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Active site interactions in the bifunctional aspartokinase I-homoserine dehydrogenase I of Escherichia coli K12
Biochimica et Biophysica Acta, O0 (1984) 98-100
98
Elsevier
BBA Report BBA 30079
ACTIVE SITE I N T E R A C T I O N S IN T H E B I F U N C T I O N A L A S P A R T O K I N A S E IH O M O S E R I N E D E H Y D R O G E N A S E I OF E S C H E R I C H I A C O L t KI2 L E L A N D P. VICKERS and ROBERT MOBLEY, Jr.
Department of Chemistry and the Laboratory of Microbial and Biochemical Sciences, Georgia State University, Atlanta, GA
30303 (U.S.A.)
(Received May 1st, 1984)
Key words: Aspartokinase I," Homoserine dehydrogenase 1," Active site," (E. coli K12)
The combination of L-aspartate and the magnesium ion complex of A D P results in an activation of the dehydrogenase activity of aspartokinase I-homoserine dehydrogenase I. This activation is not seen with M g X D P alone and is greater than the activation by L-aspartate alone. Attempts to fill with other anions that gap between aspartate and M g A D P which would normally be filled with the terminal phosphate of A T P did not result in further activation. The results are consistent with the proposal that the utility of this bifunctional enzyme includes both the inter-site communication and inhibition of both activities by threonine.
Aspartokinase I-homoserine dehydrogenase I from Escherichia coli is a tetrameric enzyme composed of identical subunits, each of which is bifunctional. The two activities appear to reside in regions of the polypeptide chain which unfold [1] and refold [2] independently. However, there is evidence that the two active sites are not completely independent in the active enzyme. In this paper we present additional evidence that the binding of substrates in the kinase site is communicated to the dehydrogenase site. The enzyme was isolated from E. coli K12 strain TIR-8 [3], which was provided to us by Dr. Umbarger. The bacteria were grown and harvested and the enzyme purified by a modification of a previously described procedure [4]. Significant differences were the use of a New Brunswick Scientific IF130 fermentor and cell disruption by the use of a Gaulin homogenizer. The reverse direction dehydrogenase assay (hom o s e r i n e ~ aspartate fl-semialdehyde) was employed in these studies. The initial velocity was measured by following the appearance of N A D P H spectrophotometrically at 340 nm with a Spec0167-4838/84/$03.00 © 1984 Elsevier Science Publishers B.V.
tronic 2000 with cuvet holders maintained at 25 o C. Reaction mixtures had a total volume of 3 ml and contained 0.1 M Tris-HC1 buffer (pH 8.9), 330 # M N A D P +, 25 mM oL-homoserine, and other reagents as described. The salt concentration was maintained constant by addition of CsC1 to make the total of KC1 and CsC1 equal 0.1 M. Fig. 1A shows the effect of the M g A D P complex on the dehydrogenase initial velocity. A D P and MgC12 were added in equimolar amounts and the concentration of the complex was calculated from published stability constants [5]. The highest concentration (1.2 mM MgADP) results from the mixture of 2 mM MgC12 and 2 mM ADP. In the absence of L-aspartate the M g A D P complex has no effect. However, in the presence of 25 mM L-aspartate, M g A D P is an activator with maximal activation being reached at approximately 1 mM. Fig. 1B shows the optimal concentration of Laspartate for the activation by MgADP. Decreases in the aspartate activation at concentrations above 30 mM may be due to complexation of magnesium ion by aspartate and the consequent reduction in M g A D P concentration. This effect would also
99
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100
B .1 O O
0
"~ 0.03 x.
"~ 8 0
~
>
o
o
E_ 6o
<~ 0.02'
x
E 40
0.01 c
o
0.4 0.8 I'MgADP], mM
1.2
10 20 30 40 EEL-asp], mM
20
a.
I
0 Fig. 1. A. Effect of M g A D P o n the reverse d i r e c t i o n d e h y d r o g e n a s e initial velocity. The filled circles are assay m i x t u r e s c o n t a i n i n g no L-aspartate a n d the open circles c o n t a i n 25 m M L-aspartate. The K + c o n c e n t r a t i o n was 10 raM. The o r d i n a t e is initial velocity as c h a n g e in a b s o r b a n c e at 340 nm. B. Effect of L-aspartate (L-asp) c o n c e n t r a t i o n on the reverse d i r e c t i o n dehyd r o g e n a s e initial velocity in the presence of 2 m M MgCI 2 a n d A D P . The K + c o n c e n t r a t i o n was 10 raM.
lower the M g A D P c o n c e n t r a t i o n s p l o t t e d in Fig. 1A on the abscissa. F u r t h e r studies were c a r r i e d o u t at 25 m M L - a s p a r t a t e a n d 2 m M M g C I 2 a n d ADP. Fig. 2 illustrates the lack of a n effect o f M g A D P a l o n e on the d e h y d r o g e n a s e assay a n d the m a r k e d effect o f the c o m b i n a t i o n o f L-aspartate a n d M g A D P . T h e curve in the presence of b o t h effectors (circles) follows M i c h a e l i s - M e n t e n kinetics with a K m of 5.0 ___1.0 m M for K +. N o n c o o p e r a tive kinetics were also r e p o r t e d in the presence of M g A T P [6], a n d a K m o f 4.6 + 0.3 m M was calcul a t e d b y Ogilvie et al [7]. F i t t i n g was d o n e b y a n o n l i n e a r m o d i f i e d G a u s s - N e w t o n procedure. A s s a y d a t a in the presence of L-aspartate alone has b e e n r e p o r t e d [6] a n d lies b e t w e e n the two curves in Fig. 2. A s well as being n o n c o o p e r a t i v e l y a c t i v a t e d b y K + in the presence o f L-aspartate a n d M g A D P , the e n z y m e also has a m a x i m a l velocity which is a b o u t 20% higher t h a n the e n z y m e in the a b s e n c e of effectors, as is the case with M g A T P [7]. T h e initial velocity d a t a in the presence of M g A D P (crosses) fall o n the s a m e curve as the d a t a in the a b s e n c e of effectors (squares). F i t s to these two d a t a sets i n d i v i d u a l l y gave a K + conc e n t r a t i o n for h a l f - m a x i m a l activity of 17 m M with no effectors p r e s e n t a n d 14 m M in the pres-
I
20
I
I
I
40
I
I
60 FK +
"1,
I
80
I
I
100
mM
Fig. 2. Effect of K + concentration on the reverse direction dehydrogenase initial velocity. Assay conditions are described in the text. No added effectors (O), 2 raM MgCI2 and ADP (×), and 25 mM L-aspartate plus 2 mM MgCI2 and ADP (O). Initial velocity is plotted as a percentage of the maximal velocity for the assays in the presence of L-aspartate and MgADP.
ence of M g A D P . T h e Hill coefficients were calcul a t e d to be 1.2 a n d 1.3, respectively Both sets gave the same best-fit m a x i m a l velocity. T h e lower solid line in Fig. 2 was d r a w n with a Hill coefficient of 1.25 a n d a K0. 5 of 15.5 m M K +. The velocity was expressed as a fraction o f the m a x i m a l velocity in the presence o f M g A D P a n d aspartate. It has b e e n r e p o r t e d that a n u m b e r of a n i o n s can b i n d to the active site of creatine k i n a s e [8] a n d i n h i b i t this enzyme. A p a r t i c u l a r l y effective c o m p l e x is the b i n d i n g of a p l a n a r a n i o n such as S C N - or NO3-- b e t w e e n creatine a n d A D P , thus t a k i n g the p o s i t i o n o f the t e r m i n a l p h o s p h a t e of A T P [9]. W e m a d e n u m e r o u s a t t e m p t s to observe a n effect of a d d e d S C N - or N O 3 on the activation of h o m o s e r i n e d e h y d r o g e n a s e b y a s p a r t a t e a n d a s p a r t a t e plus M g A D P , b u t saw none, u p to c o n c e n t r a t i o n s of 10 m M K S C N a n d K N O 3. T h e conclusion m u s t b e that either these anions d o n o t f o r m an active site c o m p l e x with t h i s kinase or t h a t M g A D P plus L-aspartate exert the m a x i m a l effect on the d e h y d r o g e n a s e site. M e c h a n i s t i c similarities o r differences b e t w e e n creatine kinase a n d a s p a r t o k i n a s e I are n o t known, except that neither e n z y m e utilizes a covalent e n z y m e - b o u n d phosp h a t e i n t e r m e d i a t e . H i g h e r c o n c e n t r a t i o n s of
100 KSCN effect an irreversible dissociation of the enzyme to monomers [4]. All structural information available on aspartokinase I-homoserine dehydrogenase I indicates that the two active sites reside in distant regions of the primary sequence [10] and in structural domains which unfold [1] and refold [2] independently. However, there is also evidence that the two active sites communicate with each other. For example, it is known that a dehydrogenase substrate can protect both activities to heat inactivation [11] and that MgATP can affect the monovalent cation activation of the dehydrogenase [6,7,12]. Additionally, at low K ÷ concentration threonine inhibition of the dehydrogenase activity differs in the absence and presence of MgATP [12]. The studies presented in this publication indicate that specific parts of the kinase active site appear to have more effect in facilitating a conformational change which is communicated to the sites of K ÷ binding a n d / o r the dehydrogenase active site. MgATP has an effect on the dehydrogenase activity, but M g A D P does not, indicating an essential role for the terminal phosphate. However, the addition of aspartate to the MgADP" enzyme complex produces the same effect as MgATP alone. Thus, aspartokinase substrates and products differ markedly in their ability to alter the monovalent cation activation of homoserine dehydrogenase, presumably due to different interactions with subsites of the kinase active site. These studies indicate that this bifunctional enzyme which cannot channel substrates has adap-
tive advantages beyond the fact that both activities share a common feedback inhibitor, L-threonine and common repressors of their synthesis. The presence of kinase substrates and products can modulate the dehydrogenase activity. Differential scanning calorimetry studies are presently being carried out to determine the effect of these kinase substrates on the unfolding of both the kinase and dehydrogenase domains. We acknowledge the support of the Research Corporation and the Petroleum Research Fund of the American Chemical Society.
References 1 Mailer, K. and Garel, J.-R. (1984) Biochemistry23, 651-654 2 Dautry-Varsat, A. and Garel, J.-R. (1981) Biochemistry20, 1396-1401 3 Szentirmai, A., Szentirmai, M. and Umbarger, H.E. (1968) J. Bacteriol. 95, 1672-1679 4 Vickers, L.P., Ackers, G.K. and Ogilvie,J.W. (1978) J. Biol. Chem. 253, 2155-2160 5 O'Sullivan, W.J. and Smithers, G.W. (1979) Methods Enzymol. 63, 294-336 6 Broussard, L., Cunningham,G.N., Starnes, W.L. and Shire, W. (1972) Biochem. Biophys Res. Commun.46, 1181-1186 70gilvie, J.W., Vickers, L.P., Clark, R.B. and Jones, M.M. (1975) J. Biol. Chem. 250, 1242-1260 8 Watts, D.C. (1973) The Enzymes8, 383-455 9 Reed, G.H., Barlow, C.H. and Burns, R.A., Jr. (1978) J. Biol. Chem. 253, 4153-4158 10 Fazel, A., Mi~ller, K., LeBras, G., Garel, J.-R., V6ron, M. and Cohen, G.N. (1983) Biochemistry22, 158-165 11 Patte, J.-C., Truffa-Bachi, P. and Cohen, G.N. (1966) Biochim. Biophys. Acta 128, 426-439 12 Truffa-Bachi, P., Costrejean, J.-M., Py, M.-C. and Cohen, G.N. (1974) Biochimie, 56, 215-219
98
Elsevier
BBA Report BBA 30079
ACTIVE SITE I N T E R A C T I O N S IN T H E B I F U N C T I O N A L A S P A R T O K I N A S E IH O M O S E R I N E D E H Y D R O G E N A S E I OF E S C H E R I C H I A C O L t KI2 L E L A N D P. VICKERS and ROBERT MOBLEY, Jr.
Department of Chemistry and the Laboratory of Microbial and Biochemical Sciences, Georgia State University, Atlanta, GA
30303 (U.S.A.)
(Received May 1st, 1984)
Key words: Aspartokinase I," Homoserine dehydrogenase 1," Active site," (E. coli K12)
The combination of L-aspartate and the magnesium ion complex of A D P results in an activation of the dehydrogenase activity of aspartokinase I-homoserine dehydrogenase I. This activation is not seen with M g X D P alone and is greater than the activation by L-aspartate alone. Attempts to fill with other anions that gap between aspartate and M g A D P which would normally be filled with the terminal phosphate of A T P did not result in further activation. The results are consistent with the proposal that the utility of this bifunctional enzyme includes both the inter-site communication and inhibition of both activities by threonine.
Aspartokinase I-homoserine dehydrogenase I from Escherichia coli is a tetrameric enzyme composed of identical subunits, each of which is bifunctional. The two activities appear to reside in regions of the polypeptide chain which unfold [1] and refold [2] independently. However, there is evidence that the two active sites are not completely independent in the active enzyme. In this paper we present additional evidence that the binding of substrates in the kinase site is communicated to the dehydrogenase site. The enzyme was isolated from E. coli K12 strain TIR-8 [3], which was provided to us by Dr. Umbarger. The bacteria were grown and harvested and the enzyme purified by a modification of a previously described procedure [4]. Significant differences were the use of a New Brunswick Scientific IF130 fermentor and cell disruption by the use of a Gaulin homogenizer. The reverse direction dehydrogenase assay (hom o s e r i n e ~ aspartate fl-semialdehyde) was employed in these studies. The initial velocity was measured by following the appearance of N A D P H spectrophotometrically at 340 nm with a Spec0167-4838/84/$03.00 © 1984 Elsevier Science Publishers B.V.
tronic 2000 with cuvet holders maintained at 25 o C. Reaction mixtures had a total volume of 3 ml and contained 0.1 M Tris-HC1 buffer (pH 8.9), 330 # M N A D P +, 25 mM oL-homoserine, and other reagents as described. The salt concentration was maintained constant by addition of CsC1 to make the total of KC1 and CsC1 equal 0.1 M. Fig. 1A shows the effect of the M g A D P complex on the dehydrogenase initial velocity. A D P and MgC12 were added in equimolar amounts and the concentration of the complex was calculated from published stability constants [5]. The highest concentration (1.2 mM MgADP) results from the mixture of 2 mM MgC12 and 2 mM ADP. In the absence of L-aspartate the M g A D P complex has no effect. However, in the presence of 25 mM L-aspartate, M g A D P is an activator with maximal activation being reached at approximately 1 mM. Fig. 1B shows the optimal concentration of Laspartate for the activation by MgADP. Decreases in the aspartate activation at concentrations above 30 mM may be due to complexation of magnesium ion by aspartate and the consequent reduction in M g A D P concentration. This effect would also
99
0.04
100
B .1 O O
0
"~ 0.03 x.
"~ 8 0
~
>
o
o
E_ 6o
<~ 0.02'
x
E 40
0.01 c
o
0.4 0.8 I'MgADP], mM
1.2
10 20 30 40 EEL-asp], mM
20
a.
I
0 Fig. 1. A. Effect of M g A D P o n the reverse d i r e c t i o n d e h y d r o g e n a s e initial velocity. The filled circles are assay m i x t u r e s c o n t a i n i n g no L-aspartate a n d the open circles c o n t a i n 25 m M L-aspartate. The K + c o n c e n t r a t i o n was 10 raM. The o r d i n a t e is initial velocity as c h a n g e in a b s o r b a n c e at 340 nm. B. Effect of L-aspartate (L-asp) c o n c e n t r a t i o n on the reverse d i r e c t i o n dehyd r o g e n a s e initial velocity in the presence of 2 m M MgCI 2 a n d A D P . The K + c o n c e n t r a t i o n was 10 raM.
lower the M g A D P c o n c e n t r a t i o n s p l o t t e d in Fig. 1A on the abscissa. F u r t h e r studies were c a r r i e d o u t at 25 m M L - a s p a r t a t e a n d 2 m M M g C I 2 a n d ADP. Fig. 2 illustrates the lack of a n effect o f M g A D P a l o n e on the d e h y d r o g e n a s e assay a n d the m a r k e d effect o f the c o m b i n a t i o n o f L-aspartate a n d M g A D P . T h e curve in the presence of b o t h effectors (circles) follows M i c h a e l i s - M e n t e n kinetics with a K m of 5.0 ___1.0 m M for K +. N o n c o o p e r a tive kinetics were also r e p o r t e d in the presence of M g A T P [6], a n d a K m o f 4.6 + 0.3 m M was calcul a t e d b y Ogilvie et al [7]. F i t t i n g was d o n e b y a n o n l i n e a r m o d i f i e d G a u s s - N e w t o n procedure. A s s a y d a t a in the presence of L-aspartate alone has b e e n r e p o r t e d [6] a n d lies b e t w e e n the two curves in Fig. 2. A s well as being n o n c o o p e r a t i v e l y a c t i v a t e d b y K + in the presence o f L-aspartate a n d M g A D P , the e n z y m e also has a m a x i m a l velocity which is a b o u t 20% higher t h a n the e n z y m e in the a b s e n c e of effectors, as is the case with M g A T P [7]. T h e initial velocity d a t a in the presence of M g A D P (crosses) fall o n the s a m e curve as the d a t a in the a b s e n c e of effectors (squares). F i t s to these two d a t a sets i n d i v i d u a l l y gave a K + conc e n t r a t i o n for h a l f - m a x i m a l activity of 17 m M with no effectors p r e s e n t a n d 14 m M in the pres-
I
20
I
I
I
40
I
I
60 FK +
"1,
I
80
I
I
100
mM
Fig. 2. Effect of K + concentration on the reverse direction dehydrogenase initial velocity. Assay conditions are described in the text. No added effectors (O), 2 raM MgCI2 and ADP (×), and 25 mM L-aspartate plus 2 mM MgCI2 and ADP (O). Initial velocity is plotted as a percentage of the maximal velocity for the assays in the presence of L-aspartate and MgADP.
ence of M g A D P . T h e Hill coefficients were calcul a t e d to be 1.2 a n d 1.3, respectively Both sets gave the same best-fit m a x i m a l velocity. T h e lower solid line in Fig. 2 was d r a w n with a Hill coefficient of 1.25 a n d a K0. 5 of 15.5 m M K +. The velocity was expressed as a fraction o f the m a x i m a l velocity in the presence o f M g A D P a n d aspartate. It has b e e n r e p o r t e d that a n u m b e r of a n i o n s can b i n d to the active site of creatine k i n a s e [8] a n d i n h i b i t this enzyme. A p a r t i c u l a r l y effective c o m p l e x is the b i n d i n g of a p l a n a r a n i o n such as S C N - or NO3-- b e t w e e n creatine a n d A D P , thus t a k i n g the p o s i t i o n o f the t e r m i n a l p h o s p h a t e of A T P [9]. W e m a d e n u m e r o u s a t t e m p t s to observe a n effect of a d d e d S C N - or N O 3 on the activation of h o m o s e r i n e d e h y d r o g e n a s e b y a s p a r t a t e a n d a s p a r t a t e plus M g A D P , b u t saw none, u p to c o n c e n t r a t i o n s of 10 m M K S C N a n d K N O 3. T h e conclusion m u s t b e that either these anions d o n o t f o r m an active site c o m p l e x with t h i s kinase or t h a t M g A D P plus L-aspartate exert the m a x i m a l effect on the d e h y d r o g e n a s e site. M e c h a n i s t i c similarities o r differences b e t w e e n creatine kinase a n d a s p a r t o k i n a s e I are n o t known, except that neither e n z y m e utilizes a covalent e n z y m e - b o u n d phosp h a t e i n t e r m e d i a t e . H i g h e r c o n c e n t r a t i o n s of
100 KSCN effect an irreversible dissociation of the enzyme to monomers [4]. All structural information available on aspartokinase I-homoserine dehydrogenase I indicates that the two active sites reside in distant regions of the primary sequence [10] and in structural domains which unfold [1] and refold [2] independently. However, there is also evidence that the two active sites communicate with each other. For example, it is known that a dehydrogenase substrate can protect both activities to heat inactivation [11] and that MgATP can affect the monovalent cation activation of the dehydrogenase [6,7,12]. Additionally, at low K ÷ concentration threonine inhibition of the dehydrogenase activity differs in the absence and presence of MgATP [12]. The studies presented in this publication indicate that specific parts of the kinase active site appear to have more effect in facilitating a conformational change which is communicated to the sites of K ÷ binding a n d / o r the dehydrogenase active site. MgATP has an effect on the dehydrogenase activity, but M g A D P does not, indicating an essential role for the terminal phosphate. However, the addition of aspartate to the MgADP" enzyme complex produces the same effect as MgATP alone. Thus, aspartokinase substrates and products differ markedly in their ability to alter the monovalent cation activation of homoserine dehydrogenase, presumably due to different interactions with subsites of the kinase active site. These studies indicate that this bifunctional enzyme which cannot channel substrates has adap-
tive advantages beyond the fact that both activities share a common feedback inhibitor, L-threonine and common repressors of their synthesis. The presence of kinase substrates and products can modulate the dehydrogenase activity. Differential scanning calorimetry studies are presently being carried out to determine the effect of these kinase substrates on the unfolding of both the kinase and dehydrogenase domains. We acknowledge the support of the Research Corporation and the Petroleum Research Fund of the American Chemical Society.
References 1 Mailer, K. and Garel, J.-R. (1984) Biochemistry23, 651-654 2 Dautry-Varsat, A. and Garel, J.-R. (1981) Biochemistry20, 1396-1401 3 Szentirmai, A., Szentirmai, M. and Umbarger, H.E. (1968) J. Bacteriol. 95, 1672-1679 4 Vickers, L.P., Ackers, G.K. and Ogilvie,J.W. (1978) J. Biol. Chem. 253, 2155-2160 5 O'Sullivan, W.J. and Smithers, G.W. (1979) Methods Enzymol. 63, 294-336 6 Broussard, L., Cunningham,G.N., Starnes, W.L. and Shire, W. (1972) Biochem. Biophys Res. Commun.46, 1181-1186 70gilvie, J.W., Vickers, L.P., Clark, R.B. and Jones, M.M. (1975) J. Biol. Chem. 250, 1242-1260 8 Watts, D.C. (1973) The Enzymes8, 383-455 9 Reed, G.H., Barlow, C.H. and Burns, R.A., Jr. (1978) J. Biol. Chem. 253, 4153-4158 10 Fazel, A., Mi~ller, K., LeBras, G., Garel, J.-R., V6ron, M. and Cohen, G.N. (1983) Biochemistry22, 158-165 11 Patte, J.-C., Truffa-Bachi, P. and Cohen, G.N. (1966) Biochim. Biophys. Acta 128, 426-439 12 Truffa-Bachi, P., Costrejean, J.-M., Py, M.-C. and Cohen, G.N. (1974) Biochimie, 56, 215-219