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A high-performance liquid chromatography method for the simultaneous assay of diaminopimelate epimerase and decarboxylase
ANALYTICALBIOCHEMISTRY 1 8 0 , 2 9 8 - 3 0 2 (1989)
A High-Performance Liquid Chromatography Method for the Simultaneous Assay of Diaminopimelate Epimerase and Decarboxylase A. N. C. Weir,* C. B u c k e , t G. H o l t , t M. D. Lilly,$ a n d A. T. Bull *'1 The Institute for Biotechnological Studies, *University of Kent at Canterbury, Canterbury, Kent CT2 7PD, United Kingdom; t School of Biotechnology, Polytechnic of Central London, 115 New Cavendish Street, London WIM 8JS, United Kingdom; and $SERC Centre for Biochemical Engineering, Department of Chemical and Biochemical Engineering, University College London, Torrington Place, London WCIE 7JE, United Kingdom
Received November 4, 1988
A s e n s i t i v e and c o m p a r a t i v e l y s i m p l e m e t h o d for the assay of diaminopimelate (DAP) decarboxylase, which s i m u l t a n e o u s l y m o n i t o r s D A P e p i m e r a s e a c t i v i t y , in t h e r e v e r s e o f the b i o s y n t h e t i c d i r e c t i o n , is d e s c r i b e d . T h e s u b s t r a t e , m e s o - D A P and p r o d u c t s L L - D A P and Ll y s i n e a r e d e r i v a t i z e d w i t h o - p h t h a l d i a l d e h y d e and res o l v e d b y r e v e r s e d - p h a s e h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h y . S e p a r a t i o n is a c h i e v e d on a S p h e r i s o r b Cls c o l u m n u s i n g a g r a d i e n t e l u t i o n s y s t e m . T h i s t e c h n i q u e offers a h i g h d e g r e e o f s e n s i t i v i t y as the det e c t i o n m e t h o d d e s c r i b e d c a n m e a s u r e p i c o m o l e quantit i e s o f s u b s t r a t e and p r o d u c t s . © 1989 AcademicPress,Inc.
The enzyme diaminopimelate (DAP) 2 decarboxylase (meso-2,6-diaminopimelate carboxylyase, EC 4.1.1.20) catalyzes the final step in the lysine biosynthetic pathway in bacteria (1,2). Both the decarboxylase and the DAP epimerase (LL-2,6-diaminopimelate 2-epimerase, EC 5.1.1.7), which catalyzes the interconversion of the LL- and meso-isomers of DAP (Fig. 1), are of interest with regard to the regulation of the lysine pathway. Study of these enzymes is, in part, associated with their potential role in satisfying the changing demands for one or other DAP isomers and lysine which may result from changing physiological states, especially in sporulating organisms (3). In another quite separate field both the decarboxylase and the epimerase enzymes have been recognized as po1 To whom correspondence should be addressed. 2 Abbreviations used: diaminopimelate; OPA, o-phthaldialdehyde; SDS, sodium dodecyl sulfate.
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tential targets for antimicrobial agents (4). The absence of these enzymes in mammals means that any such agents would be selectively antimicrobial. The procedures currently available for the assay of both enzymes have a number of drawbacks. Assay of the epimerase requires the quantitative determination of one of the DAP isomers involved in the pathway (LL o r meso) in the presence of the other. Semiquantitative measurements of the isomer ratio which would allow estimation of epimerase activity can be performed using paper chromatography (5). The procedure described by White et al. (6) requires a purified decarboxylase preparation to specifically decarboxylase the meso-isomer. This reaction is followed by manometric measurement of the evolution of CO2 or by colorimetric determination of the lysine formed. The lysine may also be determined enzymatically as in the decarboxylase assay described by Asada et al. (2). One problem associated with the manometric method, which applies to the assay of both the epimerase and the decarboxylase enzymes, is the retention of CO2 in solution at optimal assay pH values. Moreover lysine decarboxylase activity may also be present in crude preparations and by decarboxylating the lysine, formed as a result of DAP decarboxylation to cadaverine, this enzyme would be responsible for a higher rate of meso-DAP-dependent C02 evolution than that attributable to DAP decarboxylase alone. The method described by Bartlett and White (7) provides a spectrophotometric system for the assay of epimerase activity under anaerobic conditions. However, this method also requires the partial purification of an additional enzyme, diaminopimelate dehydrogenase, to specifically quantify the meso-DAP produced. In this paper we describe a method, using meso-DAP as a substrate, to simultaneously assay DAP epimerase 0003-2697/89 $3.00 Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
C H R O M A T O G R A P H I C ASSAY OF D I A M I N O P I M E L A T E E P I M E R A S E AND DECARBOXYLASE
299
Preparation of Enzyme Extract
LL-DAP <
~--meso- DAP DAP eplmerase
F I G . 1.
J
0 r--L- L y s l n e
DAP decarboxylase
Metabolic relationship between DAP isomers and lysine.
and DAP decarboxylase activities by monitoring the rates of production of LL-DAP and lysine, respectively. The nature of the assay requires t h a t the epimerase be assayed in the reverse of the biosynthetic direction for simultaneous assay of the decarboxylase and t h a t only one enzyme may be assayed at its pH optimum. Very low (picomole) amounts of certain primary amino compounds (in this case, amino acids) may be quantified using reversed-phase H P L C of o-phthaldialdehyde (OPA) derivatives (8). As meso-DAP and LL-DAP are diastereomers they may be resolved chromatographically. These factors are exploited in a highly sensitive assay for both enzymes which (a) may be used on crude cell free preparations, (b) removes the need for a separate purified decarboxylase or dehydrogenase, and (c) avoids the problems associated with the measurement of C02 release.
Cell free extracts were prepared from lyophilized bacteria and resuspended in potassium phosphate buffer (100 mM, pH 7.0) containing 6 mM 2-mercaptoethanol. The cell suspension was passed twice through a French pressure cell (SLM Instruments, Urbana, IL) at 20,000 psi and cell debris was removed by centrifugation at 35,000g for 30 min; both procedures were carried out at 4°C. The supernatant was desalted on a Sephadex G-25 column and eluted with the above buffer. Protein determinations were made on desalted preparations using the dye-binding method (11) and bovine serum albumin as a standard.
Enzyme Assay
All reagents used were analytical grade unless otherwise stated. H P L C grade methanol, water, and sodium acetate were purchased from FSA Laboratory Supplies (Loughborough, England), o-phthaldialdehyde was purchased from Fluorochem Ltd. (Glossop, Derby, England), meso-Diaminopimelate was isolated from the commercial mixture of isomers by fractional recrystallisation using the procedure of Work (9).
The assay mixture was similar to t h a t described by Vogel and Hirvonen (12) except t h a t norvatine was included as an internal standard for the final H P L C analy~ sis. Each incubation mixture (1.5 ml) normally contained 15 #mol recrystallized meso-DAP, 0.1 #mol pyridoxal 5-phosphate, 3.75 t~mol norvaline, and approximately 0.5 mg protein as desalted cell free extract, in 100 mM potassium phosphate buffer, pH 7.0. The reaction was carried out at 37°C and started by addition of the cell free extract equilibrated at the required temperature. Samples (500 ttl) were taken immediately before and after this incubation period (normally 15 min). Initial studies on the linearity of LL-DAP and lysine production with respect to incubation time involved sam~ pling up to 120 min at 5- or 10-min intervals and therefore required larger starting volumes of reaction mixture. The reaction was stopped and the majority of protein removed by the method described by Unnithan et al. (13) whereby samples were quickly transferred to 10-ml Pyrex tubes each containing 1.0 ml sodium acetate buffer (1.2 M, pH 5.2) held in a boiling water bath. The tubes were boiled for a further 5 min and the contents centrifuged in a MSE microcentaur (MSE Scientific Instruments, Crawley, England). An aliquot of the supernatant was diluted 20-fold in water and used for subsequent H P L C analysis.
Organisms
Chromatographic Conditions and Analysis
A lysine overproducing m u t a n t of Bacillus subtilis NCIB 3610 was isolated from a nonsporing m u t a n t strain selected as resistant to S-(2-aminoethyl)-Lcysteine. This was grown in carbon-limited chemostat culture at 37°C, pH 7.0, a n d D = 0.25 h -1 on the minimal salts medium described by Lang et al. (10) and used to provide enzyme material for the assay development. The assay system was also tested on the wild-type B. subtilis strain NCIB 3610.
The o-phthaldialdehyde/2-mercaptoethanol derivatizing solution was prepared and maintained as described by U n n i t h a n et al. (13). Aliquots (200 ttl) of the diluted deproteinized assay samples were mixed with 200 ~1 of a sodium dodecyl sulfate (SDS) solution (2%, w/v) in sodium borate buffer (400 mM, pH 9.5) and the derivatizing reagent (400 #1) at room temperature. The SDS was added to improve the stability of the lysineOPA derivative (8). A 20-~1 sample of this mixture was
MATERIALS AND METHODS
Reagents
300
W E I R ET AL.
8
3
2
I
I
-10 Loglo
i
I
-9
-8
A m i n o acid ( m o l e s )
FIG. 3. Fluorescenceresponse (Ex 340 nm, Em 455 nm) for lysine (O) and meso-DAP (C) derivatives. 0
10 Time
20
30
(min)
FIG. 2. HPLC analysis of an enzyme reaction mixture after 30 min of incubation (see Materials and Methods for experimental details). The peaks are o-phthaldialdehyde derivatives of LL-DAP (1); m e s o DAP (2); norvaline (3); lysine (4).
injected onto the column after exactly 1 min derivatization. Separation of the derivatized reactants was achieved on a Spherisorb C18 ODS reversed-phase column (250 × 4.5 mm i.d.; particle size 5 t~m, P e r k i n - E l m e r , Beaconsfield, England) eluted with a linear gradient from 100% solvent A (30% methanol, 70% sodium acetate buffer, p H 5.9, 50 mM) to 30% solvent A and 70% methanol over a period of 35 min. A P e r k i n - E l m e r series A solvent delivery system was used and injections were made using a Rheodyne 7125 sample injector fitted with a 20 #l-loop. Eluted derivatives were detected with a Model LS 5B luminescence spectrometer (Perkin-E1mer) fitted with a L.C. flow cell, measuring fluorescence, (excitation at 340 nm, emission at 455 nm). T he chromatographic data were processed using a LC100 computing integrator (Perkin-Elmer). Enzyme activities are expressed as nanomoles product formed per minute per milligram protein. One unit of activity is defined as nanomoles product formed per minute. RESULTS An H P L C gradient elution program, previously used to resolve OPA derivatives of amino acids in fermentation broths, produced good separation of the derivatives of substrates and products from the enzyme assay (Fig. 2). Attempts to shorten the elution program resulted in
a loss of resolution of the DAP isomers. Therefore, the 35-min gradient elution program was used in which the last peak, the derivative of lysine, was eluted after approximately 30 min. T he reproducibilities of resolution, retention times, and peak areas were in keeping with those previously accepted for this method of separating and quantifying amino acids (8). T h e detector response also was found to be linear, within the range tested of 50 pmol to 5 nmol injected derivative of m e s o - D A P and lysine (Fig. 3). Although a preparation of LL-DAP was not available, the combined peak areas produced by standard concentrations of DAP after successive recrystallizations were similar. This result suggests t hat derivatives of the DAP isomers produce similar detector responses. Figure 4 describes the linear rates of lysine and LLDAP accumulation during the initial 30-min incubation period. When reaction mixtures were incubated for longer periods of time the rate of LL-DAP production 2.5
1.5 o
0.5 0 0
I
I
l
10
20
30
Time (min) F I G . 4. Lysine and LL-DAP formation as a function of incubation time, lysine (m) and LL-DAP (@). Volume refers to desalted, cell free extract.
CHROMATOGRAPHIC ASSAY OF DIAMINOPIMELATE EPIMERASE AND DECARBOXYLASE
__
//
eo
E
_~ 40
OI 0
i
0.2
o14
o.~
Protein (mg/ml) 60
g 20
0-
0
0
0.2
0.4
0.6
Protein (mg/ml)
FIG. 5. Rates of LL-DAP and lysine formation with respect to the amount of enzyme preparation in the reaction mixtures. Top, DAP decarboxylase; bottom, DAP epimerase. Concentration refers to milligram protein per milliliter desalted, cell free extract.
declined as this i s o m e r was utilized; i.e., t h e r a t e of conversion of LL-DAP b a c k to m e s o - D A P a p p r o a c h e s a n d t h e n exceeds the r a t e of c o n v e r s i o n of t h e m e s o to t h e LL-form. T h i s latter s i t u a t i o n is due to the c o n t i n u a l rem o v a l of m e s o - D A P b y the action of the decarboxylase, a reaction which goes to c o m p l e t i o n as lysine is not carb o x y l a t e d to f o r m D A P . T h e r a t e of p r o d u c t i o n of lysine r e m a i n e d c o n s t a n t for c o n s i d e r a b l y longer t h a n t h a t of LL-DAP for all the s a m p l e s assayed. Figure 5 shows t h a t the r e l a t i o n s h i p s b e t w e e n t h e rates of a c c u m u l a t i o n of LL-DAP a n d of lysine a n d the a m o u n t of e n z y m e p r e p a r a t i o n were linear w i t h i n the range tested. W h e n a n e n z y m e r e a c t i o n m i x t u r e was i n c u b a t e d for an e x t e n d e d period (ca. 6 h), it was f o u n d t h a t a l m o s t all t h e D A P was utilized a n d could be a c c o u n t e d for as lysine. T h e a s s a y m e t h o d was f o u n d to w o r k equally well for t h e assay of D A P d e c a r b o x y l a s e activity in desalted cell free e x t r a c t s of the lysine o v e r p r o d u c i n g m u t a n t a n d for t h e assay of d e c a r b o x y l a s e activity in two o t h e r organisms tested. T h e wild-type B. subtilis N C I B 3610 gave D A P decarboxylase activities similar to t h o s e o b t a i n e d for the m u t a n t derived f r o m it, generally a b o u t 110 n m o l m i n -1 m g -1 protein. B o t h s t r a i n s gave D A P e p i m e r a s e activities of a p p r o x i m a t e l y 85 n m o l rain -~ m g ~protein. DISCUSSION T h e a p p l i c a t i o n of H P L C t e c h n i q u e s to e n z y m e assays is now relatively w i d e s p r e a d (14). In c e r t a i n cases
301
the resolving p o w e r a n d sensitivity available offer dist i n c t a d v a n t a g e s over other, s o m e t i m e s m o r e t i m e - c o n suming, intricate, a n d less sensitive, m e t h o d s . One principal a d v a n t a g e of the a s s a y s y s t e m described here is the ability to m e a s u r e directly t h e p r i n c i p a l s u b s t r a t e s a n d p r o d u c t s of the two e n z y m e s concerned. Direct m e a s u r e m e n t of b o t h s u b s t r a t e a n d p r o d u c t s also m a k e s it possible to confirm t h e s t o i c h i o m e t r y of t h e reactions and, therefore, to e l i m i n a t e t h e possibility of o t h e r e n z y m e s such as lysine d e c a r b o x y l a s e or lysyl t R N A s y n t h e t a s e affecting the o b s e r v e d rate of lysine a c c u m u l a t i o n . W h e r e the m e t h o d is used to assay e p i m e r a s e a n d dec a r b o x y l a s e activity s i m u l t a n e o u s l y t h e required subs t r a t e is m e s o - D A P a n d t h e r e f o r e the e p i m e r a s e is ass a y e d in the reverse of the b i o s y n t h e t i c direction. T h e a s s a y e d rate in the reverse direction m a y be different to t h a t f o u n d in the f o r w a r d direction (6). H o w e v e r in our experience it s e e m s likely t h a t the LL-DAP i s o m e r (15) could be used as a s u b s t r a t e to a s s a y e p i m e r a s e activities in the b i o s y n t h e t i c direction. E x c l u s i o n of p y r i d o x a l p h o s p h a t e f r o m the r e a c t i o n m i x t u r e was sufficient to p r e v e n t d e c a r b o x y l a t i o n of m e s o - D A P b y desalted p r e p arations; this m i g h t be a u g m e n t e d b y the inclusion of h y d r a z i n e which inhibits the decarboxylase. T h i s H P L C a s s a y p r o c e d u r e has b e e n t e s t e d a n d used on a s y s t e m where the a m o u n t of s a m p l e available was n o t a limiting factor. H o w e v e r , the v o l u m e of the reaction m i x t u r e m a y be scaled down c o n s i d e r a b l y where only small q u a n t i t i e s of m a t e r i a l are available. W h e r e widely differing e p i m e r a s e a n d decarboxylase activities are p r e s e n t it m a y n o t be possible to simultaneously a s s a y b o t h enzymes. In c o m m o n w i t h certain o t h e r a p p l i c a t i o n s of H P L C t e c h n o l o g y to e n z y m e analysis the a s s a y principle a n d reaction m i x t u r e for b o t h the d e c a r b o x y l a s e a n d the e p i m e r a s e e n z y m e s r e m a i n similar to those previously described (6,12). As it is only the m e t h o d of q u a n t i f y i n g the p r o d u c t s of e n z y m e action t h a t is different f r o m these o t h e r p r o v e n a s s a y m e t h o d s , the n u m b e r of experim e n t s required to initially t e s t t h e validity of this H P L C m e t h o d was significantly reduced. ACKNOWLEDGMENTS The authors thank the Department of Trade and Industry, Glaxo Group Research, Grand Metropolitan, ICI Biological Products, Perkin-Elmer, Rhone-Poulenc, Shell Research, and Unilever Research for support of the research program of which the work described here was part. REFERENCES 1. White, P. J., and Kelly, B. (1965) Biochem. J. 96, 75-84. 2. Asada, Y., Tanizawa, K., Kawabata, Y., Misono, H., and Soda, K. (1981) Agric. Biol. Chem. 45, 1513-1514. 3. Rao, A. S. (1985) Arch. Microbiol. 141,143-150.
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4: Girodeau, J.-M., Agouridas, C., Masson, M., Pineau, R., and Le Goffic, F. (1986) J. Med. Chem. 29, 1023-1030. 5. Antia, M., Hoare, D. S., and Work, E. (1957) Biochem. J. 65,448459. 6. White, P. J., Lejeune, B., and Work, E. (1969) Biochem. J. 113, 589-601. 7. Bartlett, A. T. M., and White, P. J. (1985) J. Gen. Microbiol. 131, 2145-2152. 8. Jones, B. N., P~i~bo, S., and Stein, S. (1981) J. Liq. Chromatogr. 4,565-586. 9. Work, E. (1963) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., Eds.), Vol. 6, pp. 113-149, Academic Press, New York.
10. Lang, W. K., Glassey, K., and Archibald, A. R. (1982) J. Bacteriol. 151,367-375. 11. Bradford, M. M. (1976) Anal. Biochem. 72,248-254. 12. Vogel, H. J., and Hirvonen, A. P. (1971) in Methods in Enzymology (Tabor, H., and Tabor, C. W., Eds.), Vol. 17, pp. 146-150, Academic Press, New York. 13. Unnithan, S., Moraga, D. A., and Schuster, S. M. (1984) Anal. Biochem. 136, 195-201. 14. Rossomando, E. F. (1987) High Performance Liquid Chromatography in Enzymic Analysis: Applications to the Assay of Enzymatic Activity, Wiley, New York. 15. Saleh, F., and White, P. J. (1976) J. Gen. Microbiol. 96,253-261.
A High-Performance Liquid Chromatography Method for the Simultaneous Assay of Diaminopimelate Epimerase and Decarboxylase A. N. C. Weir,* C. B u c k e , t G. H o l t , t M. D. Lilly,$ a n d A. T. Bull *'1 The Institute for Biotechnological Studies, *University of Kent at Canterbury, Canterbury, Kent CT2 7PD, United Kingdom; t School of Biotechnology, Polytechnic of Central London, 115 New Cavendish Street, London WIM 8JS, United Kingdom; and $SERC Centre for Biochemical Engineering, Department of Chemical and Biochemical Engineering, University College London, Torrington Place, London WCIE 7JE, United Kingdom
Received November 4, 1988
A s e n s i t i v e and c o m p a r a t i v e l y s i m p l e m e t h o d for the assay of diaminopimelate (DAP) decarboxylase, which s i m u l t a n e o u s l y m o n i t o r s D A P e p i m e r a s e a c t i v i t y , in t h e r e v e r s e o f the b i o s y n t h e t i c d i r e c t i o n , is d e s c r i b e d . T h e s u b s t r a t e , m e s o - D A P and p r o d u c t s L L - D A P and Ll y s i n e a r e d e r i v a t i z e d w i t h o - p h t h a l d i a l d e h y d e and res o l v e d b y r e v e r s e d - p h a s e h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h y . S e p a r a t i o n is a c h i e v e d on a S p h e r i s o r b Cls c o l u m n u s i n g a g r a d i e n t e l u t i o n s y s t e m . T h i s t e c h n i q u e offers a h i g h d e g r e e o f s e n s i t i v i t y as the det e c t i o n m e t h o d d e s c r i b e d c a n m e a s u r e p i c o m o l e quantit i e s o f s u b s t r a t e and p r o d u c t s . © 1989 AcademicPress,Inc.
The enzyme diaminopimelate (DAP) 2 decarboxylase (meso-2,6-diaminopimelate carboxylyase, EC 4.1.1.20) catalyzes the final step in the lysine biosynthetic pathway in bacteria (1,2). Both the decarboxylase and the DAP epimerase (LL-2,6-diaminopimelate 2-epimerase, EC 5.1.1.7), which catalyzes the interconversion of the LL- and meso-isomers of DAP (Fig. 1), are of interest with regard to the regulation of the lysine pathway. Study of these enzymes is, in part, associated with their potential role in satisfying the changing demands for one or other DAP isomers and lysine which may result from changing physiological states, especially in sporulating organisms (3). In another quite separate field both the decarboxylase and the epimerase enzymes have been recognized as po1 To whom correspondence should be addressed. 2 Abbreviations used: diaminopimelate; OPA, o-phthaldialdehyde; SDS, sodium dodecyl sulfate.
298
tential targets for antimicrobial agents (4). The absence of these enzymes in mammals means that any such agents would be selectively antimicrobial. The procedures currently available for the assay of both enzymes have a number of drawbacks. Assay of the epimerase requires the quantitative determination of one of the DAP isomers involved in the pathway (LL o r meso) in the presence of the other. Semiquantitative measurements of the isomer ratio which would allow estimation of epimerase activity can be performed using paper chromatography (5). The procedure described by White et al. (6) requires a purified decarboxylase preparation to specifically decarboxylase the meso-isomer. This reaction is followed by manometric measurement of the evolution of CO2 or by colorimetric determination of the lysine formed. The lysine may also be determined enzymatically as in the decarboxylase assay described by Asada et al. (2). One problem associated with the manometric method, which applies to the assay of both the epimerase and the decarboxylase enzymes, is the retention of CO2 in solution at optimal assay pH values. Moreover lysine decarboxylase activity may also be present in crude preparations and by decarboxylating the lysine, formed as a result of DAP decarboxylation to cadaverine, this enzyme would be responsible for a higher rate of meso-DAP-dependent C02 evolution than that attributable to DAP decarboxylase alone. The method described by Bartlett and White (7) provides a spectrophotometric system for the assay of epimerase activity under anaerobic conditions. However, this method also requires the partial purification of an additional enzyme, diaminopimelate dehydrogenase, to specifically quantify the meso-DAP produced. In this paper we describe a method, using meso-DAP as a substrate, to simultaneously assay DAP epimerase 0003-2697/89 $3.00 Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
C H R O M A T O G R A P H I C ASSAY OF D I A M I N O P I M E L A T E E P I M E R A S E AND DECARBOXYLASE
299
Preparation of Enzyme Extract
LL-DAP <
~--meso- DAP DAP eplmerase
F I G . 1.
J
0 r--L- L y s l n e
DAP decarboxylase
Metabolic relationship between DAP isomers and lysine.
and DAP decarboxylase activities by monitoring the rates of production of LL-DAP and lysine, respectively. The nature of the assay requires t h a t the epimerase be assayed in the reverse of the biosynthetic direction for simultaneous assay of the decarboxylase and t h a t only one enzyme may be assayed at its pH optimum. Very low (picomole) amounts of certain primary amino compounds (in this case, amino acids) may be quantified using reversed-phase H P L C of o-phthaldialdehyde (OPA) derivatives (8). As meso-DAP and LL-DAP are diastereomers they may be resolved chromatographically. These factors are exploited in a highly sensitive assay for both enzymes which (a) may be used on crude cell free preparations, (b) removes the need for a separate purified decarboxylase or dehydrogenase, and (c) avoids the problems associated with the measurement of C02 release.
Cell free extracts were prepared from lyophilized bacteria and resuspended in potassium phosphate buffer (100 mM, pH 7.0) containing 6 mM 2-mercaptoethanol. The cell suspension was passed twice through a French pressure cell (SLM Instruments, Urbana, IL) at 20,000 psi and cell debris was removed by centrifugation at 35,000g for 30 min; both procedures were carried out at 4°C. The supernatant was desalted on a Sephadex G-25 column and eluted with the above buffer. Protein determinations were made on desalted preparations using the dye-binding method (11) and bovine serum albumin as a standard.
Enzyme Assay
All reagents used were analytical grade unless otherwise stated. H P L C grade methanol, water, and sodium acetate were purchased from FSA Laboratory Supplies (Loughborough, England), o-phthaldialdehyde was purchased from Fluorochem Ltd. (Glossop, Derby, England), meso-Diaminopimelate was isolated from the commercial mixture of isomers by fractional recrystallisation using the procedure of Work (9).
The assay mixture was similar to t h a t described by Vogel and Hirvonen (12) except t h a t norvatine was included as an internal standard for the final H P L C analy~ sis. Each incubation mixture (1.5 ml) normally contained 15 #mol recrystallized meso-DAP, 0.1 #mol pyridoxal 5-phosphate, 3.75 t~mol norvaline, and approximately 0.5 mg protein as desalted cell free extract, in 100 mM potassium phosphate buffer, pH 7.0. The reaction was carried out at 37°C and started by addition of the cell free extract equilibrated at the required temperature. Samples (500 ttl) were taken immediately before and after this incubation period (normally 15 min). Initial studies on the linearity of LL-DAP and lysine production with respect to incubation time involved sam~ pling up to 120 min at 5- or 10-min intervals and therefore required larger starting volumes of reaction mixture. The reaction was stopped and the majority of protein removed by the method described by Unnithan et al. (13) whereby samples were quickly transferred to 10-ml Pyrex tubes each containing 1.0 ml sodium acetate buffer (1.2 M, pH 5.2) held in a boiling water bath. The tubes were boiled for a further 5 min and the contents centrifuged in a MSE microcentaur (MSE Scientific Instruments, Crawley, England). An aliquot of the supernatant was diluted 20-fold in water and used for subsequent H P L C analysis.
Organisms
Chromatographic Conditions and Analysis
A lysine overproducing m u t a n t of Bacillus subtilis NCIB 3610 was isolated from a nonsporing m u t a n t strain selected as resistant to S-(2-aminoethyl)-Lcysteine. This was grown in carbon-limited chemostat culture at 37°C, pH 7.0, a n d D = 0.25 h -1 on the minimal salts medium described by Lang et al. (10) and used to provide enzyme material for the assay development. The assay system was also tested on the wild-type B. subtilis strain NCIB 3610.
The o-phthaldialdehyde/2-mercaptoethanol derivatizing solution was prepared and maintained as described by U n n i t h a n et al. (13). Aliquots (200 ttl) of the diluted deproteinized assay samples were mixed with 200 ~1 of a sodium dodecyl sulfate (SDS) solution (2%, w/v) in sodium borate buffer (400 mM, pH 9.5) and the derivatizing reagent (400 #1) at room temperature. The SDS was added to improve the stability of the lysineOPA derivative (8). A 20-~1 sample of this mixture was
MATERIALS AND METHODS
Reagents
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8
3
2
I
I
-10 Loglo
i
I
-9
-8
A m i n o acid ( m o l e s )
FIG. 3. Fluorescenceresponse (Ex 340 nm, Em 455 nm) for lysine (O) and meso-DAP (C) derivatives. 0
10 Time
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30
(min)
FIG. 2. HPLC analysis of an enzyme reaction mixture after 30 min of incubation (see Materials and Methods for experimental details). The peaks are o-phthaldialdehyde derivatives of LL-DAP (1); m e s o DAP (2); norvaline (3); lysine (4).
injected onto the column after exactly 1 min derivatization. Separation of the derivatized reactants was achieved on a Spherisorb C18 ODS reversed-phase column (250 × 4.5 mm i.d.; particle size 5 t~m, P e r k i n - E l m e r , Beaconsfield, England) eluted with a linear gradient from 100% solvent A (30% methanol, 70% sodium acetate buffer, p H 5.9, 50 mM) to 30% solvent A and 70% methanol over a period of 35 min. A P e r k i n - E l m e r series A solvent delivery system was used and injections were made using a Rheodyne 7125 sample injector fitted with a 20 #l-loop. Eluted derivatives were detected with a Model LS 5B luminescence spectrometer (Perkin-E1mer) fitted with a L.C. flow cell, measuring fluorescence, (excitation at 340 nm, emission at 455 nm). T he chromatographic data were processed using a LC100 computing integrator (Perkin-Elmer). Enzyme activities are expressed as nanomoles product formed per minute per milligram protein. One unit of activity is defined as nanomoles product formed per minute. RESULTS An H P L C gradient elution program, previously used to resolve OPA derivatives of amino acids in fermentation broths, produced good separation of the derivatives of substrates and products from the enzyme assay (Fig. 2). Attempts to shorten the elution program resulted in
a loss of resolution of the DAP isomers. Therefore, the 35-min gradient elution program was used in which the last peak, the derivative of lysine, was eluted after approximately 30 min. T he reproducibilities of resolution, retention times, and peak areas were in keeping with those previously accepted for this method of separating and quantifying amino acids (8). T h e detector response also was found to be linear, within the range tested of 50 pmol to 5 nmol injected derivative of m e s o - D A P and lysine (Fig. 3). Although a preparation of LL-DAP was not available, the combined peak areas produced by standard concentrations of DAP after successive recrystallizations were similar. This result suggests t hat derivatives of the DAP isomers produce similar detector responses. Figure 4 describes the linear rates of lysine and LLDAP accumulation during the initial 30-min incubation period. When reaction mixtures were incubated for longer periods of time the rate of LL-DAP production 2.5
1.5 o
0.5 0 0
I
I
l
10
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30
Time (min) F I G . 4. Lysine and LL-DAP formation as a function of incubation time, lysine (m) and LL-DAP (@). Volume refers to desalted, cell free extract.
CHROMATOGRAPHIC ASSAY OF DIAMINOPIMELATE EPIMERASE AND DECARBOXYLASE
__
//
eo
E
_~ 40
OI 0
i
0.2
o14
o.~
Protein (mg/ml) 60
g 20
0-
0
0
0.2
0.4
0.6
Protein (mg/ml)
FIG. 5. Rates of LL-DAP and lysine formation with respect to the amount of enzyme preparation in the reaction mixtures. Top, DAP decarboxylase; bottom, DAP epimerase. Concentration refers to milligram protein per milliliter desalted, cell free extract.
declined as this i s o m e r was utilized; i.e., t h e r a t e of conversion of LL-DAP b a c k to m e s o - D A P a p p r o a c h e s a n d t h e n exceeds the r a t e of c o n v e r s i o n of t h e m e s o to t h e LL-form. T h i s latter s i t u a t i o n is due to the c o n t i n u a l rem o v a l of m e s o - D A P b y the action of the decarboxylase, a reaction which goes to c o m p l e t i o n as lysine is not carb o x y l a t e d to f o r m D A P . T h e r a t e of p r o d u c t i o n of lysine r e m a i n e d c o n s t a n t for c o n s i d e r a b l y longer t h a n t h a t of LL-DAP for all the s a m p l e s assayed. Figure 5 shows t h a t the r e l a t i o n s h i p s b e t w e e n t h e rates of a c c u m u l a t i o n of LL-DAP a n d of lysine a n d the a m o u n t of e n z y m e p r e p a r a t i o n were linear w i t h i n the range tested. W h e n a n e n z y m e r e a c t i o n m i x t u r e was i n c u b a t e d for an e x t e n d e d period (ca. 6 h), it was f o u n d t h a t a l m o s t all t h e D A P was utilized a n d could be a c c o u n t e d for as lysine. T h e a s s a y m e t h o d was f o u n d to w o r k equally well for t h e assay of D A P d e c a r b o x y l a s e activity in desalted cell free e x t r a c t s of the lysine o v e r p r o d u c i n g m u t a n t a n d for t h e assay of d e c a r b o x y l a s e activity in two o t h e r organisms tested. T h e wild-type B. subtilis N C I B 3610 gave D A P decarboxylase activities similar to t h o s e o b t a i n e d for the m u t a n t derived f r o m it, generally a b o u t 110 n m o l m i n -1 m g -1 protein. B o t h s t r a i n s gave D A P e p i m e r a s e activities of a p p r o x i m a t e l y 85 n m o l rain -~ m g ~protein. DISCUSSION T h e a p p l i c a t i o n of H P L C t e c h n i q u e s to e n z y m e assays is now relatively w i d e s p r e a d (14). In c e r t a i n cases
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the resolving p o w e r a n d sensitivity available offer dist i n c t a d v a n t a g e s over other, s o m e t i m e s m o r e t i m e - c o n suming, intricate, a n d less sensitive, m e t h o d s . One principal a d v a n t a g e of the a s s a y s y s t e m described here is the ability to m e a s u r e directly t h e p r i n c i p a l s u b s t r a t e s a n d p r o d u c t s of the two e n z y m e s concerned. Direct m e a s u r e m e n t of b o t h s u b s t r a t e a n d p r o d u c t s also m a k e s it possible to confirm t h e s t o i c h i o m e t r y of t h e reactions and, therefore, to e l i m i n a t e t h e possibility of o t h e r e n z y m e s such as lysine d e c a r b o x y l a s e or lysyl t R N A s y n t h e t a s e affecting the o b s e r v e d rate of lysine a c c u m u l a t i o n . W h e r e the m e t h o d is used to assay e p i m e r a s e a n d dec a r b o x y l a s e activity s i m u l t a n e o u s l y t h e required subs t r a t e is m e s o - D A P a n d t h e r e f o r e the e p i m e r a s e is ass a y e d in the reverse of the b i o s y n t h e t i c direction. T h e a s s a y e d rate in the reverse direction m a y be different to t h a t f o u n d in the f o r w a r d direction (6). H o w e v e r in our experience it s e e m s likely t h a t the LL-DAP i s o m e r (15) could be used as a s u b s t r a t e to a s s a y e p i m e r a s e activities in the b i o s y n t h e t i c direction. E x c l u s i o n of p y r i d o x a l p h o s p h a t e f r o m the r e a c t i o n m i x t u r e was sufficient to p r e v e n t d e c a r b o x y l a t i o n of m e s o - D A P b y desalted p r e p arations; this m i g h t be a u g m e n t e d b y the inclusion of h y d r a z i n e which inhibits the decarboxylase. T h i s H P L C a s s a y p r o c e d u r e has b e e n t e s t e d a n d used on a s y s t e m where the a m o u n t of s a m p l e available was n o t a limiting factor. H o w e v e r , the v o l u m e of the reaction m i x t u r e m a y be scaled down c o n s i d e r a b l y where only small q u a n t i t i e s of m a t e r i a l are available. W h e r e widely differing e p i m e r a s e a n d decarboxylase activities are p r e s e n t it m a y n o t be possible to simultaneously a s s a y b o t h enzymes. In c o m m o n w i t h certain o t h e r a p p l i c a t i o n s of H P L C t e c h n o l o g y to e n z y m e analysis the a s s a y principle a n d reaction m i x t u r e for b o t h the d e c a r b o x y l a s e a n d the e p i m e r a s e e n z y m e s r e m a i n similar to those previously described (6,12). As it is only the m e t h o d of q u a n t i f y i n g the p r o d u c t s of e n z y m e action t h a t is different f r o m these o t h e r p r o v e n a s s a y m e t h o d s , the n u m b e r of experim e n t s required to initially t e s t t h e validity of this H P L C m e t h o d was significantly reduced. ACKNOWLEDGMENTS The authors thank the Department of Trade and Industry, Glaxo Group Research, Grand Metropolitan, ICI Biological Products, Perkin-Elmer, Rhone-Poulenc, Shell Research, and Unilever Research for support of the research program of which the work described here was part. REFERENCES 1. White, P. J., and Kelly, B. (1965) Biochem. J. 96, 75-84. 2. Asada, Y., Tanizawa, K., Kawabata, Y., Misono, H., and Soda, K. (1981) Agric. Biol. Chem. 45, 1513-1514. 3. Rao, A. S. (1985) Arch. Microbiol. 141,143-150.
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4: Girodeau, J.-M., Agouridas, C., Masson, M., Pineau, R., and Le Goffic, F. (1986) J. Med. Chem. 29, 1023-1030. 5. Antia, M., Hoare, D. S., and Work, E. (1957) Biochem. J. 65,448459. 6. White, P. J., Lejeune, B., and Work, E. (1969) Biochem. J. 113, 589-601. 7. Bartlett, A. T. M., and White, P. J. (1985) J. Gen. Microbiol. 131, 2145-2152. 8. Jones, B. N., P~i~bo, S., and Stein, S. (1981) J. Liq. Chromatogr. 4,565-586. 9. Work, E. (1963) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., Eds.), Vol. 6, pp. 113-149, Academic Press, New York.
10. Lang, W. K., Glassey, K., and Archibald, A. R. (1982) J. Bacteriol. 151,367-375. 11. Bradford, M. M. (1976) Anal. Biochem. 72,248-254. 12. Vogel, H. J., and Hirvonen, A. P. (1971) in Methods in Enzymology (Tabor, H., and Tabor, C. W., Eds.), Vol. 17, pp. 146-150, Academic Press, New York. 13. Unnithan, S., Moraga, D. A., and Schuster, S. M. (1984) Anal. Biochem. 136, 195-201. 14. Rossomando, E. F. (1987) High Performance Liquid Chromatography in Enzymic Analysis: Applications to the Assay of Enzymatic Activity, Wiley, New York. 15. Saleh, F., and White, P. J. (1976) J. Gen. Microbiol. 96,253-261.