Conformation of Pepsin and Pepsinogen - Advances in Chemistry

19 Conformation of Pepsin and Pepsinogen GERTRUDE E. PERLMANN

Downloaded by TUFTS UNIV on June 2, 2018 | https://pubs.acs.org Publication Date: January 1, 1967 | doi: 10.1021/ba-1967-0063.ch019

Rockefeller University, New York, Ν. Y.

From the optical rotatory properties of pepsin and pepsinogen as functions of urea, temperature, and pH, it was shown that the macromolecular conformation of the zymogen differs markedly from that of the enzyme. Pepsin is essentially stabilized by hydrophobic interactions, and the fraction of amino acid residues present in a helical configuration is negligible. In contrast, the configuration of pepsinogen is stabilized by side chain interaction of an electrostatic nature between the basic amino acid residues of the peptide chain segment that are released during activation of pepsinogen to pepsin and some of the dicarboxylic acids of the pepsin moiety. Only a rough relation exists between the con formational changes observed and the susceptibility of the zymogen to activation.

ne of t h e m a j o r aims of p r o t e i n chemists is to u n d e r s t a n d t h e r e l a t i o n between t h e b i o l o g i c a l f u n c t i o n of p r o t e i n s a n d t h e i r s t r u c t u r e a n d t o define t h e t y p e of forces w h i c h g o v e r n t h e specific f o l d i n g of t h e p o l y p e p t i d e c h a i n or chains of t h e molecule. I n a c u r r e n t project of t h i s l a b o r a t o r y directed t o w a r d establishing the conformational a n d functional determi n a n t s of t h e p r o t e o l y t i c e n z y m e , p e p s i n , a n d i t s z y m o g e n , pepsinogen, c h e m i c a l , p h y s i c o c h e m i c a l , a n d e n z y m i c techniques are used t o e s t a b l i s h the f u n c t i o n - s t r u c t u r e r e l a t i o n s h i p . B e f o r e t u r n i n g t o t h e s t r a t e g y a d o p t e d i n o u r w o r k , a few of t h e c h a r a c teristics of t h e t w o proteins are r e v i e w e d . T h e p h y s i c a l a n d c h e m i c a l p r o p erties of these p r o t e i n s h a v e been discussed i n d e t a i l (11, 14). A s first s h o w n b y L a n g l e y , p e p s i n is present i n i t s i n a c t i v e f o r m , pepsinogen, i n t h e gastric m u c o s a . A t a c i d p H , t h e z y m o g e n is t r a n s f o r m e d b y a n a u t o c a t alytic reaction into pepsin, a proteolytic enzyme w i t h a n activity o p t i m u m at p H 2.0 a n d a w i d e specificity (9, 10, 19). P e p s i n is r a p i d l y i n a c t i v a t e d a b o v e p H 6.0, whereas pepsinogen is stable a t n e u t r a l i t y (14). E n d group analyses of pepsinogen, a p r o t e i n w i t h a m o l e c u l a r w e i g h t of 40,000, revealed one ΛΓ-terminal a m i n o a c i d , leucine, a n d one C - t e r m i n a l a m i n o a c i d , a l a n i n e (13,21). I n contrast, p e p s i n of 35,000 m o l e c u l a r w e i g h t 268 Porter and Johnson; Ordered Fluids and Liquid Crystals Advances in Chemistry; American Chemical Society: Washington, DC, 1967.

19.

Pepsin

PERLMANN

and

269

Pepsinogen


has isoleucine as t h e i V - t e r m i n a l a n d a l a n i n e as t h e C - t e r m i n a l a m i n o a c i d . T h e s e results s u p p o r t the concept t h a t pepsinogen a n d p e p s i n are s i n g l e c h a i n proteins w h i c h , as i l l u s t r a t e d i n F i g u r e 1, are c r o s s l i n k e d b y three disulfide bonds. T h e dashed l i n e of F i g u r e 1 indicates t h a t m o s t of t h e basic a m i n o a c i d residues of pepsinogen are near the i V - t e r m i n a l e n d of t h e p o l y p e p t i d e c h a i n , whereas t h e d i c a r b o x y l i c acids, s h o w n b y t h e d o t t e d line, are d i s t r i b u t e d over t h e m a j o r p o r t i o n of the molecule. Below p H 6.0, pepsinogen is a c t i v a t e d t o p e p s i n w i t h a c o n c o m i t a n t release of s e v e r a l basic peptides f r o m t h e i V - t e r m i n a l e n d of t h e p o l y p e p t i d e c h a i n , t h u s l e a v i n g a n acidic p r o t e i n (11, 1^).

C00H

> pH 5.0

C00H COOH

40,000

35,000

Il 3 5

Lys His Arg

46

Asp

44

Asp

30

Glu

30

Glu

I Lys 1 His 2 Arg

Figure 1. Conversion of pepsinogen to pepsin

Table I.

Properties of Pepsinogen and Pepsin (1,7)

Molecular weight Nitrogen content Amino acid distribution Acidic (Asp, Glu) Basic (His, Lys, Arg) Nonpolar (Gly, Val, Leu, lieu, Ala, Met) Hydroxy (Ser, Thr) Aromatic (Tyr, T r y , Phe) Proline y Cys (-S-S) 2

Phosphorus

Pepsinogen

Pepsin

40,000

35,000

15.0

14.8

No. Residues per Molecule 78

74

Θ

137

148 74 39 19 6

72 38 15 6

1

A closer e x a m i n a t i o n of t h e a m i n o a c i d d i s t r i b u t i o n of the t w o p r o teins g i v e n i n T a b l e I reveals t h a t d u r i n g a c t i v a t i o n of t h e z y m o g e n , t e n lysines, t w o h i s t i d i n e s , a n d three arginines are r e m o v e d , i n a d d i t i o n t o 2 5 n o n p o l a r a m i n o acids, t h e r e b y r e d u c i n g t h e n u m b e r of basic residues i n p e p s i n t o f o u r — o n e l y s i n e , one h i s t i d i n e , a n d t w o arginines (1, 7 ) . I t is,

Porter and Johnson; Ordered Fluids and Liquid Crystals Advances in Chemistry; American Chemical Society: Washington, DC, 1967.

270

ORDERED FLUIDS A N D LIQUID CRYSTALS

therefore, clear t h a t t h i s u n u s u a l a m i n o a c i d d i s t r i b u t i o n m u s t influence t h e f o l d i n g of the p o l y p e p t i d e c h a i n . H e n c e , these t w o proteins m u s t differ i n their tertiary structure. I n 1958 we h a d r e p o r t e d t h a t i n contrast to m o s t g l o b u l a r p r o t e i n s , the specific o p t i c a l r o t a t i o n , [a], the h y d r o d y n a m i c properties, a n d , m o s t of a l l , t h e e n z y m i c a c t i v i t y of p e p s i n r e m a i n u n a l t e r e d if t h e p r o t e i n is d i s s o l v e d i n concentrated u r e a s o l u t i o n or i n g u a n i d i n e h y d r o c h l o r i d e , or i f t h e s o l u t i o n is heated to 60°C. H o w e v e r , if t h e t e m p e r a t u r e is raised to 70°C., t h e r o t a t o r y dispersion constant, X increases f r o m 216 t o 236 πΐμ (15, 18). A l t h o u g h h y d r o g e n bonds of t h e t y p e C = Ο . . . Η — Ν a n d those i n v o l v i n g t h e phenolic h y d r o x y l s of t y r o s i n e a n d the c a r b o x y l a t e ions of t h e a c i d i c a m i n o a c i d residues—i.e., Downloaded by TUFTS UNIV on June 2, 2018 | https://pubs.acs.org Publication Date: January 1, 1967 | doi: 10.1021/ba-1967-0063.ch019

C.,

~0

Ο h a v e been s h o w n t o exist i n p e p s i n (8), t h e y m u s t be r e l a t i v e l y u n i m p o r t a n t i n m a i n t a i n i n g t h e c o n f o r m a t i o n necessary for t h e e n z y m i c a c t i v i t y of t h i s p r o t e i n . F r o m these studies a n d f r o m t h e f a c t t h a t 7 0 % of t h e m o l e c u l e is m a d e u p of n o n p o l a r a m i n o a c i d residues w h i c h m u s t be i n v a n der W a a l s contact w i t h t h e i r neighbors, w e c o n c l u d e d t h a t t h e f r a c t i o n of residues present i n α-helical configuration m u s t be v e r y l o w a n d almost negligible. T h u s p e p s i n is essentially s t a b i l i z e d b y h y d r o p h o b i c i n t e r a c t i o n s . T h i s " a p p a r e n t l a c k " of h e l i c i t y i n p e p s i n f u r t h e r follows f r o m t h e fact t h a t t h i s p r o t e i n has a h i g h content of d i c a r b o x y l i c a m i n o acids w h i c h , i n t h e p H range of greatest s t a b i l i t y of t h e e n z y m e — i . e . , p H 4.0 t o 5.0—are i o n i z e d , a n d t h r o u g h electrostatic r e p u l s i o n w o u l d p r e v e n t h e l i x f o r m a t i o n . The increase of t h e r o t a t o r y d i s p e r s i o n constant, X observed o n h e a t i n g of p e p s i n solutions a b o v e 60° reflects c o n f o r m a t i o n a l t r a n s i t i o n s h i t h e r t o n o t y e t described, w h i c h m a y be d u e i n p a r t t o t h e presence of t h e h y d r o x y a m i n o a c i d s — i . e . , serine a n d threonine. T h u s the presence of t h e h y d r o x y a m i n o acids i n t h i s p r o t e i n m a y w e l l f a v o r a c o n f o r m a t i o n s u c h as a 0 - s t r u c t u r e , a n d t h e c o n f o r m a t i o n a l change observed o n h e a t i n g a b o v e 6 0 ° C . m a y be a 0-sheet —> c o i l t r a n s i t i o n . C.,

I n contrast, however, we h a v e d e m o n s t r a t e d t h a t t h e m a c r o m o l e c u l a r c o n f o r m a t i o n of t h e z y m o g e n differs m a r k e d l y f r o m t h a t of t h e e n z y m e (17). T h u s , if pepsinogen is transferred f r o m a n aqueous s o l u t i o n t o c o n c e n t r a t e d u r e a , t h e specific r o t a t i o n , [ a ] 6 , decreases f r o m —200° t o —320° i n t h e c o n c e n t r a t i o n range of 1.5 to 4 . 0 M u r e a , a n d t h e r o t a t o r y d i s p e r s i o n constant, \ decreases f r o m 236 t o 216 ηΐμ. A s s h o w n i n F i g u r e 2, w h i c h also includes t h e results o b t a i n e d w i t h p e p s i n , t h i s change reflects a c o n figurational t r a n s i t i o n , s i m i l a r i n sharpness t o t h e t r a n s i t i o n f r o m a n a36

C.,


19.

Pepsin

PERLMANN

and

271

Pepsinogen

h e l i c a l c o n f o r m a t i o n t o r a n d o m c o i l as observed for p o l y - a - a m i n o acids o n change of solvent c o m p o s i t i o n . F i g u r e 3 shows t h a t o n h e a t i n g of a pepsinogen s o l u t i o n t o 70° or 75°C., t h e l e v o r o t a t i o n of pepsinogen increases w i t h i n the n a r r o w t e m p e r a t u r e range of 45° t o 53°C. w i t h a t r a n s i t i o n t e m p e r a t u r e T = 49.5°C. Between 55° a n d 60°C., [a] 66 r e m a i n s constant. A b o v e 60°C., the l e v o r o t a t i o n f u r t h e r increases b u t never exceeds t h e 4 0 % change of t h a t observed w h e n pepsinogen is d i s s o l v e d i n u r e a . I n F i g u r e 3 i t is f u r t h e r i n d i c a t e d t h a t a decrease of the r o t a t o r y d i s p e r s i o n c o n s t a n t f r o m 236 t o 216 πΐμ occurs i n Q


3

Cone, urea ( M ) Figure 2.

Dependence of specific optical rotation on urea concentration Pepsinogen Pepsin

the same n a r r o w t e m p e r a t u r e range of 45° t o 53°C. H o w e v e r , o n r a i s i n g t h e t e m p e r a t u r e f r o m 60° t o 75°C., X increases to 226 πΐμ. T h i s increase of 10 t o 14 πΐμ is of e x a c t l y t h e same order of m a g n i t u d e as has been o b c

served w i t h p e p s i n (18). Therefore, we feel t h a t the changes of the o p t i c a l r o t a t o r y properties observed d u r i n g t h e first step of the t r a n s i t i o n reflect a c o n f o r m a t i o n a l p a t t e r n r e m i n i s c e n t of a h e l i x - c o i l t r a n s i t i o n — o r , let m e r a t h e r s a y , i t is a t r a n s i t i o n f r o m a " b o n d e d " t o a n " u n b o n d e d " state. I t is t h e p o l y p e p t i d e c h a i n segment t h a t is released d u r i n g t h e a c t i v a t i o n of t h e z y m o g e n t o the e n z y m e w h i c h is responsible for e s t a b l i s h i n g the s t r u c -


272

ORDERED

FLUIDS AND

LIQUID

CRYSTALS

t u r a l p a t t e r n i n pepsinogen n o t present i n pepsin. T h e second step, h o w ever, represents a c o n f o r m a t i o n a l change i n t h e p e p s i n m o i e t y . I n v i e w of t h e fact t h a t t h e n u m b e r of basic a m i n o a c i d residues i n pepsinogen exceeds t h a t present i n p e p s i n , a n d these residues m a y f u n c t i o n as c o n f o r m a t i o n a l d e t e r m i n a n t s , t h e dependence of the specific o p t i c a l r o t a t i o n , [a] 66, o n t h e p H of t h e s o l u t i o n h a d to be considered. I f the p H of t h e s o l u t i o n is altered f r o m 6.5 to 11.5, t h e l e v o r o t a t i o n increases m a r k e d l y i n the p H range of 9.2 t o 10.8 w i t h t h e m i d - p o i n t at p H 10.0. T h i s v a l u e a p p r o x i m a t e s the a p p a r e n t p K of t h e e-amino group of the l y s y l residues if present i n p e p t i d e l i n k a g e .


3

R e c e n t l y , i n c o l l a b o r a t i o n w i t h S. B e y c h o k of C o l u m b i a U n i v e r s i t y , a n i n v e s t i g a t i o n of the o p t i c a l properties of p e p s i n a n d pepsinogen i n the far u l t r a v i o l e t was i n i t i a t e d , u s i n g c i r c u l a r d i c h r o i s m a n d o p t i c a l r o t a t o r y d i s persion. W i t h b o t h proteins a negative t r o u g h of t h e C o t t o n effect at 227 ηΐμ was observed, w h i c h , i n t h e case of pepsinogen b u t n o t w i t h p e p s i n , is abolished i n t h e presence of u r e a ( F i g u r e 4). A s first s h o w n b y P o l l o c k (14) a n d also r e p o r t e d b y B l o u t et al (£), o n h e a t i n g of the p r o t e i n solutions or o n a l t e r i n g of t h e p H , the m i n i m u m of t h e t r o u g h is s h i f t e d f r o m 227 to 232 ηΐμ a n d b o t h t h e residue r o t a t i o n a n d t h e m o l e c u l a r e l l i p t i c i t y become less negative. A n o t a b l e o b s e r v a t i o n , however, is t h e occurrence of s m a l l C o t t o n effects i n t h e w a v e l e n g t h range of 260 t o 290 ηΐμ. I n F i g u r e 4, these C o t t o n effects are set u p o n r a t h e r large a n d steeply c h a n g i n g b a c k -


19.

P E R L M A N N

Pepsin

and

Pepsinogen

273

i.o

Pepsin

Pepsinogen

0

-1.0 ro Ο

χ Ζ '


Ε

-2.0

-3.0 Acetate

pH 4 . 6

8 Μ urea Glycine

-4.0 220

240

260

pH

280

11.7

300

220

240

260

280

300

W a v e l e n g t h (m/x) Figure 4-

Dependence of optical rotatory dispersion of pepsinogen and pepsin in far-ultraviolet on pH and urea concentration

grounds. T h u s i t is difficult t o specify e x a c t l y t h e i r l o c a t i o n a n d signs f r o m the o p t i c a l r o t a t o r y dispersion measurements alone. T h e s e p o i n t s are therefore more c l e a r l y i l l u s t r a t e d w i t h t h e a i d of t h e d i c h r o i c spectra of p e p sinogen a t p H values between p H 7.7 a n d 11.6 a n d of p e p s i n a t p H 4.6 i n the w a v e l e n g t h i n t e r v a l of 250 t o 300 ιημ ( F i g u r e 5). T h e e l l i p t i c i t y b a n d s of p e p s i n a t p H 4.6 a n d of pepsinogen between p H 7.7 a n d 9.5, w h i l e of opposite s i g n , h a v e t h e i r m a x i m a a t essentially t h e same w a v e l e n g t h — i . e . , 280 ιημ. T h e m a g n i t u d e of t h e m o l e c u l a r e l l i p t i c i t i e s , o n a m e a n residue w e i g h t basis, are a p p r o x i m a t e l y e q u a l (cf. F i g u r e 5). T h e second n o t e w o r t h y feature of t h e pepsinogen c i r c u l a r d i c h r o i s m s p e c t r a is t h a t as t h e p H of t h e p r o t e i n s o l u t i o n is raised a b o v e p H 9.8, t h e w a v e l e n g t h b a n d a t 280 ιημ passes t h r o u g h zero a n d changes sign. Above p H 10.6 a b r o a d w a v e l e n g t h m a x i m u m between 265 a n d 275 ιημ is o b served, d i s t i n c t l y different f r o m t h a t of p e p s i n a n d of pepsinogen a t p H v a l u e s below p H 9.5. I t is of course t e m p t i n g t o speculate as t o t h e n a t u r e of t h e residues responsible for these bands w h i c h , as e v i d e n c e d b y t h e s m o o t h dispersion curves between 250 a n d 300 ιημ i n 8 . 0 M u r e a , are c l e a r l y c o n f o r m a t i o n dependent. B o t h t y r o s i n e a n d t r y p t o p h a n are k n o w n t o h a v e e l l i p t i c i t y b a n d s between p H 1.0 a n d 13.0 w i t h a m a x i m u m near 265 ιημ. F o r t y r o s ine a b o v e p H 11.0, t h e p o s i t i o n of t h e b a n d is s h i f t e d t o 295 πΐμ (3, 4 5> 20). S i n c e w e h a v e s h o w n t h a t a t t h i s p H a l l t y r o s i n e residues of p e p }


274

ORDERED FLUIDS A N D LIQUID CRYSTALS

Pepsinogen

80

_ Pepsin

60 40 20

-


0 -20

_

'

-40 -60

1

260

1

270

1

280

PH • 7.7 ° 8.4 ο 9.4 * 9.8 • 10.6 Δ 1 1.7 8 M urea ι ι 290 300

-

260

270

ι 280

290

ι 300

Wavelength (m/x) Figure 5. Buffers.

Circular dichroism of pepsinogen and pepsin Na phosphate-NaCl, pH 7.7, Y/2 = 0.15 Ν a glycinate-NaCl in pH range 8.0 to 12.0 0.1N Na acetate, pH A.6

sinogen are t i t r a t a b l e a n d the a b s o r p t i o n m a x i m u m of t h e p r o t e i n is at 295 ηΐμ (16) j t h e l o c a t i o n of t h e e l l i p t i c i t y m a x i m u m at 270 ηΐμ ( F i g u r e 5), c a n n o t b e assigned t o t y r o s i n e . T e n t a t i v e l y , we s h o u l d l i k e t o propose t h a t i n pepsinogen below p H 9.8 b o t h t y p e s of residues are o p t i c a l l y a c t i v e . A t h i g h e r p H values a t w h i c h t h e p r o t e i n is u n f o l d e d , t y r o s i n e a c t i v i t y is abolished a n d the o p t i c a l a c t i v i t y of the t r y p t o p h a n residues is the m a j o r c o n t r i b u t i n g factor. H o w t h i s m a y be related t o the c o n f o r m a t i o n a l fea tures i n p e p s i n to give rise to a p o s i t i v e b a n d at p H 4.6 is u n c e r t a i n . I t is clear, h o w e v e r , t h a t i n b o t h proteins t h e r i g i d i t y of t h e molecule a n d t h e s p a t i a l arrangement of charges r e l a t i v e t o t h e chromophores m u s t p l a y a m a j o r role. W h a t d o these results indicate? A s s h o w n earlier, pepsinogen c o n t a i n s a n appreciable n u m b e r of basic a m i n o a c i d residues a l l clustered w i t h i n a r e l a t i v e l y s m a l l p a r t of t h e molecule, whereas t h e acidic groups are d i s t r i b u t e d over t h e p e p s i n m o i e t y . A t n e u t r a l p H a significant n u m b e r of acidic side chains are n e u t r a l i z e d b y t h e p r o x i m i t y of t h e e-amino groups. T h u s these charged side chains p a r t i c i p a t e i n some m u t u a l i n t e r a c t i o n ,


19.

Pepsin

PERLMANN

275

and Pepsinogen

t h e r e b y s t a b i l i z i n g t h e p r o t e i n molecule a n d l o c k i n g i t i n t o i t s m o s t stable conformation. T o s u s t a i n t h i s v i e w , t w o p o l y p e p t i d y l pepsinogens were p r e p a r e d b y p o l y m e r i z a t i o n of t h e p r o t e i n w i t h t h e ΛΓ-carboxy-a-amino a c i d a n h y d r i d e of a l a n i n e a n d t y r o s i n e , r e s p e c t i v e l y (2). H e r e t h e €-amino group w i t h a n a p p a r e n t p K of 10.4 is replaced b y a n α-amino group of t h e a m i n o a c i d a t t a c h e d , h a v i n g t h e l o w e r p K of 7.8. I n a n o t h e r set of experiments p e p T a b l e II.

P r o p e r t i e s of M o d i f i e d P e p s i n o g e n s Type of Modification


Pepsinogen Number of lysine residues reacted Potential pepsin activity, % Optical rotatory dispersion constant, X τημ Transition temperature, °C. a

C.,

a

Tyrosylpepsinogen

Alanylpepsinogen

Succinylpepsinogen

none

3

2

10

100

37

30

0

236

219

224

48.5

36.5

34.0

218 Not determined

Potential pepsin activity of untreated pepsinogen taken as 100.

280

260 CO

ω ro

Ξ

240

220

7 Figure 6.

8

9 PH

10

II

Dependence of specific optical rotation on pH

• Pepsinogen A Alanylpepsinogen Ο Pepsin Buffers. Na phosphate-NaCl, Y/2 = 0.15, pH 6.5 to 8.0 Ν a glycinate-NaCl, pH 8.0 to 11.0


276

ORDERED

FLUIDS AND

LIQUID CRYSTALS

sinogen was m a d e to react w i t h succinic a c i d a n h y d r i d e to f o r m a s u c c i n y l d e r i v a t i v e i n w h i c h t h e e-amino groups of the lysines are t r a n s f o r m e d i n t o acidic side chains. A s s h o w n i n T a b l e I I , the o p t i c a l r o t a t o r y d i s p e r s i o n constant, X h a d decreased f r o m 236 ηΐμ to 219, 224, a n d 218 ηΐμ for the p o l y p e p t i d y l a n d s u c c i n y l d e r i v a t i v e s , respectively. F u r t h e r m o r e , t h e t r a n s i t i o n t e m p e r a t u r e is lowered c o n s i d e r a b l y , a n d as i l l u s t r a t e d w i t h the a i d of F i g u r e 6, the p H t r a n s i t i o n of the a l a n y l p e p s i n o g e n has been s h i f t e d to a lower p H w i t h a m i d - p o i n t at p H 8.8. A l t h o u g h u r e a s t i l l has some effect o n the o p t i c a l r o t a t o r y properties of the p o l y p e p t i d y l d e r i v a t i v e s , the s u c c i n y l pepsinogen is n o t affected b y t h i s reagent. Therefore, these r e sults corroborate t h e existence of side c h a i n interactions as a n i m p o r t a n t f a c t o r i n m a i n t a i n i n g the m a c r o m o l e c u l a r c o n f o r m a t i o n of pepsinogen. Downloaded by TUFTS UNIV on June 2, 2018 | https://pubs.acs.org Publication Date: January 1, 1967 | doi: 10.1021/ba-1967-0063.ch019

C.,

T o assess f u r t h e r the n a t u r e a n d t h e r e a c t i v i t y of the basic groups w h i c h c o n t r i b u t e to the s t a b i l i z a t i o n of pepsinogen, s p e c t r o p h o t o m e t r i c

7.0

8.0

9.0

10.0

i 1.0

PH

Figure 7.

Dependence of potentiometric titration of pepsinogen in NaCl of various concen trations on temperature

a n d p o t e n t i o m e t r i c t i t r a t i o n s were p e r f o r m e d i n the p H range of 6.0 t o 11.5 i n 0.02 to 0 . 5 M s o d i u m c h l o r i d e solutions a n d at v a r i o u s t e m p e r a t u r e s i n the range of 20° to 60°C. where c o n f o r m a t i o n a l changes occur. A b o v e p H 11.0, where t h e p r o t e i n is u n f o l d e d , a l l 17 tyrosines ionize n o r m a l l y w i t h t h e s t a n d a r d heat of i o n i z a t i o n , AH = 6.3 k c a l . per mole. I n the p H range of 7.0 t o 9.5, t w o residues w i l l c o n t r i b u t e to t h e absorbance o n l y if t h e m a c r o m o l e c u l a r c o n f o r m a t i o n of the p r o t e i n is altered b y a d d i t i o n of 4 . 0 M


19.

Pepsin

PERLMANN

and

277

Pepsinogen


u r e a (16). T h e s e results suggest t h a t t w o t y r o s i n e residues are b o n d e d to some other, as y e t undefined, group w i t h i n t h e molecule. T h a t , i n t h e p H range of 6.0 to 9.0, n o t a l l of the h i s t i d i n e s do t i t r a t e n o r m a l l y is i l l u s t r a t e d w i t h t h e a i d of F i g u r e 7, i n w h i c h , i n s t e a d of t h e c o n v e n t i o n a l t i t r a t i o n c u r v e , t h e d e r i v a t i v e b' — d N a O H / d p H is p l o t t e d against p H . O n assigning the p H range of 7.0 to 9.0 t o h i s t i d i n e a n d t h a t of p H 9.5 t o 11.0 t o l y s i n e a n d t y r o s i n e , respectively, a n d a s s u m i n g t h a t t h e p H of the m a x i m u m at w h i c h s u c h a peak occurs corresponds t o t h e p K of a g i v e n group, i t becomes evident t h a t at 30°C. t h e apparent p K of t h e histidines is s h i f t e d t o a m o r e a l k a l i n e p H — i . e . , t o p H 8.7. T h u s the close v i c i n i t y of other charged groups—e.g., a c i d i c groups—influences t h e i o n i z a t i o n of the h i s t i d i n e s , hence also t h e i r p K . F u r t h e r m o r e , as i n d i cated i n F i g u r e 7, t h e lower peak i n the p H range of 8.0 t o 9.0 corresponds to t h e t i t r a t i o n of the p r o t e i n i n 0 . 0 5 M s o d i u m chloride, whereas the higher one is a t i t r a t i o n i n 0.2M. Since t h e n u m b e r of t i t r a t a b l e residues c a n be d e r i v e d f r o m t h e height of t h e peak, i t becomes a p p a r e n t t h a t m o r e residues become accessible t o t i t r a t i o n if a c o n f o r m a t i o n a l change is i n d u c e d b y a n increase of the ionic s t r e n g t h of t h e m e d i u m . O n r a i s i n g t h e t e m p e r a t u r e to 45° a n d 50°C. where, as a l r e a d y s h o w n w i t h t h e o p t i c a l r o t a t i o n measure ments, t h e c o n f o r m a t i o n of pepsinogen changes, t h e h i s t i d i n e residues w i l l become u n m a s k e d , a n d t h e i r p K n o r m a l i z e d . F r o m a c o m p a r i s o n of t h e t i t r a t i o n at 30° a n d 45°C., t h e s t a n d a r d heat of i o n i z a t i o n , AH, of t h e histidines was c a l c u l a t e d a n d a v a l u e a p p r e c i a b l y higher t h a n t h a t for a n u n m a s k e d i m i d a z o l e group was o b t a i n e d .

50 0

10

ο

ο.

ω -40

> 'gLU

H50 7.0

8.0

9.0

10.0

11.0

PH

Figure 8.

Forward and back titration of pepsinogen in 0.1 Ν NaCl at 30°C.


278

ORDERED

FLUIDS AND

LIQUID CRYSTALS


I n v i e w of the anomalous b e h a v i o r of these a m i n o a c i d residues, the q u e s t i o n arises w h e t h e r these t i t r a t i o n s are reversible. F i g u r e 8 shows t h a t r e v e r s i b i l i t y c o u l d n o t be d e m o n s t r a t e d o n b a c k t i t r a t i o n f r o m p H 11.5 w i t h a c i d . T h e lower s o l i d c u r v e represents the f o r w a r d t i t r a t i o n , the u p p e r one is the b a c k t i t r a t i o n , whereas the dashed curves are those of the second f o r w a r d a n d b a c k t i t r a t i o n . I t is clear t h a t there is a n increase i n the n u m b e r of t i t r a t a b l e groups o n b a c k t i t r a t i o n a n d the a p p a r e n t p K of t h e i m i d a z o l e residues has n o w been s h i f t e d t o a l o w e r v a l u e — e . g . , t o p H 6.9. T h u s the i r r e v e r s i b i l i t y of these t i t r a t i o n s c a n be t a k e n as a reflection t h a t a n irreversible c o n f i g u r a t i o n a l change occurs o n exposure of t h e p r o tein to alkaline p H . T u r n i n g n o w t o a c o n s i d e r a t i o n of the a c t i v a t i o n of pepsinogen to p e p s i n a n d a t t e m p t s to correlate the b i o l o g i c a l a c t i v i t y w i t h the c o n f o r m a t i o n a l characteristics, one sees i m m e d i a t e l y t h a t o n l y a r o u g h c o r r e l a t i o n

2.0

4.0

C o n e , u r e a (M)

Figure 9.

6.0

20

40

60

80

Temperature(°C)

Dependence of specific optical rotation ( ) and potential pepsin activity ( ) of pepsinogen on urea concentration and temperature

exists between t h e c o n f o r m a t i o n a l changes observed a n d t h e s u s c e p t i b i l i t y of the z y m o g e n to a c t i v a t i o n . ( P o t e n t i a l p e p s i n a c t i v i t y of pepsinogen r e fers t o proteolysis of h e m o g l o b i n at p H 2.0 after a c t i v a t i o n w i t h 0.12V h y d r o c h l o r i c a c i d at 37°C. for 10 minutes.) I n some cases—i.e., i n the h e a t i n g experiments i n t h e t e m p e r a t u r e range of 45° t o 62°C. or o n c h a n g i n g the p H of a pepsinogen s o l u t i o n f r o m 7.0 t o 1 2 . 0 — t h e decrease of the p o t e n t i a l p e p s i n a c t i v i t y a n d t h e c o n f i g u r a t i o n a l changes p a r a l l e l each other closely. T h i s is n o t the case i n t h e u r e a experiments, where t h e onset of t h e c o n f o r m a t i o n a l change precedes t h e loss of a c t i v i t y ( F i g u r e 9). S i m i l a r l y , the degree of reversal differs. I f a pepsinogen s o l u t i o n m a i n t a i n e d at 60° to 65°C. for 10 to 20 m i n u t e s is cooled to 2o°C., the



19.

PERLMANN

Pepsin

and

279

Pepsinogen

Figure 10. Reversal of specific optical rotation ( ) and potential pepsin activity (— after repeated heating to 60°C. for 15 minutes, followed by rapid cooling to 25°C.

l e v o r o t a t i o n decreases s l o w l y b u t never r e t u r n s t o its o r i g i n a l v a l u e .

How

ever, t h e p o t e n t i a l p e p s i n a c t i v i t y has been restored c o m p l e t e l y . F i g u r e 10 shows t h a t if t h e cycle of h e a t i n g a n d c o o l i n g is repeated several t i m e s , t h e l e v o r o t a t i o n o n c o o l i n g a l w a y s r e t u r n s t o t h e c o n s t a n t v a l u e — i . e . , [a]m =

—220°.

However, the potential pepsin a c t i v i t y de

creases progressively a n d after five times of h e a t i n g a n d c o o l i n g is o n l y 6 0 % of its o r i g i n a l v a l u e .

T h u s i t appears t h a t after each u n f o l d i n g t h e

p r o t e i n molecule resumes a s o m e w h a t different m a c r o m o l e c u l a r c o n f o r m a tion.

T h e molecule, however, has been refolded sufficiently to restore t h e

a c t i v e site a n d to p e r m i t a c t i v a t i o n of t h e z y m o g e n t o t h e e n z y m e .

From

t h i s one m u s t infer t h a t a l t h o u g h a " s p e c t r u m of configurât ions' ' is a v a i l able to pepsinogen, a c e r t a i n r i g i d i t y of t h e m o l e c u l e is essential t o ensure s t a b i l i t y of t h e z y m o g e n i n t h e n e u t r a l p H range where p e p s i n is r a p i d l y inactivated. A s a l r e a d y foreshadowed

b y the potentiometric titration and the

hysteresis p h e n o m e n o n observed o n b a c k - t i t r a t i o n , t h e effect of p H a n d t i m e o n pepsinogen is m o r e d r a s t i c . irreversible ( F i g u r e 11).

I n a c t i v a t i o n occurs r a p i d l y a n d is

A l l i n a l l , one observes a process of a g i n g .

The

p r o t e i n molecule grows o l d r a p i d l y a n d l i k e a l l h u m a n beings, i t loses its memory. W e m a y n o w ask, w h a t is t h e s t r u c t u r e w h i c h has emerged w i t h p r o gressively i n c r e a s i n g c l a r i t y f r o m these experiments?

Both

pepsinogen

a n d p e p s i n are folded i n a c o m p l e x a n d u n s y m m e t r i c a l f o r m , a n d t h e i r c o n f o r m a t i o n is e x t r e m e l y c o m p a c t .

B u t w h a t forces are responsible for m a i n

t a i n i n g t h e i n t e g r i t y of the w h o l e s t r u c t u r e ?

I n pepsin the major contribu

t i o n comes f r o m t h e v a n der W a a l s forces b e t w e e n n o n p o l a r residues w h i c h m a k e u p the b u l k — i . e . , 7 0 % — o f the protein.

I n pepsinogen there are a

n u m b e r of charge i n t e r a c t i o n s between p o l a r residues o n t h e surface of t h e molecule.

H o w e v e r , t h a t does n o t exclude t h e fact t h a t t h e p e p t i d e c h a i n



280

ORDKRED

FLUIDS A N D

LIQUID

CRYSTALS

PH Figure 11.

Dependence of potential pepsin activity of pepsinogen on pH and time

pH of each solution adjusted to 7.0 before activation at pH 2.0

segment, w h i c h is r e m o v e d o n a c t i v a t i o n of t h e z y m o g e n , has a s t r u c t u r e — for instance a n α - h e l i x — w h i c h is essentially different f r o m , a n d independent of, t h e r e m a i n d e r of t h e pepsinogen molecule, t h u s d i r e c t i n g o n o v e r - a l l s t r u c t u r a l p a t t e r n i n the z y m o g e n t h a t is altered o n a c t i v a t i o n to pepsin. T h e changes of t h e o p t i c a l r o t a t o r y properties observed o n h e a t i n g a n d i n u r e a resemble those of a h e l i x - c o i l t r a n s i t i o n . H o w e v e r , the 40 residues at the ΛΓ-terminal e n d of the p o l y p e p t i d e c h a i n w o u l d at best represent t e n h e l i c a l t u r n s ; s u c h a short h e l i c a l segment s h o u l d not y i e l d as sharp t r a n s i tions as those w h i c h we h a v e observed. E v e n if the entire molecule h a d entered t h r o u g h some cooperative effects, t h e n u m b e r of h e l i c a l t u r n s — i . e . , η = 100—still w o u l d n o t e x p l a i n t h e sharpness of t h e t r a n s i t i o n (12, 22). H o w e v e r , w h e n a p a t t e r n of a few crosslinkages is i n t r o d u c e d b y side c h a i n i n t e r a c t i o n of electrostatic n a t u r e , i t w o u l d c o n t r i b u t e considerably t o w a r d s t r e n g t h e n i n g t h e configuration as a whole. T h u s t h e change f r o m one c o n f o r m a t i o n t o another m a y be s h a r p a n d resemble a phase t r a n s i t i o n . F u r t h e r m o r e , s u c h a s t r u c t u r a l p a t t e r n w o u l d also e x p l a i n t h e changes of t h e o p t i c a l r o t a t o r y properties observed if t h e p H of t h e s o l u t i o n is altered f r o m p H 8.0 to 11.0. Therefore, we suggest t h a t side c h a i n interactions between t h e p o s i t i v e l y charged e-amino groups of t h e lysines a n d t h e n e g a t i v e l y charged c a r b o x y l s of t h e d i c a r b o x y l i c a m i n o acids are the essential features i n s t a b i l i z i n g the c o n f o r m a t i o n of pepsinogen a n d m a y be super i m p o s e d o n a ^ - s t r u c t u r e , p r e v a l e n t i n pepsin. T h i s t y p e of m a c r o m o l e c u l a r c o n f o r m a t i o n has h i t h e r t o n o t been described for proteins.


19.

PERLMANN

Acknowledgmen

Pepsin and Pepsinogen

281

t

The work carried out in our laboratory on the macromolecular con formation of pepsin and pepsinogen and its relation to the biological func tion would hardly have been possible without the active contribution of some of my collaborators. I acknowledge the help of S. Beychok in the dichroism experiments and my sincere thanks go to William F . Harrington and Aharon Katchalsky for many helpful discussions which made a vital contribution to this investigation. Most of all, I like to remember the friendship of the late K . Linderstrom-Lang.


Literature

Cited

(1) Arnon, R., Perlmann, G. E., J. Biol. Chem. 238, 563 (1963). (2) Becker, R. R., Stahmann, Μ. Α., J. Biol. Chem. 204, 745 (1956). (3) Beychock, S., Proc. Natl. Acad. Sci. U.S. 53, 999 (1965). (4) Beychock, S., Science, in press. (5) Beychock, S., Fasman, G. D., Biochemistry 3, 1675 (1964). (6) Blout, E. R., Pollock, E . J., Parrish, J. R., "Abstracts of Papers," 150th Meeting, ACS, September 1965, 23C. (7) Blumenfeld, O. O., Perlmann, G. E., J. Gen. Physiol. 42, 553 (1959). (8) Ibid., p. 563. (9) Fruton, J. S., Bergmann, M . , J. Biol. Chem. 127, 627 (1939). (10) Harington, C. R., Pitt-Rivers, R. V., Biochem. J. 38, 417 (1944). (11) Herriott, R. M . , J. Gen. Physiol. 45, 57 (1962). (12) Lifson, S., Roig, Α., J. Chem. Phys. 34, 1963 (1961). (13) Lokshina, L. Α., Orekhovich, V. N., Biokhimiya 22, 699 (1957). (14) Northrop, J. H., Herriott, R. M . , ''Crystalline Enzymes," 2nd ed., pp. 28, 77, Columbia University Press, New York, 1948. (15) Perlmann, G. E., Fourth International Congress on Biochemistry, Vol. VIII, p. 32, 1959. (16) Perlmann, G. E., J. Biol. Chem. 239, 3762 (1964). (17) Perlmann, G. E., J. Mol. Biol. 6, 452 (1963). (18) Perlmann, G. E., Proc. Natl. Acad. Sci. U.S. 42, 596 (1959). (19) Sanger, F., Tuppy, H., Biochem. J. 49, 481 (1951). (20) Velluz, L., Legrand, M . , Angew. Chem. (Intern. Ed.) 4, 838 (1965). (21) Van Vunakis, H., Herriott, R. M . , Biochim. Biophys. Acta 23, 600 (1957). (22) Zimm, Β. H., Bragg, J. K., J. Chem. Phys. 31, 526 (1959). RECEIVED March 1, 1966. Garvan Award Lecture. Work supported in part by the U. S. Public Health Service, Grant AM-02449, and by the National Science Foundation, Grant GB-2419.


Conformation of Pepsin and Pepsinogen - Advances in Chemistry

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