s c r i b e d b y t h e A r r h e n i u s e q u a t i o n , k = Ae~ */RT, E

w h e r e k represents t h e rate constant, A is a constant ( t h e

frequency factor), a n d E

a

is t h e energy of a c t i v a t i o n .

Consequently, b y

i n i t i a t i n g t h e r e a c t i o n at a sufficiently l o w t e m p e r a t u r e ,

interconversion

of t h e i n t e r m e d i a t e s m a y b e effectively s t o p p e d a n d t h e y m a y b e a c c u m u l a t e d a n d s t a b i l i z e d i n d i v i d u a l l y . A l t h o u g h t h e focus of this a r t i c l e i s o n the a p p l i c a t i o n of this l o w - t e m p e r a t u r e a p p r o a c h t o t h e s t u d y of e n z y m e catalysis, that i s , c r y o e n z y m o l o g y , t h e t e c h n i q u e is p o t e n t i a l l y o f m u c h wider biological application

(1,2,3).

0-8412-0484-5/79/33-180-035$5.00/0 © 1979 American Chemical Society Fennema; Proteins at Low Temperatures Advances in Chemistry; American Chemical Society: Washington, DC, 1979.

36

PROTEINS A T L O W

TEMPERATURES

B e c a u s e of t h e i r c a t a l y t i c f u n c t i o n , w h i c h p r o v i d e s

one

with

an

a d d i t i o n a l " h a n d l e " or p r o b e f o r d e t e c t i n g s t r u c t u r a l effects, e n z y m e s are p a r t i c u l a r l y w e l l s u i t e d f o r s t u d y i n g t h e b e h a v i o r of p r o t e i n s at

low

t e m p e r a t u r e s . I n this a r t i c l e t h e e m p h a s i s w i l l b e o n i l l u s t r a t i n g t h e effect of subzero t e m p e r a t u r e o n b o t h the s t r u c t u r a l a n d c a t a l y t i c p r o p e r t i e s of the e n z y m e s a n d the a b i l i t y to a c c u m u l a t e , s t a b i l i z e , a n d c h a r a c t e r i z e i n t e r m e d i a t e s o n the c a t a l y t i c r e a c t i o n p a t h w a y w i t h v e r y l o w t e m p e r a tures. B e c a u s e t h e l o w - t e m p e r a t u r e effects are i n t i m a t e l y r e l a t e d to t h e cryosolvents u s e d , a b r i e f d i s c u s s i o n of t h e effects of t h e o r g a n i c

cosol-

vents is i n c l u d e d . A l t h o u g h the examples u s e d to i l l u s t r a t e t h e p o i n t s of this a r t i c l e h a v e been d r a w n mostly from relatively simple hydrolytic enzymes studied i n t h e a u t h o r s l a b o r a t o r y , o v e r 25 different e n z y m e s h a v e c u r r e n t l y b e e n s u b j e c t e d to c r y o e n z y m o l o g i c a l i n v e s t i g a t i o n s at s e v e r a l laboratories

(6).

A l t h o u g h t h e first reports of e n z y m e - c a t a l y z e d reactions at s u b z e r o t e m p e r a t u r e s i n fluid aqueous o r g a n i c solvents a p p e a r e d 25 years ago, i t is o n l y since t h e p i o n e e r i n g studies of D o u z o u , b e g i n n i n g a b o u t 10 years ago, t h a t m o r e c o m p r e h e n s i v e investigations h a v e b e e n p e r f o r m e d .

Con-

s i d e r a b l e i m p e t u s to the d e v e l o p m e n t of t h e a p p r o a c h w a s g i v e n b y the elegant studies of D o u z o u a n d c o w o r k e r s (2)

o n the h o r s e r a d i s h p e r o x i -

dase system, w h i c h d e m o n s t r a t e d t h e f e a s i b i l i t y of " t e m p o r a l l y r e s o l v i n g " discrete e n z y m e - s u b s t r a t e i n t e r m e d i a t e s a n d the a c c u m u l a t i o n a n d c h a r a c t e r i z a t i o n of i n d i v i d u a l i n t e r m e d i a t e s . A enzyme

comprehensive

qualitative and

catalysis has b e e n

quantitative understanding

a l o n g - s t a n d i n g g o a l of

biochemistry.

of A

necessary r e q u i r e m e n t to a c h i e v e t h i s is a d e t a i l e d k n o w l e d g e of a l l t h e i n t e r m e d i a t e s a n d transition-state structures a l o n g t h e r e a c t i o n p a t h w a y . T h e t e c h n i q u e of c r y o e n z y m o l o g y has t h e p o t e n t i a l to p r o v i d e m u c h of this i n f o r m a t i o n . B e c a u s e several recent r e v i e w s c o v e r i n g v a r i o u s aspects of t h e t e c h n i q u e are a v a i l a b l e ( 2 , 4-9),

t h i s a r t i c l e w i l l e m p h a s i z e some

of t h e effects of s u b z e r o t e m p e r a t u r e s o b s e r v e d

on the catalytic and

s t r u c t u r a l p r o p e r t i e s of select e n z y m e s . C r y o e n z y m o l o g y u t i l i z e s the f o l l o w i n g features of e n z y m e c a t a l y s i s : t h e existence

o n the c a t a l y t i c r e a c t i o n p a t h w a y

of

several

enzyme-

substrate ( o r p r o d u c t ) i n t e r m e d i a t e species, t y p i c a l l y separated b y e n e r g y barriers w i t h enthalpies of a c t i v a t i o n of 7 to 20 k c a l m o l " ; a n d t h e f a c t 1

t h a t the energies

(enthalpies)

of a c t i v a t i o n f o r t h e i n d i v i d u a l steps i n

t h e o v e r a l l c a t a l y t i c p a t h w a y are u s u a l l y s i g n i f i c a n t l y different. F o r s u c h e l e m e n t a r y steps t e m p e r a t u r e s of — 1 0 0 ° C w i l l r e s u l t i n rate r e d u c t i o n s o n the o r d e r of 1 0

5

to 1 0

1 1

c o m p a r e d to those at 25 o r 3 7 ° C ( 5 ) .

t h e o r e t i c a l basis of c r y o e n z y m o l o g y w h e r e ( 5 , 7, 9, 1 0 ) .

The

has b e e n p r e s e n t e d i n d e t a i l else-

I f the r e a c t i o n is i n i t i a t e d b y m i x i n g e n z y m e a n d

substrate at a s u i t a b l y l o w t e m p e r a t u r e , o n l y the i n i t i a l n o n c o v a l e n t E S

Fennema; Proteins at Low Temperatures Advances in Chemistry; American Chemical Society: Washington, DC, 1979.

3.

Enzyme-Catalyzed

FINK

37

Reactions

c o m p l e x w i l l b e f o r m e d . T h i s is a r e s u l t of there b e i n g insufficient e n e r g y a v a i l a b l e to o v e r c o m e t h e e n e r g y b a r r i e r to t h e s u b s e q u e n t i n t e r m e d i a t e . If t h e t e m p e r a t u r e is g r a d u a l l y i n c r e a s e d , a p o i n t w i l l b e r e a c h e d w h e r e E S is t r a n s f o r m e d i n t o t h e f o l l o w i n g i n t e r m e d i a t e ( I i ) .

Maintenance or

r e d u c t i o n of the t e m p e r a t u r e w i l l a l l o w this i n t e r m e d i a t e to b e t r a p p e d . F u r t h e r r a i s i n g of the t e m p e r a t u r e w i l l r e s u l t i n t r a n s f o r m a t i o n of I i to a s u b s e q u e n t i n t e r m e d i a t e ( I ) , a n d so o n u n t i l t h e o v e r a l l r a t e - l i m i t i n g 2

step is r e a c h e d , at w h i c h p o i n t t u r n o v e r w i l l o c c u r .

A n y intermediate

w h o s e rate of f o r m a t i o n is m o r e r a p i d t h a n its rate of b r e a k d o w n m a y b e a c c u m u l a t e d i n this m a n n e r . T h e m a j o r advantages

u n i q u e to c r y o e n z y m o l o g y

stem f r o m

the

p o t e n t i a l to a c c u m u l a t e essentially a l l of t h e e n z y m e i n t h e f o r m of a p a r t i c u l a r i n t e r m e d i a t e . T h e l a r g e rate r e d u c t i o n s a l l o w t h e most specific substrates to b e u s e d a n d h e n c e p r o v i d e t h e most accurate m o d e l f o r t h e i n v i v o c a t a l y z e d reactions. V i r t u a l l y a l l t h e s t a n d a r d c h e m i c a l a n d b i o physical techniques used i n studying proteins a n d enzymes under n o r m a l c o n d i t i o n s m a y b e u s e d at s u b z e r o t e m p e r a t u r e s .

T h e m a i n limitations

of t h e t e c h n i q u e are t h e necessity to use a q u e o u s o r g a n i c

cryosolvent

systems to p r e v e n t the i n h e r e n t r a t e - l i m i t i n g e n z y m e - s u b s t r a t e d i f f u s i o n of f r o z e n solutions, a n d the p o s s i b i l i t y t h a t t h e p o t e n t i a l - e n e r g y

surface

for the reaction m a y be such that conditions i n w h i c h a n intermediate accumulates cannot be attained. The

general approach

laboratory for

t h a t has b e e n d e v e l o p e d

cryoenzymological

i n the

author's

studies i n v o l v e s t h e f o l l o w i n g :

(1)

S e l e c t i o n of a s u i t a b l e c r y o s o l v e n t a n d d e m o n s t r a t i o n t h a t i t causes no adverse

effects o n e i t h e r the c a t a l y t i c or s t r u c t u r a l p r o p e r t i e s of

enzyme

(e.g., 11);

perature b y enzyme,

(2)

T h e d e t e c t i o n of i n t e r m e d i a t e s at subzero t e m -

m o n i t o r i n g s u i t a b l e s p e c t r a l p r o b e s i n the

a n d the k i n e t i c a n d t h e r m o d y n a m i c

i n t e r m e d i a t e s (e.g., 12);

the

substrate

c h a r a c t e r i z a t i o n of

or the

a n d ( 3 ) T h e a c q u i s i t i o n of s t r u c t u r a l l y r e l a t e d

d a t a c o n c e r n i n g t h e t r a p p e d i n t e r m e d i a t e s , b y t e c h n i q u e s s u c h as X - r a y diffraction a n d n m r (nuclear magnetic resonance)

(e.g., J 3 ) .

Details

c o n c e r n i n g the e x p e r i m e n t a l m e t h o d o l o g y f o r e x p e r i m e n t s u s i n g p r o t e i n s at subzero temperatures m a y b e f o u n d i n t h e f o l l o w i n g references: 5, 6, 8 , 9 , I I , 12.

Cryosolvents A t present the most versatile m e t h o d of o b t a i n i n g a fluid s o l u t i o n of p r o t e i n at s u b z e r o

temperatures

seems to b e

t h a t of

using aqueous

o r g a n i c solvent m i x t u r e s . A n u m b e r of s u c h solvent systems are k n o w n w i t h f r e e z i n g p o i n t s i n t h e v i c i n i t y of — 1 0 0 ° C . 60-80%

of the o r g a n i c c o m p o n e n t .

These usually contain

T h e most u s e f u l cryosolvents


are

38

PROTEINS A T L O W T E M P E R A T U R E S

Table I.

Physical-Chemical Properties of Freez-

Cryosolvents"

pH*

at

™9 . Dielectric Constant at ζ ° , 2{ Point pH 4.75 (°C) 0° -50° -100°CAcetate* 2 (

Cryosolvent D i m e t h y l sulfoxide 50% 65% Methanol 50% 70% 80%

s.c/ s.c.

84 79

105 98

132 124

5.40 6.7

68 57 49

— 71 66

— — 88

5.45 5.95 6.1

67 59

88 79

—

5.60 6.20

-100

56

70

86

6.9

-44

72

93

—

5.25

d

-49 -85 -100

Ethylene glycolmethanol 40%:20% 10%:60%

-71 s.c.

e

T 7

.

Viscosity (cps)

38 @ 48 @

-40° -60°

420 @ - 6 0 ° 76 @ -60°

Dimethyl formamide 80% Ethylene glycol 50%

125 @

-40°

Based on Ref. 29 and 80. The apparent p H of the mixed aqueous organic solvent when the aqueous com ponent was p H 4.75. s.c. = super cooled. Fluid to < - 9 0 ° C . β

b

0

d

those b a s e d o n m e t h a n o l , e t h a n o l , d i m e t h y l s u l f o x i d e , d i m e t h y l f o r m a m i d e , a n d e t h y l e n e g l y c o l - m e t h a n o l . E x t e n s i v e p h y s i c a l - c h e m i c a l studies of these solvents h a v e b e e n c a r r i e d out b y D o u z o u a n d c o w o r k e r s 15).

(14,

I n g e n e r a l the v i s c o s i t y , the p H * ( t h e a p p a r e n t p H i n the c r y o s o l

v e n t ) , a n d the d i e l e c t r i c constant increase w i t h d e c r e a s i n g t e m p e r a t u r e . Representative data for some c o m m o n

c r y o s o l v e n t systems are

shown

i n T a b l e I. Cosolvent

Effects

Structural.

E x p e r i m e n t a l l y , one

of t h e m o s t n o t i c e a b l e

features

c a u s e d b y the presence of o r g a n i c cosolvents o n p r o t e i n s t r u c t u r e is t h e decrease i n the t e m p e r a t u r e at w h i c h d e n a t u r a t i o n occurs.

Interestingly,

f o r most of the e n z y m e s t h u s f a r s t u d i e d , at the 60 to 8 0 %

cosolvent

c o n c e n t r a t i o n r e q u i r e d i n t h e cryosolvents, t h e m i d p o i n t of the t h e r m a l d e n a t u r a t i o n t r a n s i t i o n is u s u a l l y i n t h e — 1 0 ° to + 1 0 ° C r a n g e i n the p H * r e g i o n of c a t a l y t i c a c t i v i t y . T h i s means t h a t i n s u c h solutions t h e enzymes

are u s u a l l y d e n a t u r e d at r o o m t e m p e r a t u r e , b u t are i n t h e i r


3.

Enzyme-Catalyzed

FINK

39

Reactions

n a t i v e states at subzero t e m p e r a t u r e s . T h e effect of i n c r e a s i n g c o s o l v e n t c o n c e n t r a t i o n o n the m i d p o i n t of t h e t h e r m a l d e n a t u r a t i o n of r i b o n u c l e a s e A is g i v e n i n T a b l e I I . A s t h e c o n c e n t r a t i o n of e t h a n o l increases the T m o v e s to i n c r e a s i n g l y l o w e r temperatures. typical

m

T h i s b e h a v i o r seems q u i t e

(3).

I n g e n e r a l the effect of cosolvent o n the s t r u c t u r e of a p r o t e i n m a y b e d e t e r m i n e d b y e x a m i n i n g t h e i n t r i n s i c u v s p e c t r a l p r o p e r t i e s of t h e p r o t e i n as the cosolvent c o n c e n t r a t i o n increases.

Smooth monotonie

l i n e a r c u r v e s reflect solvent effects o n the exposed

aromatic

or

residues,

w h e r e a s s h a r p breaks i n s u c h plots o c c u r i f a s t r u c t u r a l p e r t u r b a t i o n occurs

(Figure 1).

F o r e x a m p l e , i f the effect of i n c r e a s i n g d i m e t h y l

s u l f o x i d e c o n c e n t r a t i o n o n β-galactosidase is e x a m i n e d at 0 ° C , p H * 7.0, b y either

fluorescence

or a b s o r p t i o n spectroscopy,

smooth

curves

are

o b s e r v e d u p to a n d i n c l u d i n g 5 0 % d i m e t h y l s u l f o x i d e , w h e r e a s a sharp b r e a k i n these curves b e c o m e s a p p a r e n t at h i g h e r c o n c e n t r a t i o n s 1)

(IS).

T h e intrinsic

fluorescence,

(Figure

absorbance, a n d circular dichroism

s p e c t r a are most c o n v e n i e n t f o r s u c h studies (11, 16, 17, 18).

Additional

t e c h n i q u e s that c a n b e u s e d f o r this p u r p o s e i n c l u d e p r o t o n n m r [e.g., the n m r spectrum for subtilisin i n 6 5 % r i b o n u c l e a s e A i n 50 o r 7 0 % aqueous

solution under otherwise

unpublished observations)]

d i m e t h y l sulfoxide, a n d

for

m e t h a n o l , is essentially the same as i n similar conditions

(Fink

and Kar,

a n d X - r a y c r y s t a l l o g r a p h y , vide infra

(13).

B a s e d o n the a c c u m u l a t e d d a t a f o r s o m e 15 e n z y m e s e x a m i n e d i n t h e a u t h o r s l a b o r a t o r y , w e are a b l e to state t h a t for e a c h e n z y m e i n v e s t i g a t e d t h e r e is c o n s i d e r a b l e e v i d e n c e to i n d i c a t e t h a t f o r s u i t a b l e cryosolvents the cosolvent has l i t t l e detectable effect o n the s t r u c t u r e at a p p r o p r i a t e l y l o w t e m p e r a t u r e s a n d d e f i n i t e l y causes n o m a j o r changes i n t h e c o n f o r m a t i o n of t h e p r o t e i n . monomelic

T h e s e statements a p p l y to o l i g o m e r i c as w e l l as

enzymes,

for

example,

β-galactosidase, β-glucosidase,

Table II. Effect of Cosolvents on the Midpoint ( T ) Reversible Native *± Denatured Transition of Ribonuclease A at p H * 2.8 m

of

a

Solvent

T

(v/v)

re)

m

Aqueous

40.0 ±

0.5

Ethanol 15% 30% 45% 60%

38.5 31.5 20.5 9.0

0.5 0.5 0.5 0.5

Methanol 70%

15.0 db 1.0

"Determined by absorbance change at 286 nm

± ± ± ±

(5).


the

and

40


J Ο

I 10

I 20

1 30

1 40

ί50

%DMS0( / ) V

v

Biochemistry

Figure 1. The effect of dimethyl sulfoxide on the structure of β-gàlactosidase, as monitored by: (A) the intrinsic fluorescence; (B) the intrinsic uv absorption. Conditions: 0°C, pH* 7.0, excitation at 285 nm, χ = 300 nm, Δ = 290 nm, 0=280. (IS) g l u c o s e oxidase.

H o w e v e r , as n o t e d b e l o w , t e m p e r a t u r e - i n d u c e d

struc

t u r a l i s o m e r i z a t i o n s h a v e b e e n seen i n a f e w cases. T h e a b o v e statements a p p l y o n l y t o those e n z y m e - c r y o s o l v e n t

systems i n w h i c h t h e e n z y m e i s

a c t i v e . I t m u s t also b e n o t e d t h a t f o r m a n y e n z y m e s o n l y one o f s e v e r a l c r y o s o l v e n t s e x a m i n e d has b e e n f o u n d t o b e satisfactory.

F o r example,

m a n y e n z y m e s are p a r t i c u l a r l y sensitive t o m e t h a n o l . Catalytic.

T y p i c a l l y t h e effects o f t h e c r y o s o l v e n t o n t h e c a t a l y t i c

p a r a m e t e r s m a y b e d e t e r m i n e d b y e x a m i n i n g t h e effect o f i n c r e a s i n g concentration of t h e cosolvent

o n k i n e t i c p a r a m e t e r s , s u c h asfc at>^m> C

a n d rate constants c o r r e s p o n d i n g t o m o r e e l e m e n t a r y steps i n t h e o v e r a l l r e a c t i o n s u c h as fc yiation, fcdegaiactosyiation, as w e l l as i n h i b i t i o n aC

constants

( e . g . , K i ) a n d p H - r a t e profiles. I n g e n e r a l , o n l y t h e e x p e c t e d effects o n


3.

Enzyme-Catalyzed

FINK

fccat a n d p H d e p e n d e n c e

41

Reactions

are f o u n d (11,

H o w e v e r , for

16, 17, 18).

a n d Κι one f r e q u e n t l y finds a n e x p o n e n t i a l increase as t h e c o n c e n t r a t i o n increases

( I I , 17).

K

m

cosolvent

A l t h o u g h this w a s a t t r i b u t e d to

a

c o m b i n a t i o n of d i e l e c t r i c a n d c o m p e t i t i v e i n h i b i t i o n effects i n e a r l i e r p u b l i c a t i o n s (16, effect (18)

i t is m o r e l i k e l y d u e to a h y d r o p h o b i c

17, 19, 20),

i n w h i c h h y d r o p h o b i c substrates p a r t i t i o n m o r e f a v o r a b l y to

t h e b u l k c r y o s o l v e n t t h a n to the a c t i v e site, c o m p a r e d w i t h b u l k w a t e r as t h e solvent.

Effects of Low

Temperature

on

Structure

S i n c e m o s t p r o t e i n s are i n a d e l i c a t e l y b a l a n c e d e q u i l i b r i u m w i t h solvent w a t e r , l a r g e concentrations of o r g a n i c solvents m i g h t be

expected

to cause s u b s t a n t i a l p e r t u r b a t i o n s . W e a t t r i b u t e t h e d e m o n s t r a t e d sta b i l i t y of e n z y m e s a n d p r o t e i n s i n h i g h concentrations of o r g a n i c solvent at s u b z e r o t e m p e r a t u r e s to t h e f o l l o w i n g m a i n features: t h e increase of d i e l e c t r i c constant w i t h d e c r e a s i n g t e m p e r a t u r e hydrogen-bond

strength w i t h

t h e increase

(21);

decreasing temperature

of

opposing

(22);

effects o n h y d r o p h o b i c i n t e r a c t i o n s w h i c h t e n d to c a n c e l as t h e t e m p e r a t u r e d r o p s ( 5 ) ; a n d the " t r a p p i n g " of t h e n a t i v e state d u e to the h i g h a c t i v a t i o n energy for d e n a t u r a t i o n , as w e l l as the s t a b i l i z i n g influence of substrate or other b o u n d l i g a n d s ( 5 ) . B e c a u s e o u r c u r r e n t u n d e r s t a n d i n g of the i n t e r a c t i o n s b e t w e e n w a t e r a n d p r o t e i n s is r e l a t i v e l y p o o r i t is not p o s s i b l e to g i v e a d e t a i l e d , q u a n t i t a t i v e e x p l a n a t i o n of t h e a d d i t i o n a l effects a n d c o m p l i c a t i o n s c a u s e d b y a n a d d e d o r g a n i c cosolvent

a n d subzero temperatures.

I t is p o s s i b l e ,

h o w e v e r , to m a k e some q u a l i t a t i v e estimates of the a n t i c i p a t e d effects a n d to r a t i o n a l i z e to some extent t h e o b s e r v e d conditions.

stability under

such

A d v e r s e effects o n t h e e n z y m e structure c o u l d arise f r o m

d e s t a b i l i z a t i o n of t h e n a t i v e c o n f o r m a t i o n

or f r o m

non-native noncatalytically active (denatured) the f o r m e r is of p r i m e interest.

A

more

s t a b i l i z a t i o n of

a

state. I n t h i s d i s c u s s i o n

d e t a i l e d d i s c u s s i o n of

the

a n t i c i p a t e d effects of l o w t e m p e r a t u r e s a n d o r g a n i c cosolvents o n p r o t e i n structure has b e e n g i v e n elsewhere

(5).

W e h a v e o b s e r v e d three types of effects o n t h e structures of e n z y m e s as the t e m p e r a t u r e is l o w e r e d i n c r y o s o l v e n t s : ( 1 ) no a p p a r e n t c h a n g e i n the protein conformation, mobility

of

the

surface

usually marked by

w i t h the possible

side

chains;

(2)

exception

of

decreased

conformational transitions,

l i t t l e effect o n the c a t a l y t i c p r o p e r t i e s ; a n d

(3)

i n c r e a s e d association of s u b u n i t s . I n most cases n o d e t e c t a b l e effects of d e c r e a s i n g t e m p e r a t u r e o n t h e enzyme's structure h a v e b e e n b y such procedures

as m o n i t o r i n g t h e i n t r i n s i c

i n t r i n s i c v i s i b l e absorbance

i n t h e case of

flavin

fluorescence enzymes

detected (16),

or

(Fink

and


42


A h m e d , i n p r e p a r a t i o n ) , or i n t h e p r o t o n n m r s p e c t r u m (e.g., s u b t i l i s i n ) ( F i n k a n d Tsai, i n preparation). I n addition, the structure of crystalline elastase has b e e n c o m p a r e d a t 25 ° C i n aqueous

solution w i t h that at

— 5 5 ° C i n 7 0 % m e t h a n o l . E x c e p t f o r changes o n t h e p r o t e i n s surface, due t o decreased

m o b i l i t y o f surface s i d e c h a i n s , t h e structures a r e

identical (13). W h e n β-galactosidase i s c o o l e d i n e i t h e r d i m e t h y l s u l f o x i d e , m e t h a n o l , o r e t h y l e n e g l y c o l - m e t h a n o l cryosolvents, u n d e r c e r t a i n c o n d i t i o n s of e n z y m e c o n c e n t r a t i o n a n d i o n i c e n v i r o n m e n t , a n increase i n t h e u v a b s o r p t i o n o f the e n z y m e is n o t e d at t e m p e r a t u r e s b e l o w — 3 0 ° C . T h e k i n e t i c s o f this r e a c t i o n are

first-order

a n d t h e rates are s i m i l a r i n a l l

t h r e e cryosolvents ( I S ) . T h e c a t a l y t i c a c t i v i t y r e m a i n s u n c h a n g e d d u r i n g t h e progress o f t h i s r e a c t i o n . T h e increase i n a b s o r b a n c e is a t t r i b u t e d t o a

temperature-induced conformational

change

i n t h e /?-galactosidase

s u b u n i t s , w h i c h does n o t affect the active-site o r c a t a l y t i c a c t i v i t y . A similar phenomenon

has b e e n n o t e d also w i t h a - c h y m o t r y p s i n

(Fink

and Good, unpublished observations). B e c a u s e m a n y m e t a b o l i c a l l y significant e n z y m e s are o l i g o m e r i c i t is of p a r t i c u l a r interest t o d e t e r m i n e the effects o f cryosolvents a n d s u b z e r o t e m p e r a t u r e s o n t h e state o f association o f m u l t i s u b u n i t e n z y m e s . A t appropriately l o w temperatures ( b e l o w the denaturation transition) w e find n o e v i d e n c e o f s u b u n i t d i s s o c i a t i o n i n t h e case o f β-galactosidase i n a q u e o u s d i m e t h y l s u l f o x i d e ( 1 8 ) , glucose oxidase i n aqueous m e t h a n o l ethylene glycol ( F i n k a n d A h m e d , i n preparation), a n d liver alcohol dehydrogenase formamide

(LADH)

i n aqueous

d i m e t h y l sulfoxide o r d i m e t h y l

( F i n k a n d Geeves, u n p u b l i s h e d observations).

However i n

the case of β-glucosidase ( a l m o n d ) t h e t e t r a m e r i c e n z y m e dissociates a t 25°C i n 5 0 %

d i m e t h y l s u l f o x i d e , as d e t e r m i n e d b y loss o f c a t a l y t i c

activity, a n d gel-filtration chromatography

(24).

A s t h e temperature

decreases the degree o f association increases: f r o m 1 3 % a t 2 5 ° C , p H * 7.0 to 1 0 0 % a t - 1 7 ° C (see T a b l e I I I ) (24). C o n f i r m a t i o n t h a t i n a c t i v e m o n o m e r s p r e d o m i n a t e at h i g h e r temperatures i n this c r y o s o l v e n t comes Table I I I .

T h e Effect of Temperature on the Dissociation of β-Glucosidase i n 5 0 % Dimethyl Sulfoxide 0

Solvent

(v/v)

Aqueous 5 0 % D i m e t h y l sulfoxide

Temperature 25 25 0 -17

(°C)

%

Tetramer

b

100 13 ± 5 76 ± 7 100

pH*7.0. * Using exclusion chromatography on CPG-glycophase porous glass beads, 100 X 1 cm column . Biochemistry e


3.

Enzyme-Catalyzed

FINK

f r o m measurements

43

Reactions

of t h e a m o u n t

of p - n i t r o p h e n o l l i b e r a t e d i n

r e a c t i o n w i t h p - n i t r o p h e n y l - / ? - D - g l u c o s i d e (24, 29), active

tetramer

-5°C

at

— 25 ° C

and

below,

and

the

w h i c h indicate 100%

only

30%

tetramer

at

(24).

Effects of Low

Temperature

on the Kate of

Catalysis

T h e effect of t e m p e r a t u r e o n t h e c a t a l y t i c r e a c t i o n is m o s t d e t e r m i n e d w i t h A r r h e n i u s plots. t h e r a t e of

individual

steps

easily

S u c h plots of the t u r n o v e r rate, or of

i n the

catalytic reaction

^-deglycosylation ) c a n b e v e r y u s e f u l i n c o m p a r i s o n s

(e.g., fcacyiation,

between the catalytic

r e a c t i o n i n the c r y o s o l v e n t at subzero t e m p e r a t u r e a n d t h e r e a c t i o n u n d e r n o r m a l conditions. T y p i c a l effects of t e m p e r a t u r e o n catalysis w i l l b e i l l u s t r a t e d w i t h t h e r e a c t i o n of β-galactosidase a n d o- a n d p - n i t r o p h e n y l galactosides T h e m i n i m u m reaction pathway m a y be represented b y

(18).

Scheme

1 in

w h i c h E S is t h e n o n c o v a l e n t M i c h a e l i s c o m p l e x , a n d E G represents a n enzyme-galactose intermediate (25,

26).

Scheme 1 E + S « ± E S - * E G - » E + + Pi From

k i n e t i c considerations

g a l a c t o s i d e substrate, a n d k

3

G

is r a t e - l i m i t i n g f o r

k

2

the

p-nitrophenyl

is r a t e - d e t e r m i n i n g f o r t h e o-nitro-substrate

at 2 5 ° C , a q u e o u s s o l u t i o n (27).

F o r b o t h substrates t h e A r r h e n i u s p l o t s

at s u b z e r o t e m p e r a t u r e s w e r e f o u n d to b e l i n e a r ( F i g u r e s 2 A a n d

B)

i n d i c a t i n g n o c h a n g e i n the r a t e - l i m i t i n g step, a n d no significant s t r u c t u r a l p e r t u r b a t i o n of

the protein

substrates w o u l d b e e x p e c t e d

(18).

The

affinity of

enzymes

to increase w i t h d e c r e a s i n g

for

their

temperature

b e c a u s e t h e r e a c t i o n i n t h e f o r w a r d d i r e c t i o n is d i f f u s i o n c o n t r o l l e d , a n d h e n c e has a l o w e n e r g y of a c t i v a t i o n c o m p a r e d to t h e d i s s o c i a t i o n r e a c tion.

Because

the

enzyme-substrate

c o m p o n e n t of the K

m

dissociation

constant

is a

major

t e r m , o n e w o u l d e x p e c t t h a t K , i n m a n y cases,

w o u l d also decrease w i t h d e c r e a s i n g t e m p e r a t u r e .

m

T h i s is i n d e e d

s e r v e d , for e x a m p l e , i n t h e a b o v e - m e n t i o n e d β-galactosidase case

ob

(18).

W e h a v e n o t i c e d for s e v e r a l systems e x a m i n e d t h a t extrapolations of t h e A r r h e n i u s plots f o r i n t e r m e d i a t e f o r m a t i o n to a m b i e n t t e m p e r a t u r e s suggest that the rates of i n t e r m e d i a t e i n t e r c o n v e r s i o n w i l l b e of t h e same o r d e r of m a g n i t u d e at t h e t e m p e r a t u r e s at w h i c h the e n z y m e s n o r m a l l y operate

(12).

T h e c o n v e r g e n c e o f A r r h e n i u s p l o t s is s h o w n

for

the

p a p a i n - c a t a l y z e d h y d r o l y s i s of N - c a r b o b e n z o x y - L - l y s i n e p - n i t r o a n i l i d e i n Figure 3

(28).


44


/

{

T

(°Κ'

1

Χ ΙΟ ) 3

Biochemistry

Figure 2A. Arrhenius plot for the reaction of β-galactosidase with p-nitrophenyl^-O-galactoside in 60% aqueous dimethyl sulfoxide, pH* 7.0. Ratelimiting step is degalactosylation. (18)


3.

FINK

Enzyme-Catalyzed

3.8

3.9

45

Reactions

4.0

4.1 l

/

T

4.2

(°K

4.3

4.4

4.5

4.6

X I0 ) 3

Biochemistry

Figure 2B. Arrhenius plot for the reaction of β-galactosidase with p-nifrophenyl-fi-O-gahctoside in 60% dimethyl sulfoxide, pH* 7.0. Rate-determining step is formation of the galactose-enzyme intermediate. (IS)


46

PROTEINS A T L O W T E M P E R A T U R E S 0

-10

3.7

3.8

-20

3.9

-30

40

4.1

-40

4.2

°C

4.3

'/j X I 0 ° K " ' + 3

Figure 3. Arrhenius plots for the rates of formation of intermediates in the reaction of papain with N -carbobenzoxy-i.-lysine p-nitroanilide. The solvent was 60% dimethyl sulfoxide, pH* 6.1, E = 3.0 Χ ΙΟ M, S = 3.0 X 10 M (32). Reactions 2 and 3 correspond to enzyme isomerization (32), Reaction 4 corresponds to the formation of the tetrahedral intermediate (32). a

6

0

Table I V . Enzyme Elastase Trypsin Papain Lysozyme β-Glucosidase Subtilisin Ribonuclease A

Substrate* AcAlaProAlaPNA CBZ-AlaPNP CBZ-LysPNP CBZ-LysPNA CBZ-LysPNP NAG PNPGlu BzPheValArgPNA 2'3'CMP 6

s

0

Conditions Necessary Cryosolvent^ 70% 70% 65% 60% 60% 70% 50% 70% 50%

MeOH MeOH DMSO DMSO DMSO MeOH DMSO MeOH MeOH

P N A = p-nitroanilide; P N P = p-nitrophenyl ester; N A G e = hexamer of Nacetyl-glucosamine; C B Z = 2V -carbobenzoxy-; G l u = glucoside; Bz = benzoyl; 2'3' cyclic cytidine monophosphate. e

a


3.

Enzyme-Catalyzed

FINK

Conditions

47

Reactions

Necessary to Stop

Turnover

T h e t e m p e r a t u r e necessary t o effectively stop t h e t u r n o v e r r e a c t i o n varies g r e a t l y d e p e n d i n g o n t h e p a r t i c u l a r e n z y m e - s u b s t r a t e s y s t e m , t h e cryosolvent, t h e p H * , a n d t h e enzyme concentration.

I n some cases,

w h e r e t h e e n e r g y o f a c t i v a t i o n o f t h e r a t e - d e t e r m i n i n g step i s l a r g e , as i n t h e glycosidases, t e m p e r a t u r e s as h i g h as — 2 0 ° C a t t h e p H o p t i m u m are sufficient (24, 29, 30, 31). B y c h o o s i n g a n o n o p t i m a l p H * t h e t u r n o v e r r e a c t i o n f o r m o s t e n z y m e s c a n b e b r o u g h t t o a h a l t a t — 50° C o r higher.

Some representative data are given i n T a b l e I V . I n general the

o f t e n l a r g e effect o f t h e cosolvent o n K

m

means that substantial a d d i

t i o n a l r a t e r e d u c t i o n s c a n b e effected b y w o r k i n g w i t h n o n s a t u r a t i n g substrate c o n c e n t r a t i o n s ( 5 ) . F o r reactions i n w h i c h a c h r o m o p h o r i c p r o d u c t i s r e l e a s e d p a r t w a y t h r o u g h t h e c a t a l y t i c r e a c t i o n — f o r e x a m p l e , i n protease c a t a l y s i s w h e r e a n a c y l - e n z y m e i n t e r m e d i a t e i s f o r m e d — i t i s o f t e n p o s s i b l e t o see t h e release of a n e q u i v a l e n t a m o u n t o f t h e p r o d u c t f o r m e d c o n c u r r e n t l y t o t h e f o r m a t i o n of t h e e n z y m e - s u b s t r a t e i n t e r m e d i a t e . F o r e x a m p l e , as s h o w n i n F i g u r e 4, i n t h e r e a c t i o n o f p a p a i n w i t h N - c a r b o b e n z o x y - L a

l y s i n e p - n i t r o p h e n y l ester i n 6 0 % d i m e t h y l s u l f o x i d e at p H * 6.1 ( t h e p H o p t i m u m ) a stoichiometric "burst" of p-nitrophenol is observed at t e m p e r a t u r e s b e l o w — 40 ° C as t h e a c y l - e n z y m e i s f o r m e d , f o l l o w e d b y n o f u r t h e r release o f p - n i t r o p h e n o l , i n d i c a t i n g t h a t n o t u r n o v e r i s o c c u r r i n g (12).

to Effectively Stop T u r n o v e r pH*

Ε · (M)

9.2 7.2 7.7 6.1 7.0 5.8 7.1 9.5 2.1

3.8 X 1 0 1.3 Χ 10" 2.8 Χ 10" 3.0 X 1 0 1.4 χ 10" 3.6 Χ 10" 4.7 Χ 10" 2.4 X 10" 8.0 Χ 10"

'MeOH

Temperature e

5

5

e

5

7

5

e

5

5.0 3.2 1.0 3.0 1.0 2.0 1.0 6.9 3.2

Χ 10" Χ 10" X 10" X10 X 10" Χ 10" X 10" Χ 10" Χ 10"

4

3

3

s

3

5

2

5

4

-60 -40 -45 -15 -70 -20 -20 -21 -40

methanol; D M S O = dimethyl sulfoxide.

American Chemical Society Library 1155 16th St. N. W.

Fennema; Proteins at Low Temperatures Advances in Chemistry; Washington. American Chemical D. C. Society: 20036Washington, DC, 1979.

(°C)


48

1.0

ο χ Ο 5

0

8

0.4 0.2

Oh 0

100

200

300

400

500

600

700

Time (secs) Figure 4. The acylation of papain by N -carbobenzoxy-^lysine ester under nonturnover conditions in 60% dimethyl sulfoxide, (See Ref. 11) a

Correspondence

Between Subzero and Normal

\>-nitrophenyl —70°C, pH* 6.8.

Temperature

Data

I n order for cryoenzymology experiments t o p r o v i d e mechanistically significant i n f o r m a t i o n , i t i s i m p o r t a n t t h a t t h e r e a c t i o n a t l o w t e m p e r a t u r e b e analogous

t o t h a t u n d e r n o r m a l c o n d i t i o n s , that i s , a q u e o u s

solution and ambient temperature.

O n e definitive w a y t o demonstrate

this i s t o t a k e t h e k i n e t i c d a t a f o r a p a r t i c u l a r i n t e r m e d i a t e a t s u b z e r o t e m p e r a t u r e s a n d t o c a l c u l a t e t h e e x p e c t e d rates o f f o r m a t i o n a n d b r e a k d o w n under normal conditions. m e n t (e.g., b y s t o p p e d - f l o w

I f these rates are accessible t o m e a s u r e

techniques)

then comparison can b e made

between the low-temperature and high-temperature data. I n t h e reaction of p a p a i n w i t h 2V -carbobenzoxy-L-lysine p-nitroa

a n i l i d e , s t u d i e d at s u b z e r o t e m p e r a t u r e s i n 6 0 % d i m e t h y l s u l f o x i d e , t h e slowest step p r e c e d i n g t h e o v e r a l l r a t e - l i m i t i n g step corresponds

to t h e

f o r m a t i o n o f a t e t r a h e d r a l i n t e r m e d i a t e (28). T h e r e a c t i o n appears as a n increase i n a b s o r b a n c e i n the 3 6 0 - 4 0 0 n m r e g i o n ( F i g u r e 5 ) , a n d at h i g h p H * ( a b o v e 9 ) essentially a l l t h e e n z y m e c a n b e t r a p p e d i n the f o r m o f t h i s i n t e r m e d i a t e . B y c o r r e c t i o n f o r the effect o f cosolvent o n t h e r a t e , a n d e x t r a p o l a t i o n t o 25 ° C u s i n g the A r r h e n i u s p l o t , a v a l u e o f 6 5 db 10 s" w a s e s t i m a t e d f o r the o b s e r v e d r a t e o f f o r m a t i o n u n d e r a g i v e n set 1

of e n z y m e a n d substrate c o n d i t i o n s . readily observable u s i n g stopped-flow

T h e r e a c t i o n s h o u l d therefore b e spectrophotometry,

b e t h e case. T h e m e a s u r e d rate w a s f o u n d t o b e 7 0 ±

as p r o v e d t o 4 s" (32). I n 1


3.

Enzyme-Catalyzed

FINK

49

Reactions

addition, at the l o w temperatures the concentration of the tetrahedral intermediate that could b e accumulated was p H * - d e p e n d e n t , increasing to a m a x i m u m at h i g h p H * . E x a c t l y the same r e l a t i o n s h i p w a s f o u n d i n t h e stopped-flow

studies.

S i m i l a r l y the rate o f f o r m a t i o n o f t h e i n t e r -

mediate was p H * - d e p e n d e n t tures

b o t h at subzero

and ambient

tempera-

(32).

S u c h correlations are n o t a l w a y s p o s s i b l e b e c a u s e t h e e s t i m a t e d rates at 2 5 ° C m a y b e too fast t o m e a s u r e w i t h r a p i d - m i x i n g t e c h n i q u e s a n d b e c a u s e t h e rates o f f o r m a t i o n o f m o r e t h a n one i n t e r m e d i a t e m a y b e o f t h e same o r d e r o f m a g n i t u d e at t h e h i g h e r t e m p e r a t u r e

300

320

340

360

380

400

420

440

(12).

460

480

500

WAVELENGTH nm Biochemistry

Figure S. Formation of the tetrahedral intermediate in with N -carbobenzoxy^-lysine p-nitroanilide in 60% —3°C, pH 9.3. The kinetics were followed by repetitive the intermediate is at 361 nm. The value of k is 2.4 X Χ ΙΟ- M , S = 2.7 X 10' M (32;. a

o 6 s

5

0

the reaction of papain dimethyl sulfoxide at spectral scans, X of 10' sec' for Ê = 7.5 maw

5

1

5


0

50


Change in Rate-Determining

Step at Low

Temperature

A n a d d i t i o n a l p o t e n t i a l a d v a n t a g e of c r y o e n z y m o l o g y is t h a t reactions i n w h i c h intermediates

cannot

be

detected

at a m b i e n t

temperature,

b e c a u s e t h e y b r e a k d o w n m o r e r a p i d l y t h a n t h e y are f o r m e d , m a y u n d e r g o changes i n t h e r a t e - d e t e r m i n i n g step s u c h t h a t t h e i n t e r m e d i a t e b r e a k d o w n b e c o m e s s l o w e r t h a n its rate of f o r m a t i o n at l o w t e m p e r a t u r e s . T h e m i n i m u m reaction p a t h w a y for most h y d r o l y t i c enzymes c a n be represented

b y S c h e m e 2 i n w h i c h E S ' is a n e n z y m e - s u b s t r a t e

inter

m e d i a t e s u c h as a n a c y l - , p h o s p h o r y l - , or g l y c o s y l - e n z y m e . Scheme 2 ki k Ε + S *± E S - » 2

If k

2

ks ES' -» E + + Pi

P

2

is less t h a n fc at a m b i e n t t e m p e r a t u r e s E S ' c a n n o t b e a c c u m u l a t e d . 3

H o w e v e r , i f t h e energies o f a c t i v a t i o n ( E ) o f steps 2 a n d 3 are different a

and i n particular such that E

a

f o r fc is greater t h a n E 3

a

f o r k , t h e n as t h e 2

t e m p e r a t u r e is decreased t h e r e w i l l c o m e a p o i n t at w h i c h k

2

is greater

than k and E S ' w i l l accumulate. 3

A t least t w o s u c h examples are k n o w n . I n t h e r e a c t i o n of p a n c r e a t i c carboxypeptidase

w i t h a n ester substrate a c h a n g e i n r a t e - d e t e r m i n i n g

step f r o m f o r m a t i o n to b r e a k d o w n of a p o s t u l a t e d a c y l - e n z y m e i n t e r m e d i a t e occurs at — 1 0 ° C ( 3 3 ) .

T h u s the i n t e r m e d i a t e is d e t e c t a b l e

s u b z e r o t e m p e r a t u r e s b u t not u n d e r n o r m a l c o n d i t i o n s . of β-glueosidase ( a l m o n d )

at

I n the r e a c t i o n

w i t h p-nitrophenyl-£-D-glucoside a glucosyl-

e n z y m e i n t e r m e d i a t e c a n be r e a d i l y d e t e c t e d a n d s t a b i l i z e d at s u b z e r o t e m p e r a t u r e s (24, 29).

T h e i n t e r m e d i a t e has also b e e n d e t e c t e d at 2 0 ° C

u s i n g stopped-flow t e c h n i q u e s b y T a k a h a s i (34).

However, Legler

f a i l e d to d e t e c t the i n t e r m e d i a t e at 37° u s i n g s t o p p e d - f l o w

(35)

experiments.

T h e reason for these a p p a r e n t l y c o n t r a d i c t o r y results b e c o m e s c l e a r f r o m a n e x a m i n a t i o n of F i g u r e 6, w h i c h shows t h e A r r h e n i u s p l o t s o b t a i n e d f o r the g l u c o s y l a t i o n a n d d e g l u c o s y l a t i o n reactions

in 50%

dimethyl

s u l f o x i d e at s u b z e r o t e m p e r a t u r e s . T h e energies of a c t i v a t i o n f o r t h e t w o steps are q u i t e different a n d are s u c h t h a t g l u c o s y l a t i o n is faster at h i g h e r t e m p e r a t u r e s , w h e r e a s h y d r o l y s i s of the g l u c o s y l - e n z y m e is r a t e l i m i t i n g at t e m p e r a t u r e s b e l o w 0 ° C Temperature-Dependent

Changes in

(24). ΔΗ

A l t h o u g h t h e A r r h e n i u s plots at s u b z e r o t e m p e r a t u r e s f o r m a n y of t h e reactions i n v e s t i g a t e d h a v e b e e n l i n e a r (e.g., F i g u r e s 2 a n d 3 ) , some cases h a v e b e e n o b s e r v e d i n w h i c h changes i n t h e e n t h a l p y of a c t i v a t i o n


3.

Enzyme-Catalyzed

FINK

51

Reactions

V

T

(°KXI0 ) 3

Figure 6. Change in the rate-determining step in the reaction of β-glucosidase with p-nitrophenyl^-O-glucoside in 50% dimethyl sulfoxide, pH* 7.1. The rate constant k is that for formation of the enzyme-glucose intermediate; k corresponds to the rate constant for deglucosylation at temperatures below 0°C. 0l)S

cat

o c c u r (e.g., F i g u r e 6 ) . L y s o z y m e ( h e n e g g - w h i t e ) is a n o t h e r s u c h case, a n d represents s o m e i n t e r e s t i n g features ( 3 1 ) . solvents

Aqueous methanol cryo

a r e s u i t a b l e f o r l o w t e m p e r a t u r e i n v e s t i g a t i o n s of

lysozyme

catalysis (31). T h e i n t e r a c t i o n b e t w e e n l y s o z y m e a n d t h e h e x a s a c c h a r i d e of I V - a c e t y l - g l u c o s a m i n e , o r t h e c o r r e s p o n d i n g t r i s a c c h a r i d e i n h i b i t o r , c a n be f o l l o w e d b y changes i n t h e i n t r i n s i c

fluorescence

of the enzyme.

Three

i n t e r m e d i a t e i n t e r c o n v e r s i o n s c a n b e d e t e c t e d w i t h t h e substrate p r i o r to t h e r a t e - l i m i t i n g step, a n d t w o i n t e r c o n v e r s i o n s c a n b e seen i n t h e case o f t h e i n h i b i t o r (31).

B o t h t h e rate constants f o r e a c h i n d i v i d u a l

t r a n s f o r m a t i o n a n d t h e d i s s o c i a t i o n constants f o r t h e o v e r a l l n o n t u r n o v e r process

h a v e b e e n d e t e r m i n e d as a f u n c t i o n of t e m p e r a t u r e , b o t h a t

subzero temperatures conditions

(36).

(31) a n d at h i g h e r t e m p e r a t u r e s u n d e r n o r m a l

C o m p a r i s o n o f t h e t w o sets o f d a t a i n d i c a t e t h a t a

b r e a k occurs i n t h e A r r h e n i u s a n d V a n t H o f f plots i n t h e v i c i n i t y o f 15-20°C.

L y s o z y m e is k n o w n t o u n d e r g o a s t r u c t u r a l r e a r r a n g e m e n t i n

this t e m p e r a t u r e r e g i o n (37). S o m e other systems i n w h i c h n o n l i n e a r A r r h e n i u s p l o t s h a v e f o u n d at subzero

temperatures have been

been

reported b y D o u z o u ( 2 ) .

T h e r e is n o r e a s o n a t p r e s e n t t o assume t h a t t h e o b s e r v e d changes i n


52

PROTEINS A T L O W

TEMPERATURES

e n e r g y of a c t i v a t i o n at temperatures a b o v e — 1 0 0 ° C are not d u e to the same p h e n o m e n a

as cause s u c h d e v i a t i o n s at h i g h e r t e m p e r a t u r e s i n

aqueous solutions (e.g., c h a n g e i n r a t e - d e t e r m i n i n g step a n d t e m p e r a t u r e i n d u c e d c o n f o r m a t i o n a l c h a n g e i n t h e p r o t e i n ) . H o w e v e r , at t e m p e r a t u r e s closer to a b s o l u t e zero F r a u e n f e l d e r s observations r e g a r d i n g t h e k i n e t i c s of l i g a n d r e c o m b i n a t i o n w i t h h e m e p r o t e i n s suggest t h a t a d h e r e n c e t h e s i m p l e A r r h e n i u s expression is n o l o n g e r v a l i d

to

(38).

Conclusions F r o m o u r investigations of several different e n z y m e - c a t a l y z e d tions at subzero t e m p e r a t u r e s i n aqueous

reac-

o r g a n i c solvent systems

we

h a v e b e e n a b l e to m a k e a n u m b e r of g e n e r a l i z a t i o n s . T h e first is t h a t , i n m a n y cases, the r e a c t i o n p a t h w a y a n d c a t a l y t i c m e c h a n i s m seem to be u n c h a n g e d u n d e r these c o n d i t i o n s , c o m p a r e d to t h e r e a c t i o n u n d e r n o r m a l c o n d i t i o n s . T h e s e c o n c l u s i o n s are b a s e d o n a v a r i e t y of e v i d e n c e , i n c l u d i n g k i n e t i c a n d t h e r m o d y n a m i c p r o p e r t i e s , types of i n t e r m e d i a t e detecta b l e , a n d l a c k of s t r u c t u r a l changes i n t h e p r o t e i n . S e c o n d l y , i t appears t h a t i n most, i f n o t a l l , cases t h e i n i t i a l b i n d i n g of substrate to the e n z y m e is f o l l o w e d b y a n i s o m e r i z a t i o n step i n v o l v i n g at least some of t h e a c t i v e site c a t a l y t i c groups.

F u r t h e r m o r e i t seems, b a s e d o n a l i m i t e d n u m b e r

of systems, t h a t t h e e x t r a p o l a t e d rates f o r e a c h i n t e r m e d i a t e t r a n s f o r m a t i o n t o 2 5 - 3 7 ° i n d i c a t e s t h a t u n d e r n o r m a l c o n d i t i o n s t h e rates of most of the i n t e r m e d i a t e interconversions are v e r y s i m i l a r .

T h i s is e n t i r e l y

consistent w i t h t h e ideas of e n z y m e e v o l u t i o n ( c a t a l y t i c ) as e n u n c i a t e d b y K n o w l e s a n d coworkers

(39).

I m p l i c i t i n the a b o v e

findings

is t h e

f a c t t h a t the s t r u c t u r e of t h e e n z y m e at subzero t e m p e r a t u r e s , i n t h e a p p r o p r i a t e c r y o s o l v e n t , is n o t s i g n i f i c a n t l y different f r o m t h a t i n aqueous solution a n d ambient temperatures. A l t h o u g h the e n z y m e s u s e d i n this a r t i c l e to i l l u s t r a t e v a r i o u s features of c r y o e n z y m o l o g y

have been relatively simple and w e l l characterized,

t h e r a t h e r g e n e r a l a p p l i c a b i l i t y of t h e t e c h n i q u e to p r o t e i n - l i g a n d i n t e r actions is i n d i c a t e d b y successful investigations of m u c h m o r e c o m p l i c a t e d systems.

S e v e r a l b i o c h e m i c a l systems i n v o l v i n g e l e c t r o n t r a n s p o r t h a v e

b e e n s u b j e c t e d to d e t a i l e d e x a m i n a t i o n at s u b z e r o temperatures i n solvents.

F o r e x a m p l e , chloroplasts a n d some of t h e i r i n d i v i d u a l

ponents i n the photosynthetic electron-transport c h a i n have been by

Cox

(40,

41)

to b e f u n c t i o n a l , i n d i c a t i n g t h a t

fluid comfound

cryoenzymological

t e c h n i q u e s are a p p l i c a b l e to m e m b r a n e - b o u n d e n z y m e s .

I n fact D o u z o u

a n d c o w o r k e r s h a v e c a r r i e d o u t extensive i n v e s t i g a t i o n s of l i v e r m i c r o s o m a l , a n d b a c t e r i a l , oxidase chrome P o 4 5

(hydroxylating)

systems

i n v o l v i n g cyto-

(42-44).


3.

FINK

Enzyme-Catalyzed

53

Reactions

E l e c t r o n t r a n s p o r t i n m i t o c h o n d r i a l systems h a s b e e n

studied b y

C h a n c e i n some l o w - t e m p e r a t u r e ( t o 7 7 ° K ) e x p e r i m e n t s i n v o l v i n g i n t e r m e d i a t e s i n t h e o x i d a t i o n o f c y t o c h r o m e oxidase ( 4 5 , 46).

Flash photo-

lysis w a s u s e d t o dissociate t h e C O c o m p l e x u n d e r c o n d i t i o n s w h e r e t h e r e a s s o c i a t i o n w i t h o x y g e n is f a v o r e d . I n t r a m o l e c u l e e n z y m e - l i k e r e a c t i o n s , w h i c h c a n b e c a r r i e d o u t i n n o n f l u i d (i.e. glass) solvents h a v e also b e e n probed

successfully

at very l o w temperatures

(e.g., r h o d h o p s i n a n d

b a c t e r i o r h o d o p s i n (47) a n d t h e reassociation o f l i g a n d s t o h e m e p r o t e i n s i n w h i c h Fraeunfelder and coworkers very intriguing phenomena

(38, 48, 49) h a v e o b s e r v e d

some

over t h e temperature range 20 to 3 0 0 ° K ) .

O n e o t h e r i n v e s t i g a t i o n o f note is t h a t o f H a s t i n g s a n d c o w o r k e r s ( 5 0 , 51 ) i n w h i c h s e v e r a l i n t e r m e d i a t e s i n t h e r e a c t i o n o f b a c t e r i a l l u c i f erase have been trapped a n d characterized. m a y b e f o u n d elsewhere

R e v i e w s o f these

investigations

(2,8).

O n e of the u l t i m a t e goals of o u r c r y o e n z y m o l o g y studies i s t o o b t a i n detailed, high-resolution structural information about each intermediate on the reaction pathway.

T h e present m e t h o d o f c h o i c e t o a c c o m p l i s h

this g o a l is X - r a y c r y s t a l l o g r a p h y . O t h e r t e c h n i q u e s c a n p r o v i d e specific i n f o r m a t i o n i n t h e d e t a i l r e q u i r e d f o r o n l y l i m i t e d parts o f t h e s t r u c t u r e at best.

T h e first steps i n t h i s d i r e c t i o n h a v e n o w b e e n

accomplished.

F o r e x a m p l e , the f e a s i b i l i t y o f o b t a i n i n g X - r a y d i f f r a c t i o n d a t a o n t r a p p e d crystalline enzyme-substrate intermediates has been demonstrated 23)

as f o l l o w s .

(13,

C r y s t a l s o f elastase w e r e g r o w n i n t h e n o r m a l m a n n e r ,

t r a n s f e r r e d to 7 0 % m e t h a n o l c r y o s o l v e n t , a n d t h e substrate a l l o w e d to diffuse i n at — 5 5 ° C u n t i l t h e a c y l - e n z y m e w a s f o r m e d yield).

(in ^ 80%

Diffraction data were then collected, leading to t h e eventual

solution of the structure of t h e trapped acyl-enzyme

(13).

Based o n

experiments u n d e r w a y w e expect t h a t a set o f " t i m e - l a p s e " p i c t u r e s of t h e step-wise t r a n s f o r m a t i o n o f substrate to p r o d u c t w i l l b e c o m e a v a i l a b l e i n t h e near future.

Clearly the potential of cryoenzymology

i s just

b e g i n n i n g to b e c o m e a p p a r e n t . Literature Cited 1. Freed, S. Science 1965, 150, 576. 2. Douzou, P. "Cryobiochemistry"; Academic: Paris, 1977. 3. Fink, A. L . ; Gray, B. L . In "Biomolecular Structure and Function," Agris, P. F., Sykes, B., Loeppky, R., Eds.; Academic: New York, 1978, p 471. 4. Fink, A. L. Acc. Chem. Res. 1977, 10, 233. 5. Fink, A. L. J. Theor. Biol. 1976, 61, 419. 6. Fink, A. L.; Geeves, M. "Methods in Enzymol"; Academic: New York, 1979. 7. Makinent, M. W.; Fink, A. L . Ann. Rev. Biophys. Bioeng. 1977, 6, 301. 8. Douzou, P. Adv. Enzymol. 1977, 45, 157. 9. Douzou, P. Methods Biochem. Anal. 1974, 22, 401. 10. Douzou, P. Mol. Cell Biochem. 1973, 1, 15.


54 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51.

PROTEINS AT LOW TEMPERATURES

Fink, A. L.; Angelides, K. J. Biochemistry 1976, 15, 5287. Fink, A. L. Biochemistry 1976, 15, 1580. Alber, T.; Petsko, G. Α.; Tsernoglou, D. Nature 1976, 263, 297. Douzou, P.; Hui Bon Hoa, G.; Maurel, P.; Travers, F. In "Handbook of Biochemistry and Molecular Biology," Fasman, G., Ed.; Chemical Rub ber Co: Cleveland, Ohio, 1976. Hui Bon Hoa, G.; Douzou, P. J. Biol. Chem. 1973, 248, 4649. Fink, A. L. Biochemistry 1973, 12, 1736. Fink, A. L. Biochemistry 1974, 13, 277. Fink, A. L.; Magnusdottir, K. Biochemistry 1979, submitted. Clement, G. E.; Bender, M. L. Biochemistry 1963, 2, 836. Mares-Guia, M.; Figueiredo, A. F. S. Biochemistry 1972, 11, 2091. Akerlöf, G. J. Am. Chem. Soc. 1932, 54, 4125. Kavanau, J. L. J. Gen. Physiol. 1950, 34, 193. Fink, A. L.; Ahmed, A. I. Nature 1976, 263, 294. Fink, A. L.; Weber, J. Biochemistry 1979, submitted. Fink, A. L.; Angelides, K. J. Biochem. Biophys. Res. Commun. 1975, 64, 701. Wallenfels, K.; Weil, R. The Enzymes 1972, 7, 618. Sinnott, M. L.; Souchard, I. J. L . Biochem.J.1973, 133, 89. Angelides, K. J.; Fink, A. L. Biochemistry 1978, submitted. Fink, A. L.; Good, N. Biochem. Biophys. Res. Commun. 1974, 58, 126. Hui Bon Hoa, G.; Douzou, P.; Petsko, G. A. J. Mol. Biol. 1975, 96, 367. Fink, A. L.; Homer, R.; Weber, J. Biochemistry 1979, submitted. Angelides, K. J.; Fink, A. L. Biochemistry 1979, in press. Makinen, M. W.; Yamamura, G.; Kaiser, Ε. T. Proc. Nat. Acad. Sci. U.S. 1976, 73, 3882. Takahashi, K. J. Sci. Hiroshima Univ. Ser. A. Phys. Chem. 1975, 39, 237. Legler, G. ActaMicrobiol.Acad. Sci. Hung. 1975, 22, 403. Banerjee, S. K.; Holler, E.; Hess, G. P.; Rupley, J. A. J. Biol. Chem. 1975, 250, 4355. Saint-Blanchard, J.; Clochard, Α.; Cozzone, P.; Berthou, J.; Jollès, P. Biochim. Biophys. Acta 1977, 491, 354. Alberding, N.; Austin, R. H.; Chan, S. S.; Eisenstein, L.; Frauenfelder, H.; Gunsalus, I. C.; Nordlund, T. M. J. Chem. Phys. 1976, 65, 5631. Albery, W. J.; Knowles, J. R. Biochemistry 1976, 15, 5631. Cox, R. Eur. J. Biochem. 1975, 55, 625. Cox, R. Biochim. Biophys. Acta 1975, 387, 588. Debey, P.; Hui Bon Hoa, G.; Douzou, P. FEBS Lett. 1973, 70, 2633. Debey, P.; Balny, C.; Douzou, P. Proc. Nat. Acad. Sci. U.S. 1973, 70, 2633. Debey, P.; Balny,C.;Douzou, P. FEBS Lett. 1973, 35, 86. Chance, B.; Graham, N.; Legallais, V. Anal. Biochem. 1975, 67, 552. Chance, B.; Saronio,C.;Leigh, J. S. J. Biol. Chem. 1975, 250, 9226. Hess, B.; Oesterhelt, D. In "Dynamics of Energy-Translucing Membranes", Ernster, Estabrook and Slater, Eds. Elsevier: Amsterdam, 1974; p 257. Austin, R. H.; Beeson, K. W.; Eisenstein, L.; Frauenfelder, H.; Gunsalus, I. C. Biochemistry 1975, 14, 5355. Austin, R.H.;Alberding, N.; Beeson, K.; Chan, S.; Eisenstein, L.; Frauenfelder, H.; Gunsalus, I. C.; Nordlund, T. Croat. Chem. Acta 1977, 49, 287. Hastings, J. W.; Balny, C. J. Biol. Chem. 1975, 250, 7288. Balny,C.;Hastings, J. W. Biochemistry 1975, 14, 4719.

RECEIVED June 16, 1978.


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