Phosphorolytic Synthesis of Low-Molecular-Weight Amyloses with

Chapter 13

Phosphorolytic Synthesis of Low-MolecularWeight Amyloses with Modified Terminal Groups Downloaded by STANFORD UNIV GREEN LIBR on August 2, 2012 | http://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0458.ch013

Comparison of Potato Phosphorylase and Muscle Phosphorylase Β 1,4

1

2

3

C. Niemann , W. Saenger , B. Pfannemüller , W. D. Eigner , and A. Huber 3

1

Institut für Kristallographie der Freien Universität Berlin, Takustrasse 6, D-1000 Berlin 33, Germany Institut für Makromolekulare Chemie der Albert-Ludwigs-Universität Freiburg, Stefan-Meier-Strasse 31, D-7800 Freiburg, Germany Institut für Physikalische Chemie, Karl-Franzens-Universität Graz, Heinrichstrasse 28, A-8010 Graz, Austria 2

3

The preparation of low molecular weight amyloses with modified terminal groups in a DP range of 4-25 was carried out by enzymatic synthesis using e i t h e r potato phosphorylase or phosphorylase b from r a b b i t muscle. p-Nitrophenyl-α-D-maltooligomers with a minimum chain length of f i v e glucosyl residues served as primers; glucose-1-phosphate was the monomer. The i n v e s t i g a t i o n of the products by s i z e exclusion chromatography/low angle laser l i g h t s c a t t e r i n g and HPLC showed that the behaviour of the enzymes is d i f f e r e n t in the view of the d i s t r i b u t i o n of oligomers formed under the same reaction conditions. Whereas the synthesis by muscle phosphorylase leads to an expected Poisson d i s t r i b u t i o n the reaction pro ducts from potato phosphorylase are a l t e r e d by a simultaneous pH dependent d i s p r o p o r t i o n a t i o n . With both enzymes, significant amounts of the desired p-nitrophenyl-α-D-maltooligosaccharides in the DP range 10-20 can be obtained. These oligomers are of s p e c i a l interest for X-ray single crystal diffraction analyses. 4

Current address: Whistler Center for Carbohydrate Research, Smith Hall, Purdue University, West Lafayette, IN 47907 0097-6156/91/0458-0189$06.00/0 © 1991 American Chemical Society

In Biotechnology of Amylodextrin Oligosaccharides; Friedman, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Downloaded by STANFORD UNIV GREEN LIBR on August 2, 2012 | http://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0458.ch013

190

BIOTECHNOLOGY OF AMYLODEXTRIN OLIGOSACCHARIDES

Although the formation o fsupermolecular arrangements by a m y l o s e a n d t h e b r a n c h e d a m y l o p e c t i n h a v e b e e n extensively discussed, the crystal 1inity o f starch granules and d e t a i l s o f rétrogradation s t i l l remain somewhat o b s c u r e . I n o r d e r t omake p r o g r e s s i n t h i s f i e l d , l o w m o l e c u l a r w e i g h t a m y l o s e s (LMWA) w i t h d e f i n i t e c h a i n lengths c a n serve a s model substances to a n s w e r open q u e s t i o n s . Compounds w i t h a d e g r e e o f p o l y m e r i z a t i o n (DP) i n t h e range o f 10-20 g l u c o s e units p e r molecule appear t obe p a r t i c u l a r l y u s e f u l , because they represent an intermediate stage between the "low molecular" m a l t o o l i g o s a c c h a r i d e s and t h e " h i g h m o l e c u l a r " a m y l o s e a n d a m y l o p e c t i n . T h i s DP range corresponds t ot h e length o ft h e o u t e r chains o f the amylopectin molecule, which a r e probably respons i b l e f o r s t a r c h c r y s t a l 1 i n i t y ( 1 ) . On t h e o t h e r hand, in p r e l i m i n a r y s t u d i e s a s u d d e n c h a n g e o f t h e X - r a y powder d i f f r a c t i o n p a t t e r n o f r e t r o g r a d e d m i c r o c r y s t a l l i n e LMWA f r o m t h e B - t y p e t o t h e A - t y p e w a s o b s e r v e d b e t w e e n DP 1 3 a n d 12 ( 2 ) . The b e s t method t oe l u c i d a t e m o l e c u l a r s t r u c t u r e s in t h e s o l i d s t a t e i s t h e s i n g l e c r y s t a l X - r a y d i f f r a c t i o n a n a l y s i s . However, t h e p r o d u c t i o n o f l a r g e r a m o u n t s o f LMWA i n a p u r i t y a n d q u a n t i t y r e q u i r e d f o r c r y s t a l l i z a t i o n i s s t i l l a p r o b l e m . R e c e n t l y we succeeded i n t h e c r y s t a l l i z a t i o n o fa maltohexaose ( 3 ) with an α-ρ-nitropheny1 group a tt h e reducing end as i t s p o l y i o d i d e c o m p l e x e s . We a s s u m e t h a t s u c h a d e r i v a t i z a t i o n completed with complex formation f a c i l i t a t e s the growth o f s i n g l e c r y s t a l s a t l e a s t i n t h i s lower DP r a n g e . T o s t u d y t h e s t r u c t u r e o f t h e a m y l o s e s f u r t h e r , we d e c i d e d t o e x t e n d t h e s e s t u d i e s t o m o d i f i e d LMWA i n t h e r a n g e o f D P 1 0 - 2 0 . T h e u s u a l m e t h o d s t o o b t a i n LMWA, e . g . a c i d hydrolysis o fstarch (4), amylolytic degradation o f amylose (5) o rdebranching o famylopectin (6) and glycogen ( 7 ) , c a n n o t be t h e method o f c h o i c e because a subsequent chemical m o d i f i c a t i o n t oblock t h e reducing end i n one c o n f i g u r a t i o n would be d i f f i c u l t and i n e f f e c t i v e . A n a l t e r n a t i v e way i s t h e c h a i n elongation o fa s u i t a b l y modified acceptor with t h e help o fc y c l o d e x t r i n g l u c o s y l t r a n s f e r a s e and a-cyclodextrin o rby phosphorolytic synthesis with glucose-1phosphate a s t h e monomer donor. The p r e p a r a t i o n o f p - n i t r o p h e n y 1 - α - D - m a l t o o l i g o s a c c h a r i d e s by t h e former method has been r e p o r t e d by W a l l e n f e l s e ta l . ( 8 ) l e a d i n g t oo n l y low y i e l d s o f o l i g o m e r s i n t h e r a n g e b e y o n d DP 7 , b u t w i t h c o n s i d e r a b l e amounts o f n o n - s u b s t i t u t e d m a l t o o l i g o s a c c h a rides and β- and γ-cyclodextrin. Meanwhile t h e p - n i t r o p h e n y l a t e d m a l t o o l i g o m e r s ( u p t o DP 8 ) a r e c o m m e r -



13.

NIEMANN ET AL.

191

Phosphorolytic Synthesis of Amyloses

c i a l l y a v a i l a b l e a n d c a n be used as s u i t a b l e p r i m e r s for chain elongation reactions. Results of our investi g a t i o n s t o p r o d u c e LMWA w i t h m o d i f i e d t e r m i n a l g r o u p s u s i n g c y c l o d e x t r i n g l u c o s y l t r a n s f e r a s e w i l l be pub lished elsewhere. In p h o s p h o r o l y t i c s y n t h e s i s ( e q . 1 ) , p r o d u c t s o f a r a t h e r narrow c h a i n l e n g t h d i s t r i b u t i o n c a n be e x p e c t e d , a n d u n s u b s t i t u t e d o l i g o m e r s s h o u l d n o t be formed. Using t h i s method, high molecular weight a m y l o s e s (HMWA) c a r r y i n g d i f f e r e n t t e r m i n a l g r o u p s were o b t a i n e d ( 9 ) , and i n p r e l i m i n a r y s t u d i e s t h e reaction conditions t o prepare larger quantities o f LMWA w e r e o p t i m i s e d ( 1 0 ) . The a i m o f t h i s work i s t h e c o m p a r i s o n o f t w o o f the most commonly employed p h o s p h o r y l a s e s , p o t a t o phosphorylase and r a b b i t muscle phosphorylase b i n t h e l a r g e s c a l e p r o d u c t i o n o f LMWA. T h e d e c i s i v e c r i t e r i o n s h o u l d be t h e c h a i n l e n g t h d i s t r i b u t i o n o f t h e o l i g o mers formed. Results and D i s c u s s i o n P h o s p h o r o l y t i c s y n t h e s i s . I n o r d e r t o s y n t h e s i z e LMWA with a p-nitropheny1 group i n «-position a t t h e r e d u c i n g e n d , p - n i t r o p h e n y l - a - D - m a l t o t e t r a o s i d e was u s e d a s a p r i m e r . I n t h e s e e x p e r i m e n t s we c o n f i r m e d p r e v i o u s r e s u l t s o f E m m e r l i n g e t a l . ( 1 1 ) who o b s e r v e d t h a t an α - s u b s t i t u t e d m a l t o t e t r a o s i d e b e h a v e s l i k e an u n s u b s t i t u t e d m a l t o t r i o s e which i s n o t a good p r i m e r for potato phosphorylase. The d i s t r i b u t i o n pattern o f t h e p r o d u c t s showed l a r g e amounts o f n o n - c o n v e r t e d primer and low y i e l d s o f h i g h e r oligomers (12). Sub sequent s t u d i e s showed t h a t a m o d i f i e d p r i m e r s u i t a b l e for t h e reaction has t o contain at least five glucosyl units per molecule (10). phosphorylases pNP G + m 6 - 1 - P pNP G + m ? (1) n

n + m

{

pNP = p - n i t r o p h e n y l , G - 1 - P - g 1 u c o s e - 1 - p h o s p h a t e , i n o r g a n i c p h o s p h a t e , η > 5 , m = 11

P, =

We u s e d a r a t i o o f p r i m e r t o m o n o m e r o f 1 : 1 1 b e c a u s e t h e c o n v e r s i o n o f G - 1 - P a t pH 6 . 0 , t h e pH o p t i m u m f o r s y n t h e s i s , s h o u l d be a t about 90 % f o r p o t a t o phos p h o r y l a s e ( 1 3 ) . T h e same c o n d i t i o n s were used i n t h e experiments with muscle phosphorylase. P o t a t o p h o s p h o r y l a s e ( I ) was i s o l a t e d from p o t a t o t u b e r s by p r e c i p i t a t i o n w i t h ammonium s u l p h a t e a c c o r ding t o t h e method o f Z i e g a s t e t a l . ( 9 ) . F u r t h e r p u r i f i c a t i o n was c a r r i e d b u t by h y d r o p h o b i c i n t e r a c t i o n


192

BIOTECHNOLOGY OF AMYLODEXTRIN OUGOSACCHARIDES

chromatography and subsequent g e l f i l t r a t i o n ( I I ) (14) . F o r comparison w i t h t h e s e p r e p a r a t i o n s a s t i l l h i g h e r p u r i f i e d p o t a t o p h o s p h o r y l a s e ( I I I ) was used (15) . P o t a t o p h o s p h o r y l a s e I I and I I I appeared a s a s i n g l e band i n SDS g e l e l e c t r o p h o r e s i s . An improved s t a b i l i t y o b s e r v e d f o r I I I may r e s u l t from i t s p r e p a r a t i o n i n 0.1 M p h o s p h a t e b u f f e r pH 7 . 5 , w h e r e a s I a n d II were p r e p a r e d w i t h o u t a n y a d d i t i o n o fi n o r g a n i c phosphate. Muscle phosphorylase b (Sigma), c o n t a i n i n g a s m a l l amount o f i n o r g a n i c phosphate and 5 -AMP was u s e d w i t h o u t f u r t h e r p u r i f i c a t i o n . 5'-AMP a n d m e r c a p t o e t h a n o l were added t ot r a n s f o r m t h e i n a c t i v e b-form into t h e a c t i v e form ( 1 6 ) . A l l p h o s p h o r y l a s e s were u s e d i n a r a t i o o f 0.1 U per gmol p r i m e r t o r e a c h e q u i l i b r i u m w i t h i n a r e l a t i v e l y s h o r t t i m e ( 1 0 ) . T h e c o n v e r s i o n o f G-1-P, m e a s u r e d by c o l o r i m e t r i c d e t e r m i n a t i o n o f l i b e r a t e d i n o r g a n i c phosphate (17), i n d i c a t e s t h e r a t e o f r e a c t i o n . Whereas p o t a t o p h o s p h o r y l a s e shows a h i g h e r i n i t i a l r a t e o f r e a c t i o n , t h e muscle p h o s p h o r y l a s e g i v e s a somewhat higher c o n v e r s i o n a tt h e end (Table I ) .


1

Table I .

Conversion o f glucose-1-phosphate i n phosphorolytic synthesis (see text f o r explanation)

reaction time min 5 10 20 40 60 120 180 240

% conversion o f total potato phosphorylase 14.1 24.4 38.6 58.0 67.2 77.7 83.3 85.3

glucose-1-phosphate by muscle phosphorylase 7.5 15.0 26.4 43.8 57.5 78.8 88.8 91.3

The d i f f e r e n c e i n i n i t i a l r a t e o f r e a c t i o n c o u l d b e due t ot h e h i g h e r a f f i n i t y o f p o t a t o p h o s p h o r y l a s e f o r short chain maltooligosaccharides i n comparison t o muscle phosphorylase which, according t oFukui e t a l . (18), i s a s s o c i a t e d with d i f f e r e n t amino a c i d sequences at t h e s u b s t r a t e b i n d i n g s i t e s . The e f f e c t o b v i o u s l y disappears during further progress o f reaction. C h a r a c t e r i z a t i o n o ft h e products o f phosphorol y t i c s y n t h e s i s wasc a r r i e d o u t by s i z e e x c l u s i o n chromatography combined w i t h low angle l a s e r l i g h t s c a t t e r i n g a c c o r d i n g t oa technique u s u a l l y a p p l i e d t o h i g h e r m o l e c u l a r weight compounds (19,20). The s i m u l taneous d e t e c t i o n o f l a s e r s c a t t e r i n g i n t e n s i t y (LALLS d e t e c t o r ) and c o n c e n t r a t i o n (DRI-detector) p r o v i d e s



13.

NIEMANN ET A L


193

i n f o r m a t i o n a b o u t m o l e c u l a r w e i g h t a v e r a g e s Μ η , Mw, M z , p o l y d i s p e r s i t i e s Mw/Mn, M z / M n a n d m o l e c u l a r w e i g h t d i s t r i b u t i o n s (MWD) w i t h o u t a n y e x t e r n a l c a l i b r a t i o n required. The D R I - s i g n a l s w e r e o f e x c e l l e n t q u a l i t y a n d r e p r o d u c i b i l i t y . The LALLS-signa 1s were n o t as good as expected, d e s p i t e t h e averaging procedures used. This c o u l d be a s s o c i a t e d w i t h t h e i n h e r e n t l i m i t s o f t h e t e c h n i q u e f o r t h e low m o l e c u l a r weight f r a c t i o n o fo u r o l i g o m e r m i x t u r e s b e l o w DP 1 2 . I t c o u l d a l s o e x p l a i n t h e d i s c r e p a n c y o f t h e DP m a x i m a d e t e c t e d i n H P L C a n d the observed m o l e c u l a r weight averages by SEC/LALLS (Table I I ) . Table

II.

DP M a x i m a ( H P L C ) ; e x p e c t e d a n d o b s e r v e d molecular weight averages and p o l y d i s p e r s i t i e s (SEC/LALLS) o f products and phosphorolytic synthesis SEC/LALLS data M /M p h o s p h o r y l a s e DP max. a v P M w V n source (HPLC) g /mol g /mol g/mol g/mol 1.14 1.13 potato (II) 10 2700 1800 2100 2400 potato ( I I I ) 11 1950 2500 3000 1.11 1.07 2800 muscle 14 1.08 1.04 2400 2400 2700 2600 M

e x

M

2

w

T h e r e s u l t i n g MWD f o r t h e LMWA o b t a i n e d w i t h p o t a t o a n d w i t h m u s c l e p h o s p h o r y l a s e a r e g i v e n i n F i g u r e 1. The p r o d u c t s o b t a i n e d by t h e m u s c l e enzyme e x h i b i t a h i g h e r u n i f o r m i t y than t h o s e p r e p a r e d by t h e p o t a t o enzyme, independent o f t h e degree o f p u r i t y o f t h e used p o t a t o p h o s p h o r y l a s e s . S i m i l a r r e s u l t s were o b s e r v e d w i t h u n m o d i f i e d LMWA a n d w i l l b e p u b l i s h e d elsewhere. F i g u r e 2 shows t h e m o l e c u l a r weight d i s t r i b u t i o n c a l c u l a t e d f r o m HPLC ( 2 1 ) i n c o m p a r i s o n t o a c o r r e s p o n ding t h e o r e t i c a l Poisson d i s t r i b u t i o n ( 2 2 ) . F o r muscle p h o s p h o r y l a s e t h e r e i s good agreement. The s e r i o u s d e v i a t i o n shown by p o t a t o p h o s p h o r y l a s e prompted us t o investigate the development o f chain length d i s t r i b u t i o n with both enzymes as a f u n c t i o n o f r e a c t i o n time. A l i q u o t s were removed from t h e r e a c t i o n m i x t u r e s a t f i x e d t i m e s a n d t h e r e a c t i o n was t e r m i n a t e d by t r e a t ment with a mixed bed i o nexchanger t o remove s a l t s and e n z y m e s . T h e p r o d u c t s were a n a l y z e d by HPLC. F i g u r e 3 a - h a n d T a b l e s I I I a n d IV i l l u s t r a t e t h e time dependent d i f f e r e n c e s i n t h e molar composition o f the oligomer mixtures. I t i s obvious that t h e r e a c t i o n of both phosphorylases proceeds d i f f e r e n t l y . Whereas the muscle phosphorylase r e a c t s by a s t r a i g h t c h a i n elongation o f t h e primer, t h e reaction with potato p h o s p h o r y l a s e i s more c o m p l i c a t e d . A l r e a d y a f t e r f i v e


194


O.B

4.Β -


3.8 -

8.Β

O.B

-O.B 2.6

2.8

3.0

3.2

3.4

3.6

3.8

logQQ

PC-LALLS Figure

1

M o l e c u l a r w e i g h t d i s t r i b u t i o n (SEC/LALLS) of LMWA w i t h m o d i f i e d t e r m i n a l g r o u p s o b t a i n e d by p h o s p h o r o l y t i c s y n t h e s i s e i t h e r by p o t a t o p h o s p h o r y l a s e ( I I ) ( 7 2 ) and ( I I I ) ( 5 8 ) o r m u s c l e p h o s p h o r y l a s e b (62).

I.II

8.15

Figure

2

M o l a r r a t i o s np o f LMWA ( c a l c u l a t e d from HPLC) v e r s u s DP i n c o m p a r i s o n t o a t h e o r e t i c a l Poisson d i s t r i b u t i o n .


4.0

13.

Table

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Table

195

I I I . Development o f c h a i n l e n g t h d i s t r i b u t i o n o f LMWA w i t h m o d i f i e d t e r m i n a l g r o u p s d u r i n g p h o s p h o r o l y t i c s y n t h e s i s . Enzyme: p o t a t o p h o s p h o r l y a s e . C o n c e n t r a t i o n i n mol % c a l c u l a t e d from HPLC. DP Maxima w r i t t e n i n i t a l i c

DP



NIEMANN ET A L

0

5

100

2.75 21.78

10

0 100

40

2.60 5.35 39.56 86.72 18.79 10.70 21.00 27.14 17.90 11.00 6.71 6.48 10.13 8.62 5.73 2.04 5.65 4.35 8.03 2.66 5.80 8.02 1.52 8.16 3.29 6.90 0.07 2.58 6.93 8.69 7.47 1.62 4.90 0.06 3.09 5.80 4.28 2.10 3.53 1.55 2.65 0.09 2.00 1.19 0.09 0.05 2.00 11.40

IV. See T a b l e

DP 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

20

5

120

40 20 0.18 5.69 8.39 0.72 3.81 25.54 19.19 17.84 87.20 18.36 3.08 18.98 22.65 7.23 20.83 0.36 2.09 16.18 9.84 0.60 4.35 1.63 0.40 10

J

3.70 5.47 6.74 8.10 6.92 5.70 7.06 5.70 7.34 7.43 8.39 7.99 8.60

2.33 4.75 8.05

8.54

7.88 6.75 5.50 4.74 3.83 2.88 2.06 1.32 0.08 0.07 0.04

8.70

7.91 6.69 5.50 4.41 3.37 2.70 2.38 1.60 1.18 1.07 0.09

I I I . Enzyme: m u s c l e

50.59 27.10 28.27 24.92

14.59 4.39 0.30

2.05 7.18

60

180 0.04 4.59 5.97 6.79 7.38 7.81 8.25

240min 0.04 4.12 5.71 6.31 7.08 7.63

8.49

7.55 7.14 6.85 6.54 5.65 4.98 4.60 3.54 2.72 2.30 1.85 1.30 0.09 0.08

8.08 7.51 6.61 5.79 4.44 3.54 2.72 2.16 1.92 1.42 0.09 0.08 0.07

7.70

phosphorylase

180 240min 120 0 0 0 0.22 0.13 0 0.62 0.50 0.50 1.30 0.95 1.37 2.59 2.74 2.93 4.52 4.61 5.04 6.69 7.13 8.08 18.92 9.75 8.88 18.25 11.13 10.72 13.59 12.95 11.81 8.43 11.72 18.38 12.82 4.57 11.64 1163 12.25 9.57 10.70 10.67 2.05 8.80 0.89 6.55 8.39 6.34 6.63 0.23 4.39 4.49 4.00 2.59 1.57 2.59 2.84 1.57 1.34 1.10 0.82 0.90 1.04 0.74 0.48

60 0.04 0.08 1.09 5.89 11.26 15.48




196

Figure 3. Chain length distribution of LMWA during phosphorolytic synthesis after a, 5 and b, 40 min by potato phosphorylase. Continued on next page.



197


13. NIEMANN ET A L

Ο

10

20

min

Figure 3. Continued. Chain length distribution of LMWA during phosphorolytic synthesis after c, 120 and d, 240 min by potato phosphorylase. Continued on next page.


198


>

I


e

Mu005min

li_L 10

20

min

Mu040min

il 10

20

min

Figure 3. Continued. Chain length distribution of LMWA during phosphorolytic synthesis after e, 5 and f, 40 min by muscle phosphorylase. Continued on next page.


13. NIEMANN ET AL.


199


Mul20min

min

h

Mu240min _

14

-

0 0

10

20

min

Figure 3. Continued. Chain length distribution of LMWA during phosphorolytic synthesis after g, 120 and h, 240 min by muscle phosphorylase.



200


minutes a remarkable amount o fthe p - n i t r o p h e n y 1 - α - D ma l t o t e t r a o s i d e i s f o r m e d . T w o d i s t i n c t m a x i m a d e v e l o p w i t h i n a p p r o x i m a t e l y 40 m i n u t e s . T h i s can be c o r r e l a ted with twosimultaneous, independent enzymatic a c t i v i t i e s . A f t e r two h o u r s r e a c t i o n t i m e , t h e d i s t r i b u t i o n s h o w s a s i n g l e m a x i m u m i n t h e r e g i o n o f DP 1 2 . E v e n l a t e r , t h e m a x i m u m i s s h i f t e d t o DP 10 a n d s m a l l t r a c e s o f t h e m a l t o t r i o s i d e a r e d e t e c t e d . The f l a t tening o fthe d i s t r i b u t i o n continues, i n d i c a t i n g that a d i s p r o p o r t i o n a t i o n r e a c t i o n influences the chain length d i s t r i b u t i o n while synthesis i s reduced o r even discontinued under e q u i l i b r i u m conditions. These phenomena appear t o i n d i c a t e t h a t the l e s s purified potato phosphorylase (I) is contaminated by a d i s p r o p o r t i o n a t e enzyme, most probably D-enzyme, o r a s p e c i a l k i n d o f α - a m y l a s e ( 1 6 , 2 3 ) . The use o f p u r i f i e d e n z y m e d i d n o t c o n f i r m t h i s a s s u m p t i o n . On t h e contrary, the e f f e c t was rather pronounced. In o r d e r t o e x a m i n e t h e d i s p r o p o r t i o n a t i o n a c t i v i t y s e p a r a t e l y we i n c u b a t e d t h e p-nitrophenyl-a-D-maltop e n t a o s i d e w i t h t h e e n z y m e s u n d e r t h e same c o n d i t i o n s as i n s y n t h e s i s but i n t h e a b s e n c e o f g l u c o s e - 1 - p h o s p h a t e . T h e r e s u l t s o fHPLC a n a l y s i s are shown i n F i g . 4. A f t e r f o u r h o u r s a t p H 6 . 0 w i t h t h e p h o s p h a t e f r e e prepared phosphorylase preparation ( I I ) , a slow d i s p r o p o r t i o n a t i o n i s observed. A f t e r 24 hours the amounts of p - n i t r o p h e n y l - a - D - m a l t o t e t r a o s i d e and -hexaoside a r e c o n s i d e r a b l y i n c r e a s e d . A t pH 7.0 (0.1 M T r i s / H C L b u f f e r ) the d i s p r o p o r t i o n a t i o n i s even more s i g n i f i c a n t a f t e r four hours. I nc o n t r a s t , the muscle phosphorylase d o e s n o t a t t a c k t h e p r i m e r a t pH 6.0 e v e n a f t e r 2 4 h o u r s . A t pH 7.0 d i s p r o p o r t i o n a t i o n i s c o n s i d e r a b l y slower than with the potato enzyme. Incubation o fthe pentaoside with h i g h l y p u r i f i e d phosphorylase ( I I I ) c o n t a i n i n g 0.03 p m o l P i p e r m l r e a c t i o n m i x t u r e showed a more r a p i d r e a c t i o n even a t pH 5 . 5 a n d p H 6 . 0 . A f t e r t w o h o u r s m o r e t h a n 5 0 % o f t h e p r i m e r was d i s p r o p o r t i o n a t e d t o t h e t e t r a o s i d e and h i g h e r o l i g o m e r s u p t o DP 1 0 . A s i m i l a r e f f e c t was a l s o observed by Palm e ta l . (24) w i t h Ε . c o l i p h o s p h o r y l a s e . They reported that a f t e r prolonged incubation o fa r a d i o a c t i v e l y l a b e l l e d m a l t o t e t r a o s e a t pH 6.5 a s m a l l f r a c t i o n o f r a d i o a c t i v i t y w a s a l s o f o u n d b y TLC i n t h e p o s i t i o n o f m a l t o t r i o s e , m a l t o s e and a d d e d l i m i t d e x t r i n . O u r i n v e s t i g a tions with 30fold r a t i o o fpotato phosphorylase (I) t o m o d i f i e d p r i m e r s a t pH 5 . 8 r e v e a l e d a f t e r 24h a s a main product o fd i s p r o p o r t i o n a t i o n the m a l t o t r i o s i d e . The m a l t o s i d e and g l u c o s i d e a s w e l l a s t h e w h o l e s e r i e s u p t o DP 16 w e r e f o r m e d ( 1 0 ) . It should be discussed whether inorganic phosphate is a c t u a l l y involved in the g l y c o s y l t r a n s f e r r e a c t i o n . K l e i n e ta l . (25) p o s t u l a t e d a c a t a l y t i c mechanism o f


13.


NIEMANN ET AL.

201

I 0P 5

,0O

300

OPS


RoAh pH 6.0

JU

•τ

0

—

1

t(min)

10

OD300

DP

5 0P5

Hu pH

—r

Figure

4

10

2Lh

Mu Ah pH7.0

6.0

·"

_^JVJ 10

Disproportionation m a l t o p e n t a o s i d e (DP r y l a s e ( I I ) and (b) b a t pH 6.0 a n d 7.0

10

t(min)

of p-nitrophenyl-a-D5) ( a ) p o t a t o p h o s p h o muscle phosphorylase a f t e r 4 h a n d 24 h .



202


phosphorylase a c t i o n i n which a "mobile" phosphate a n i o n p l a y s a v e r s a t i l e r o l e . I t c o u l d s e r v e as a proton c a r r i e r f o r s u b s t r a t e a c t i v a t i o n , s t a b i l i z i n g t h e i n t e r m e d i a t e and a c t i n g as a n u c l e o p h i l e w h i c h c a n a c c e p t a g l y c o s y l r e s i d u e r e v e r s i b l y . However, m u s c l e p h o s p h o r y l a s e was n o t a b l e t o d i s p r o p o r t i o n a t e t h e p e n t a o s i d e a t pH 6.0 even i n t h e p r e s e n c e o f 5.5 pmol P i . O b v i o u s l y t h e h i g h amounts o f l i b e r a t e d p h o s p h a t e i n p h o s p h o r o l y t i c s y n t h e s i s (90 pmol/ml a t e q u i l i b r i u m ) d i d not impair the d i s t r i b u t i o n p a t t e r n of t h e prod u c t s . The l o w e r r a t e o f d i s p r o p o r t i o n a t i o n by phosp h o r y l a s e " ! 1 I ) and t h e c o n s i d e r a b l e i n c r e a s e o f r e a c t i o n r a t e a t e x t r e m e l y low c o n c e n t r a t i o n s o f P i s u g g e s t t h a t i n o r g a n i c p h o s p h a t e c o u l d have a s t i l l unknown f u n c t i o n i n enzyme a c t i v a t i o n , e s p e c i a l l y i n n o n - r e g u l a t e d p l a n t systems. The g l y c o s y l t r a n s f e r a c t i v i t y seems t o be more s e n s i t i v e w i t h r e g a r d t o t h e pH d e p e n d e n c e . The pHoptimum f o r b o t h enzymes i s r e p o r t e d i n t h e range between pH 4.9 and 8.7 ( 1 7 , 2 2 , 2 6 ) . P a r t i c u l a r s p e c i f i c a t i o n s o f optimum pH v a l u e s f o r s y n t h e s i s and f o r degradation d i f f e r . In g e n e r a l , p h o s p h o r o l y t i c degradat i o n s h o u l d n o t o c c u r a t pH 5.5 o r 6.0. B u t i t has t o be c o n s i d e r e d t h a t t h e d i s p r o p o r t i o n a t i o n r e a c t i o n i s due t o a s y n t h e s i s / d e g r a d a t i o n e q u i l i b r i u m e s p e c i a l l y at pH 7.0. Conclusions A s i g n i f i c a n t d i f f e r e n c e was o b s e r v e d between t h e c h a i n l e n g t h d i s t r i b u t i o n o f LMWA w i t h m o d i f i e d t e r m i n a l g r o u p s o b t a i n e d by p h o s p h o r o l y t i c s y n t h e s i s e i t h e r w i t h p o t a t o p h o s p h o r y l a s e o r m u s c l e phosphor y l a s e b. Under t h e g i v e n c o n d i t i o n s t h e s y n t h e s i s by m u s c l e p h o s p h o r y l a s e d e l i v e r s a much n a r r o w e r c h a i n l e n g t h d i s t r i b u t i o n w i t h i n t h e DP range o f 10-20. The p r o d u c t s o f t h e s y n t h e s i s by p o t a t o p h o s p h o r y l a s e a r e a l t e r e d by a d i s p r o p o r t i o n a t i o n o f t h e p r i m e r and formed o l i g o m e r s w h i c h i m p a i r s t h e d i s t r i b u t i o n p a t t e r n t o such a d e g r e e t h a t t h e y i e l d o f LMWA w i t h m o d i f i e d t e r m i n a l groups d e c r e a s e s .

Legend o f Symbols SEC LALLS DR1 M M M

^ = = = = =

s i z e e x c l u s i o n chromatography low a n g l e l a s e l i g h t s c a t t e r i n g d i f f e r e n t i a l r e f r a c t i v e index number a v e r a g e weight average z-average


13.

NIEMANN ET


203

Acknowledgments, We t h a n k D r . M a n f r e d B u e h n e r f o r h i g h l y p u r i f i e d p o t a t o p h o s p h o r y l a s e and h e l p f u l d i s c u s s i o n s . We a l s o t h a n k D r . D i e t e r Palm f o r v a l u a b l e s u g g e s t i o n s . This work was s u p p o r t e d by t h e D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t and t h e Fonds d e r C h e m i s c h e n I n d u s t r i e . Literature Cited


1.

2. 3.

4. 5. 6. 7. 8. 9. 10. 11.

12. 13. 14. 15. 16. 17.

18.

French, D. Starch, Chemistry and Technology; Whistler, R.L.; BeMiller, J.N.; Paschall, E.F.; Academic: Orlando, 1984, pp 184-247 Pfannemüller, B. Int. J. Biol. Macromol. 1987, 9, 105-108. Hinrichs, W.; Büttner, G.; Steifa, M.; Betzel, Ch.; Zabel, V.; Pfannemüller, B.; Saenger, W. Science 1987, 238, 205-208. Kikomoto, S.; French, D. J. Jpn. Starch. Sci. 1983, 30, 69-75. Emmerling, W.; Pfannemüller, B. Carbohydr. Res. 1980, 86, 321-324. Hizukuri, S. Carbohydr. Res. 1985, 141, 295-306. Gidley, M.J.; Bulpin, P.V. Carbohydr. Res. 1987. 161, 105-108. Wallenfels, K.; Föld i. D.; Niermann, H.; Bender, H.; Lindner, D. Carbohydr. Res. 1978, 61, 359-368. Ziegast, G.; Pfannemüller, Β. Carbohydr. Res. 1987, 160, 185-204. Niemann, C.; Nuck, R.; Pf annemüller, B.; Saenger, W. Carbohydr. Res. 1989, 197, 187. Emmerling, W. In Mechanism of Saccharide Polymerisation and Depolymerisation; Pfannemüller, B.; Marshall, J.J., Ed.: Academic: New York, 1980, pp 413-420. Pfannemüller, B.; Burchard, W. Makromol. Chem. 1969, 121, 1. Holló, J.; László, E.; Hoschke, A. Stärke/Starch, 1965, 17, 377-381. Niemann, C.; Goyal, B.; Beck, R.H.F.; in preparation Bühner, M. Universität Würzburg, private communication Pfannemüller, B.; Potratz, Ch. Stärke/Starch 1977, 29, 73-80. Husemann, E.; Fritz, B.; Lippert, R.; Pfannemüller, B.; Schupp. E. Makromol. Chem. 1958, 26, 181-198. Fukui, T.; Shimomura, S.; Nakano, K. Mol. Cell. Biochem. 1982, 42, 129-144.


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BIOTECHNOLOGY OF AMYLODEXTRIN OLIGOSACCHARIDES Yu, L.P.; Rollings, J.E. J. Appl. Polymer Sci. 1987, 33, 1909-1921. Eigner, W.-D.; Abuja, P.; Beck, R.H.F.; Praznik, W. Carbohydr. Res. 1988, 180, 87-95. Niemann, C.; Nuck, R.; Pfannemüller, B.; Saenger, W. Carbohydr. Res. in preparation. Flory, P.J. Principles of Polymer Chemistry; Cornell University Press: Ithaka, 1953, p 337. Linder, D. Ph.D. Thesis, Universität Freiburg, Freiburg 1978/79. Palm, D.; Blumenauer, G.; Klein, H.W.; BlancMuesser, M. Biochem. Biophys. Res. Comm. 1983, 111, 530-536. Klein, H.W.; Im, H.J.; Palm, D. Eur. J. Biochem. 1986, 157, 107-114. Lee, Y.P. Biochem. Biophys. Acta 1960, 43, 18-24.

September 9, 1990


Phosphorolytic Synthesis of Low-Molecular-Weight Amyloses with

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