122 Phospholipase A2 Hydrolysis of Phospholipids: Use of P NMR to Study the Hydrolysis, Acyl Migration, Regiospecific Synthesis, and Solubilization of Phospholipids 31
A N D R E A S P L U C K T H U N and E D W A R D A . D E N N I S
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Department of Chemistry, University of California at San Diego, La Jolla, C A 92093
Our laboratory has been studying the kinetics (1) and mechanism of action (2) of phospholipase A which catalyzes the hydrolysis of the fatty acyl chain i n the sn-2 position of phos pholipids to give the 1-acyl lyso-phospholipid product: 2
Recently, we found that phosphorylcholine-containing lipids activate the enzyme from cobra venom (Naja naja naja) toward phos phatidylethanolamine (PE) (3,4). These studies led to the suggestion of two sites for the enzyme - an activator site with minimum specificity for phosphorylcholine and a hydrophobic chain and a catalytic site with less specificity for the polar group. We have now extended these studies to synthetic phospholipids that contain short chain fatty acyl groups and which are water soluble, such as dibutyryl and dihexanoyl phosphatidylcholine (PC). These phospholipids are monomeric below their critical micelle concen tration (cmc), yet activate the enzyme. In order to carry out kinetic studies, the long chain phospholipid substrate must generally be solubilized by a detergent such as Triton X-100 which serves as an inert matrix. Further understanding of the mechanism of the activation by short-chain phospholipids requires first a quantitation of the solubilization of these compounds by detergent To solve this problem, the very high sensitivity of the P -NMR chemical shift to changes in the hydrophobicity of the environ ment has proven to be very useful. The sensitivity of the chemica shift to structural changes in the phospholipid molecule several atoms away allowed us to also examine several other mechanistic questions including the hydrolysis and specificity of phospholipas 31
0097-6156/81/0171-0591$05.00/0 © 1981 American Chemical Society Quin and Verkade; Phosphorus Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
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A2 i n m i x e d p h o s p h o l i p i d s y s t e m s and t h e k i n e t i c s and m i g r a t i o n o f t h e a c y l and p h o s p h o r u s g r o u p o f t h e l y s o - p h o s p h o l i p i d p r o d u c t s . The new 31p-NMR r e s u l t s a l s o a l l o w us t o d i r e c t l y e s t a b l i s h t h e s p e c i f i c i t y o f v a r i o u s p h o s p h o l i p a s e s by d i r e c t p r o d u c t o b s e r v a t i o n and s h o u l d a l s o be v e r y u s e f u l i n r e g i o s p e c i f i c c h e m i c a l s y n t h e s i s of p h o s p h o l i p i d s . We f o u n d t h e 31p-NMR c h e m i c a l s h i f t o f monomeric d i h e x a n o y l PC i n c r e a s e s upon t h e a d d i t i o n o f t h e n o n i o n i c d e t e r g e n t T r i t o n X-100. T h i s phenomenon was u s e d t o q u a n t i t a t e t h e s o l u b i l i z a t i o n o f t h i s p h o s p h o l i p i d by t h e d e t e r g e n t m i c e l l e s as a f u n c t i o n o f d e t e r g e n t c o n c e n t r a t i o n u s i n g a s i m p l e phase s e p a r a t i o n model ( 5 ) . S i m i l a r s t u d i e s w e r e c a r r i e d o u t on d i b u t y r y l PC. At a phospho l i p i d c o n c e n t r a t i o n o f 7 mM and 56 mM d e t e r g e n t , 85% o f t h e d i h e x a n o y l PC, b u t o n l y 3% o f t h e d i b u t y r y l PC was i n c o r p o r a t e d i n t o the m i c e l l e s . H a v i n g shown t h a t d i b u t y r y l PC i s monomeric u n d e r t h e enzyme a s s a y c o n d i t i o n s , we f o u n d t h a t t h e p h o s p h o l i p a s e A 2 , w h i c h a c t s p o o r l y on PE i n m i x e d m i c e l l e s , i s a c t i v a t e d by d i b u t y r y l PC w h i c h i s i t s e l f an e v e n p o o r e r s u b s t r a t e . 31p_NMR s p e c t r o s c o p y was em p l o y e d t o show t h a t o n l y PE i s h y d r o l y z e d i n m i x t u r e s o f v a r i o u s c o m p o s i t i o n s o f t h e s e two p h o s p h o l i p i d s . The f u l l y a c t i v a t e d e n zyme h y d r o l y z e s PE a t a s i m i l a r r a t e t o i t s o p t i m a l s u b s t r a t e , PC c o n t a i n i n g long-chain f a t t y a c i d groups. B e c a u s e d i b u t y r y l PC i s not i n c o r p o r a t e d i n t o the m i c e l l e s , these r e s u l t s are c o n s i s t e n t w i t h a m e c h a n i s m o f d i r e c t a c t i v a t i o n o f t h e enzyme by phosphorylc h o l i n e - c o n t a i n i n g l i p i d s ( e i t h e r monomeric o r m i c e l l a r ) r a t h e r t h a n a change i n t h e p r o p e r t i e s o f t h e i n t e r f a c e b e i n g r e s p o n s i b l e f o r t h e a c t i v a t i o n o f p h o s p h o l i p a s e A£. T h e r e f o r e , two f u n c t i o n a l s i t e s on t h e enzyme h a v e t o be assumed: an a c t i v a t o r s i t e and a c a t a l y t i c s i t e (6). The d e t e r m i n a t i o n o f t h e p o s i t i o n a l s p e c i f i c i t y o f p h o s p h o l i p a s e s by t h e d i r e c t o b s e r v a t i o n o f p r o d u c t f o r m a t i o n r e q u i r e s t h a t the m i g r a t i o n of the r e m a i n i n g f a t t y a c i d group i n the l y s o p h o s p h o l i p i d p r o d u c t be s l o w compared t o t h e e n z y m a t i c h y d r o l y s i s reaction. T h e r e f o r e a k i n e t i c i n v e s t i g a t i o n o f t h i s m i g r a t i o n was c a r r i e d out. One e n a n t i o m e r o f e a c h o f t h e t h r e e p o s s i b l e i s o m e r i c lyso-phosphatidylcholines (l-acyl-sn-glycero-3-phosphorylcholine, 2-acyl-sn-glycero-3-phosphorylcholine and 3-aeyl-s«-glycero-2-phosp h o r y l c h o l i n e ) were generated e n z y m a t i c a l l y . The a s s i g n m e n t o f t h e g l y c e r o l p o r t i o n i n t h e iH-NMR s p e c t r u m a l l o w e d an unequivocal i d e n t i f i c a t i o n o f t h e s e compounds. The 31p_NMR s p e c t r u m o f e a c h gave a d i s t i n c t peak. From t h e a s s i g n m e n t s , t h e 31p-NMR s p e c t r u m o f t h e l y s o p r o d u c t s c o u l d a l s o be u s e d t o show t h e s p e c i f i c i t y o f p h o s p h o l i p a s e A2 f o r t h e sn-2 p o s i t i o n and o f l i p a s e f o r t h e sn-1 position directly. T h i s e l i m i n a t e s the n e c e s s i t y of r e g i o s p e c i f i c a l l y r a d i o - l a b e l l e d p h o s p h o l i p i d s , which are d i f f i c u l t to synthe s i z e w i t h a b s o l u t e i s o m e r i c p u r i t y . 31P-NMR a l s o a l l o w e d us t o d e m o n s t r a t e t h e a b s o l u t e s p e c i f i c i t y o f c o b r a venom p h o s p h o l i p a s e A2 f o r t h e sn-2 p o s i t i o n o f monomeric d i b u t y r y l PC, f o r w h i c h t h e r e w e r e no s p e c i f i c a l l y r a d i o - l a b e l l e d p h o s p h o l i p i d s a v a i l a b l e and t h e y w o u l d be e v e n more d i f f i c u l t t o p r e p a r e b e c a u s e o f t h e f a c i l e acyl migration. The monomeric p h o s p h o l i p i d s do n o t h a v e t h e
Quin and Verkade; Phosphorus Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
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122.
PLuCKTHUN A N D DENNIS
Hydrolysis
of
593
Phospholipids
~2
-7 Q
I
ι _j ι _j ι » ι « 1
I
2
3
4
5
6
7
8
9
10
Figure 1: The pH-dependence of the acyl migration of lysophospholipids is shown. The log of the pseudo first-order rate constant kjfor the rearrangement of 2-palmitoylsn-glycero-3-phosphorylcholine into l-palmitoyl-sn-glycero-3-phosphorylcholine is plotted against the pH buffered with 50m M tris HCl (·), 50mM citrate (O), or 0.1 M HCl and 160mM Triton X-100 (A) to solubilize the reaction products at acidic pH.
Quin and Verkade; Phosphorus Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
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PHOSPHORUS CHEMISTRY
sn-2 carbonyl group preferentially exposed at the interface as do micellar phospholipids (7). Although the isomeric lyso-phospholipids can in principle be distinguished by IR, ^H-NMR, C-NMR and ^P-NMR, only the latter provides the necessary sensitivity and well-resolved single reso nance peaks that make it suitable as a kinetic tool to study the migration of the acyl-chain between the sn-1 and sn-2 position. The acyl migration was found to be both base catalyzed with a second order rate constant of k£ = 160 M~l s"l and acid catalyzed, however much more slowly, with a second order rate constant of k2 = 4 χ 10-4 M-l s l (calculated between pH 1 and 2). The pH-rate profile is shown in Figure 1. At basic pH, the hydrolysis of the fatty acyl group is approximately 6500 times slower than migration. The equilibrium mixture in base contains approximately 90% of the 1-acyl isomer and 10% of the 2-acyl isomer. The phosphoryl mi gration was found to be too slow to measure, except at very acidic pH. Under these conditions, however, a variety of hydrolytic re actions also take place on a similar time scale so that the quan titation of the phosphoryl migration is complicated. Two factors make the preparation of phospholipids that are specifically labelled in one acyl chain with a high degree of purity rather difficult: Acyl-migration during the preparation of the monoacyl intermediate and acyl migration during the reacylation in organic solvents. Basic catalysts that are used in common re acylation procedures of lyso-phospholipids also catalyze the latter migration. However, the possibility of directly observing it by 31p-NMR provides a simple and convenient method to optimize condi tions which minimize acyl migration. 31p-NMR also allows one to test the isomeric purity of a- and β-phospholipids rapidly because the chemical shift of the β-phospholipid in mixed micelles is about 0.6 ppm upfield from the α-phospholipid. These compounds have been widely used in the study of model membranes. 13
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1. 2. 3. 4. 5. 6. 7.
Deems, R.A.; Eaton, B.R.; Dennis, E.A. J. Biol. Chem. 1975, 250, 9013. Roberts, M.F.; Deems, R.A.; Dennis, E.A. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 1950. Roberts, M.F.; Adamich, M.; Robson, R . J . ; Dennis, E.A. Biochemistry 1979, 18, 3301. Adamich, M.; Roberts, M.F.; Dennis, E.A. Biochemistry 1979, 18, 3308. Plűckthun, Α.; Dennis, E.A. J . Phys. Chem. 1981, 85, 678. Plűckthun, Α.; Dennis, E.A. Fed. Proc. 1981, 40, 1805. Roberts, M.F.; Bothner-By, A.A.; Dennis, E.A. Biochemistry 1978, 17, 935.
RECEIVED
June 30, 1981.
Quin and Verkade; Phosphorus Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
