- Email: [email protected]
[52] Chemical stabilization of conformational states of dCMP deaminase
[52]
F R E E Z I N G OF d C M P
D E A M I N A S E CONFORMERS
577
that expected of the R state. These observations indicate that subtle conformational differences may be detectable using a variety of functional and structural parameters as probes. P o s s i b l e D i r e c t i o n s for F u t u r e W o r k
It would be interesting to use a bifunctional reagent similar to tartryl diazide but containing a disulfide linkage which could be cleaved without affecting enzyme activity. Furthermore, identification of the cross-links in the amino acid sequence would help to establish the structural differences between the R and T states. Finally, purification by ion-exchange or affinity chromatography might yield homogeneous derivatives. These stable derivatives could aid in the analysis by X-ray crystallography of the conformation of the R and T states of the enzyme.
[52] C h e m i c a l S t a b i l i z a t i o n o f C o n f o r m a t i o n a l S t a t e s o f dCMP Deaminase By Moss Rossl, C. A. RAIA, and C. VACCARO
Nearly all enzymes whose activity is regulated by allosteric mechanisms exhibit a quaternary structure composed of one or different types of subunits. Allosteric regulation of a particular enzyme is the result of the control of enzyme action by metabolites having a chemical structure different from either the structure of the substrate or that of the product. These metabolites exert their modulating effects by binding to secondary sites (allosteric sites) different from the substrate binding sites (isosteric sites). In certain enzymes, e.g., ribonucleotide reductase, ~ one type of subunit contains either isosteric or allosteric sites; in other enzymes, e.g., aspartate transcarbamoylase,2 one type of subunit binds the substrate and another type binds the effector(s). Several models for allosteric regulation have been proposed. Monod et al. 3 have explained the regulatory properties of some enzymes by assuming the existence of an equilbrium between at least two conformational states named the R (activated conformation) and the T (inhibited conformation) states. The R state shows affinity for the substrate and the allosteric activator; the T state shows affinity for the allosteric inhibitor. L. Thelander, J. Biol. Chem. 248, 4591 (1973). z H. K. Schachman, Harvey Lect. 68, 67 (1974). 3 j. Monod, J. Wyman, and J.-P. Changeux, J. Mol. Biol. 12, 88 (1965).
METHODS IN ENZYMOLOGY, VOL. 135
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
578
APPLICATION OF IMMOBILIZED ENZYMES/CELLS
[52J
The equilibrium is shifted toward the R or T form depending on the interaction of the enzyme with ligands. On the other hand, from the theory of induced fit, proposed by Koshland, 4,5 it is assumed that ligand binding induces different conformational states of a single molecular species resulting in allosteric regulation. In order to explore the basis for the kinetic evidence indicating the occurrence of conformational states and furthermore to obtain information on the conformers, we have tried to stabilize chemically by glutaraldehyde treatment conformers of the allosteric enzyme dCMP deaminase (dCMP aminohydrolase, EC 3.5.4.12). The enzyme catalyzes the hydrolytic deamination of dCMP to dUMP 6 and is composed of six subunits of the same or similar molecular weight (Mr of the hexameric enzyme is 120,000). The catalyzed reaction is at a branching point of the pathway leading to dCTP and dTTP, the immediate pyrimidine deoxynucleotide precursors in DNA biosynthesis, dCTP is an allosteric activator, dTTP an allosteric inhibitor, and activation or inhibition of the enzyme depends on their relative concentrations. 7 The allosteric ligands induce conformational changes in the enzyme molecule, thus modifying the affinity of the catalytic sites for the substrate without changing the molecular weight. 8 On the basis of several lines of evidence, the occurrence of at least three interconverting conformational isomers of dCMPase has been proposed. 9 The proposed conformers are the native enzyme containing no complexed ligands, the enzyme-Mg-dCTP complex (activated conformation, R form) and the enzyme-Mg-dTTP complex (inhibited conformation, T form). By treatment of the enzyme with bifunctional reagents, such as glutaraldehyde, it has been demonstrated that dCMPase can be "frozen" in both the activated and inhibited conformational state of the enzyme. 10,~1 Choice of Cross-Linking Conditions Kinetic and electrophoretic analyses of the modified protein were carried out to study the cross-linked enzyme preparations. Stabilization of 4 D. E. Koshland, G. N6m6thy, and D. Filmer, Biochemistry 5, 365 (1966). D. E. Koshland, Enzymes 342, 305 (1970). 6 G. Geraci, M. Rossi, and E. Scarano, Biochemistry 6, 183 (1967). 7 E. Scarano, G. Geraci, and M. Rossi, Biochemistry 6, 192 (i967). 8 M. Rossi, G. Geraci, and E. Scarano, Biochemistry 6, 3640 (1967). 9 M. Rossi, I. Dosseva, M. Pierro, M. Cacace, and E. Scarano, Biochemistry 10, 3060
(1971). l0 R. Nucci, C. A. Raia, C. Vaccaro, S. Sepe, E. Scarano, and M. Rossi, J. Mol. Biol. 124, 133 (1978). l~ C. A. Raia, R. Nucci, C. Vaccaro, S. Sepe, R. Rella, and M. Rossi, J. Mol. Biol. 157, 557 (1982).
[52]
FREEZINGOF dCMP DEAMINASECONFORMERS
579
the enzyme in one of its conformations should (1) decrease the enzyme's sensitivity to the effect of aUosteric ligands (at least at concentrations required for effective regulation of the native enzyme), (2) modify cooperativity toward the substrate, and (3) if intersubunit cross-linking occurs, produce a hexamer that is not dissociated by sodium dodecyl sulfate (SDS) treatment. Among the cross-linking reagents tested, glutaraldehyde was the most efficient. Diimidates of different chain length, such as dimethyl suberimidate, dimethyl pimelimidate, dimethyl adipimidate, and diethyl pyrocarbonate, only slightly modified the kinetic behavior of dCMPase; the modified protein remained sensitive to the effect of allosteric ligand binding and, in the most favorable cases, 20 to 30% of the modified enzyme was cross-linked in the hexameric form.~2 In contrast, on cross-linking using glutaraldehyde the shape of the enzyme molecule was locked either in the R or the T form, depending on the experimental conditions used. Cross-Linking of dCMPase in the Activated R Conformation The chemical stabilization of the activated R form of dCMPase was carried out in the presence of the allosteric ligand dCTP in order to induce the R conformation, and in the presence of the competitive inhibitor dAMP to protect the enzyme from inactivation, dCMPase was prepared by using a modified version of the method previously described. 6 The enzyme activity was measured at 38° with a thermostated Cary 118 spectrophotometer using dCMP or CH3dCMP as substrates. 7 Cuvettes of 10 or 1 mm light paths were used, depending on the substrate concentration employed. The dCMPase frozen in the R form was prepared in the following way: 1 mg of enzyme was dissolved at 4 ° in 50 mM sodium phosphate buffer, pH 7.5, containing 1 mM mercaptoethanol, 1 mM MgCI2, 50/xM dCTP, 1 mM dAMP, and 20% glycerol. After dissolution of the enzyme, glutaraldehyde (25% Merck, Darmstadt) was added to the solution (0.25% in a final volume of 5 ml) and the mixture was allowed to incubate at 22°. The process of enzyme freezing was followed by studying desensitization of the modified enzyme toward the effector dTTP. Figure 1 shows the time-dependent desensitization of dCMPase. At the indicated times, microliter portions were withdrawn from the reaction mixture to determine the enzyme activity at 38° in the presence and in the absence of 20/zM dTTP. When the determined activities were identical in the two cases (after reaction for 40-50 min), the mixture was cooled in an ice-bath, diluted 10 times with cold quartz-distilled water, and 0.4 ml of a suspen~2 M. Rossi, C. A. Raia, R. Nucci, C. Vaccaro, S. Sepe, and R. Rella, in "Macromolecules in the Functioning Cells'" (A. A. Bayer, ed.), Book 2, p. 52. Nauka, Moscow, 1982.
580
APPLICATION OF IMMOBILIZED ENZYMES/CELLS
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60
40j
/
--
20
40 Time {mini
v
60
FIG. 1. Time-dependent desensitization of dCMPase toward the allosteric inhibitor dTTP studied by treatment of the enzyme with glutaraldehyde in the presence of the allosteric activator dCTP. Of 25% glutaraldehyde 2/.d was added to the incubation mixture (0.2 ml) containing 10/zg of dCMPase, 50 mM sodium phosphate (pH 7.5), 20% glycerol, 1 mM MgClz, 50/zM dCTP, and 1 mM dAMP. At the times indicated, portions were taken out for enZyme activity measurements (38°) both in the presence and absence of dTTP. (©) Enzyme activity; (A) enzymatic activity measured in the presence of 10/xM dTTP and expressed as a percentage of the activity measured in the absence of dTTP. From Nucci e t al. I°
sion containing 2% (w/v) alumina gel C7 (Schuchardt) was added to the e n z y m e mixture. After magnetic stirring for 15 min, the suspension was w a s h e d twice with 20 ml portions of I0 m M Tris-HC1 (pH 7.5), 7 m M m e r c a p t o e t h a n o l , 1 m M ethylenediaminetetraacetic acid (EDTA), and 20% glycerol in order to eliminate the excess glutaraldehyde and nucleotides. P r e p a r e d g l u t a r a l d e h y d e - d C M P a s e was eluted from the alumina gel precipitate with 0.5 ml of 0.1 M sodium p h o s p h a t e buffer (pH 7.5) containing 50% glycerol. Properties of d C M P a s e Cross-Linked in the Activated R Conformation The specific d C M P a s e activity expressed in terms of micromoles (with s p e c t r o p h o t o m e t r i c assay) d C M P deaminated per minute and milligrams of protein at 38 ° is 720. T h e average specific activity of 5 preparations of g l u t a r a l d e h y d e - d C M P a s e was 400 -+ 30 /~mol/min and mg of protein. E x p e r i m e n t s carried out using deoxy[5AH]cytidine 5'-triphosphate demonstrated that the d C T P remaining bound to the e n z y m e was less than 0.1
[52]
FREEZING OF d C M P DEAMINASE CONFORMERS
581
nmol/nmol of enzyme molecules.l° The modified enzyme showed a slight cooperativity with respect to the substrate and the n value determined using Hill plots was 1.28. In addition, glutaraldehyde-dCMPase exhibited an apparent Kmfor dCMP (0.18 mM) similar to that of the native enzyme in the presence of dCTP (0.15 mM) and was no longer sensitive to the effect of low concentrations of dTTP. Electrophoretic analysis of glutaraldehyde-dCMPase did not show formation of enzyme aggregates; 90% of the enzyme migrated as the hexamer on analysis of the enzyme preparation by SDS electrophoresis.~° Cross-Linking of dCMPase in the Inhibited T Conformation dCMPase was allowed to react with glutaraldehyde in the absence of ligands and in the presence of the competitive inhibitor dGMP or the allosteric inhibitor dTTP, or both. The reason for adding dGMP was to protect the catalytic sites of the enzyme, whereas that of dTTP was to induce the inhibited T form of the enzyme, dCMPase stabilized in the T conformation was prepared as follows: 2 mg of enzyme was dissolved at 4° in 20 mM sodium phosphate buffer, pH 7.5, containing 2 mM MgC12, 0.1 mM dTTP, 1 mM dGMP, and 20% glycerol. After this step, glutaraldehyde (25% Merck, Darmstadt) was added to the solution (0.25% in a final volume of 10 ml). The mixture was allowed to incubate for 40 min at 22° and was then rapidly cooled in an ice-bath. The cooled mixture was diluted 10 times with cold quartz-distilled water and then 0.6 ml of a suspension containing 2% (w/v) alumina gel was added to the enzyme mixture. After magnetic stirring for 15 min, the suspension was centrifuged, and the supernatant discarded. The alumina gel precipitate was washed twice with 20 ml portions of 10 mM Tris-HCl (pH 7.5), 7 mM mercaptoethanol, 1 mM EDTA, and 20% glycerol to eliminate the excess glutaraldehyde and added nucleotides. The obtained glutaraldehydedCMPase preparation was eluted from the alumina gel precipitate with 0.5 ml of 0.1 M sodium phosphate buffer (pH 7.5) in 50% glycerol. On stabilization of the T form, it was found that dCMPase activity could not be assayed, since dCMP is not a substrate of the enzyme in the dTTP-induced conformation, and therefore it was not easy to analyze the results of the cross-linking procedure. This problem was circumvented by taking advantage of a peculiar kinetic effect observed when a derivative of dCMP mercurated in the 5 position of the pyrimidine ring is used as a dCMPase substrate in the presence of mercaptoethanol. The formed analog, d C M P - H g - S - C H 2 - C H 2 - O H , is a good substrate for the enzyme in the dTTP-induced conformation. This observation permitted us to follow the process of glutaraldehyde cross-linking of the enzyme in the dTTP-
582
APPLICATION OF IMMOBILIZED ENZYMES/CELLS
IS21
induced form and to study the effect of different ligands on cross-linking of the enzyme. H During the reaction with glutaraldehyde, dCMPase activity was measured by using either dCMP or d C M P - H g - S - C H 2 - C H 2 - O H as substrates. The assay using dCMP as a substrate was performed in the presence of dCTP since the enzyme-dTTP complex was not active toward dCMP, and dCTP could shift the equilibrium toward the R form only for enzyme molecules that had not been cross-linked. If the glutaraldehyde treatment had locked the enzyme in the T form, we should observe a decrease in the activity with the time of the reaction when using dCMP as a substrate. However, the activity when using d C M P - H g - S - C H z - C H z OH as a substrate should remain constant because the equilibrium can no longer be shifted toward the R form. Glutaraldehyde rapidly inactivated the enzyme activity toward dCMP (curves at the bottom of the left corner of Fig. 2) and protection by dGMP or dTTP, or both, was inefficient. Upon reaction with glutaraldehyde, however, the activity using d C M P - H g - S - C H z - C H 2 - O H as a substrate increased in all cases about 3-fold and declined at different rates as a function of time. The best protection was observed in the presence of both
30C
ot
20C
10C
3b
is
minules
FIG. 2. Effect of dGMP (1 mM), dTFP (0. ! mM), and dGMP + dTTP (1 + 0.1 raM) on the modification of dCMPase by glutaraldehyde treatment of the enzyme. Each incubation mixture (1 ml) contained 0.02 M sodium phosphate buffer (pH 7.5), 2 mM igCl2, 20% glycerol, 0.20 mg of dCMPase, and nucleotides. The reaction took place at 22°. After measurements of the activity at 0 time (100%), 0.25% glutaraldehyde was added and, at the indicated times, portions were withdrawn and assayed by using either dCMP (curves at bottom of left corner) or d C M P - H g - S - C H 2 - C H 2 - O H as substrates. (D) No addition; (D) 0.1 mM dTTP; (O) 1 mM dGMP; (A) 0.1 mM dTTP + I mM dGMP. From Raia e t al. tt
[52]
FREEZING OF d C M P DEAMINASE CONFORMERS
583
the competitive and the allosteric inhibitors. In all the cases reported in Fig. 2, in addition to enzymatic measurements, samples were withdrawn at different time intervals and were analyzed by SDS gel electrophoresis to measure the size distribution of formed cross-linked species. Figure 3 ~3 shows the kinetics of the formation of dimer, trimer, tetramer, and pentamer + hexamer in the presence of glutaraldehyde as well as the effect on the cross-linking pattern of the isosteric ligand dGMP and of the allosteric inhibitor dTTP. In the absence of complexing ligands -85% of the enzyme was cross-linked as a hexamer after reaction for about 20 min, and it showed very little or no activity either toward the natural substrate or the mercurated analog (Fig. 3a). On the other hand, in the presence of both dGMP and dTTP (Fig. 3b), about 95% of the enzyme molecules were cross-linked as hexamers after reaction for 40 min; this enzyme preparation showed almost no activity using dCMP as a substrate whereas the activity using d C M P - H g - S - C H 2 - C H 2 - O H as a substrate increased to about twice the starting value. An intermediate situation was observed when dGMP or dTTP was present in the cross-linking solutions alone. These data wee taken as evidence that the enzyme under these conditions is stabilized in the T conformation. Properties of dCMPase Cross-Linked in the Inhibited T Conformation In Table I the specific activity of the native enzyme and that of the T form-stabilized enzyme is compared by using dCMP and d C M P - H g - S CH2-CH2-OH as substrates. Also, the effects of dCTP and dTTP on the enzyme activity are listed in Table I. The specific activity of native dCMPase using a saturating concentration of dCMP as a substrate was 820 tzmol/min and mg of protein and that of the enzyme stabilized in the T form was as low as I/zmol/min and mg of protein. In both cases dCTP and dTTP had little or no effect. On the other hand, when using the mercury substrate it was found that the modified enzyme was activated and inhibited by dTTP and dCTP, respectively. However, both ligands inhibited the activity of the enzyme locked in the T conformation in a noncompetitive manner. The assays were carried out at ligand concentrations 10- to 100-fold higher than those effective in modulating the activity of the native enzyme when using dCMP as a substrate. The modified enzyme showed little substrate cooperativity with an n value of 1.5 and the presence of allosteric ligands in the assays yielded the same n value. The kinetic properties of the modified enzyme differed compared with those obtained for native enzyme in studies using d C M P - H g - S - C H 2 13 K. Weber and M. Osborn J. Biol. Chem. 244, 4406 (1969).
584
APPLICATION OF IMMOBILIZED ENZYMES/CELLS
[52]
100
.__.-..--.--..--.-1
>=
10
20
30
I
L,O
minutes
100
~6 50
E I0
20 minutes
30
~'0
FIG. 3. Time-dependent production of oligomeric species by glutaraldehyde treatment of dCMPase in the absence of effector (a) and in the presence of 0.1 mM dTTP + 1 mM dGMP (b). The incubation conditions were the same as those described in the legends to Fig. 2. At the indicated times, portions of 100/~1 (20/zg of protein) were withdrawn and added to 10/~1 of a mixture containing 10% sodium dodecyl sulfate and 10% 2-mercaptoethanol in 0.I M sodium phosphate buffer (pH 7.0). After 5 rain of time of reaction at 90° the samples were analyzed by SDS electrophoresis in 5% polyacrylamide gels according to the method of Weber and Osborn. ~3The relative amounts of monomer (O), dimer (a), trimer (1), tetramer (I-q), and pentamer + hexamer (11) were determined by means of densitometric analysis. From Raia e t al. 11
[52]
FREEZING OF d C M P DEAMINASE CONFORMERS
585
TABLE 1 SPECIFIC ACTIVITY OF NATIVE dCMPase AND GLUTARALDEHYDE-TREATED dCMPase USING dCMP AND d C M P - H g - S - C H 2 - C H ~ - O H AS SUBSTRATES"
dCMP-Hg-S-CH2-CH2-OH
Enzyme system
/zmol dCMP per min and mg of protein
/xmol
dCMPase dCMPase + dCTP (1 /xM) dCMPase + dTTP (5 txM) Glutaraldehyde dCMPase (T form) Glutaraldehyde dCMPase (T form) +
820 820 820 1 4
23 10 71 43 35
1
25
per min and mg of protein
dCTP (20/xM)
Glutaraldehyde dCMPase (T form + dTTP (50/xM) a From Raia et al. 11
CH2-OH as a substrate in the presence of dTTP. This is not surprising since about 30 lysyl residues per molecule had been modified and at least five cross-linking bridges had been formed between subunits. The formed cross-links presumably froze the modified enzyme in the T conformation in such a way that allosteric ligands could no longer shift the equilibrium between the two enzyme forms. In fact, the modified enzyme was still able to bind dCTP or dTTP, but both of them behaved as noncompetitive inhibitors, probably only inducing local conformational changes. It was not possible to stabilize the protein structure containing no ligands complexed with enzyme because, in the absence of substrate or competitive inhibitors, glutaraldehyde rapidly reacted with a reactive amino group localized in the catalytic sites and inactivated the enzyme. In this context, it is worth noting that dCMPase has been entrapped in glutaraldehyde-albumin membranes in both the presence and absence of isosteric and allosteric ligands. 14 The kinetic behavior of the enzyme immobilized in such artificial membranes is markedly different from that of free enzyme. In fact, the enzyme loses its cooperative action toward the substrate and the observed effects on the kinetics of the enzyme in the presence of allosteric ligands could in this case also be attributed to freezing of the enzyme in either of its conformations. However, physical and diffusional resistance to translocation of substrate in the membrane must also be taken into account on interpretation of the obtained kinetic data. 14 G. Iorio, E. Drioli, R. Molinari, and M. Rossi, A n n . N . Y . A c a d . Sci. 369, 235 (1981).
F R E E Z I N G OF d C M P
D E A M I N A S E CONFORMERS
577
that expected of the R state. These observations indicate that subtle conformational differences may be detectable using a variety of functional and structural parameters as probes. P o s s i b l e D i r e c t i o n s for F u t u r e W o r k
It would be interesting to use a bifunctional reagent similar to tartryl diazide but containing a disulfide linkage which could be cleaved without affecting enzyme activity. Furthermore, identification of the cross-links in the amino acid sequence would help to establish the structural differences between the R and T states. Finally, purification by ion-exchange or affinity chromatography might yield homogeneous derivatives. These stable derivatives could aid in the analysis by X-ray crystallography of the conformation of the R and T states of the enzyme.
[52] C h e m i c a l S t a b i l i z a t i o n o f C o n f o r m a t i o n a l S t a t e s o f dCMP Deaminase By Moss Rossl, C. A. RAIA, and C. VACCARO
Nearly all enzymes whose activity is regulated by allosteric mechanisms exhibit a quaternary structure composed of one or different types of subunits. Allosteric regulation of a particular enzyme is the result of the control of enzyme action by metabolites having a chemical structure different from either the structure of the substrate or that of the product. These metabolites exert their modulating effects by binding to secondary sites (allosteric sites) different from the substrate binding sites (isosteric sites). In certain enzymes, e.g., ribonucleotide reductase, ~ one type of subunit contains either isosteric or allosteric sites; in other enzymes, e.g., aspartate transcarbamoylase,2 one type of subunit binds the substrate and another type binds the effector(s). Several models for allosteric regulation have been proposed. Monod et al. 3 have explained the regulatory properties of some enzymes by assuming the existence of an equilbrium between at least two conformational states named the R (activated conformation) and the T (inhibited conformation) states. The R state shows affinity for the substrate and the allosteric activator; the T state shows affinity for the allosteric inhibitor. L. Thelander, J. Biol. Chem. 248, 4591 (1973). z H. K. Schachman, Harvey Lect. 68, 67 (1974). 3 j. Monod, J. Wyman, and J.-P. Changeux, J. Mol. Biol. 12, 88 (1965).
METHODS IN ENZYMOLOGY, VOL. 135
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
578
APPLICATION OF IMMOBILIZED ENZYMES/CELLS
[52J
The equilibrium is shifted toward the R or T form depending on the interaction of the enzyme with ligands. On the other hand, from the theory of induced fit, proposed by Koshland, 4,5 it is assumed that ligand binding induces different conformational states of a single molecular species resulting in allosteric regulation. In order to explore the basis for the kinetic evidence indicating the occurrence of conformational states and furthermore to obtain information on the conformers, we have tried to stabilize chemically by glutaraldehyde treatment conformers of the allosteric enzyme dCMP deaminase (dCMP aminohydrolase, EC 3.5.4.12). The enzyme catalyzes the hydrolytic deamination of dCMP to dUMP 6 and is composed of six subunits of the same or similar molecular weight (Mr of the hexameric enzyme is 120,000). The catalyzed reaction is at a branching point of the pathway leading to dCTP and dTTP, the immediate pyrimidine deoxynucleotide precursors in DNA biosynthesis, dCTP is an allosteric activator, dTTP an allosteric inhibitor, and activation or inhibition of the enzyme depends on their relative concentrations. 7 The allosteric ligands induce conformational changes in the enzyme molecule, thus modifying the affinity of the catalytic sites for the substrate without changing the molecular weight. 8 On the basis of several lines of evidence, the occurrence of at least three interconverting conformational isomers of dCMPase has been proposed. 9 The proposed conformers are the native enzyme containing no complexed ligands, the enzyme-Mg-dCTP complex (activated conformation, R form) and the enzyme-Mg-dTTP complex (inhibited conformation, T form). By treatment of the enzyme with bifunctional reagents, such as glutaraldehyde, it has been demonstrated that dCMPase can be "frozen" in both the activated and inhibited conformational state of the enzyme. 10,~1 Choice of Cross-Linking Conditions Kinetic and electrophoretic analyses of the modified protein were carried out to study the cross-linked enzyme preparations. Stabilization of 4 D. E. Koshland, G. N6m6thy, and D. Filmer, Biochemistry 5, 365 (1966). D. E. Koshland, Enzymes 342, 305 (1970). 6 G. Geraci, M. Rossi, and E. Scarano, Biochemistry 6, 183 (1967). 7 E. Scarano, G. Geraci, and M. Rossi, Biochemistry 6, 192 (i967). 8 M. Rossi, G. Geraci, and E. Scarano, Biochemistry 6, 3640 (1967). 9 M. Rossi, I. Dosseva, M. Pierro, M. Cacace, and E. Scarano, Biochemistry 10, 3060
(1971). l0 R. Nucci, C. A. Raia, C. Vaccaro, S. Sepe, E. Scarano, and M. Rossi, J. Mol. Biol. 124, 133 (1978). l~ C. A. Raia, R. Nucci, C. Vaccaro, S. Sepe, R. Rella, and M. Rossi, J. Mol. Biol. 157, 557 (1982).
[52]
FREEZINGOF dCMP DEAMINASECONFORMERS
579
the enzyme in one of its conformations should (1) decrease the enzyme's sensitivity to the effect of aUosteric ligands (at least at concentrations required for effective regulation of the native enzyme), (2) modify cooperativity toward the substrate, and (3) if intersubunit cross-linking occurs, produce a hexamer that is not dissociated by sodium dodecyl sulfate (SDS) treatment. Among the cross-linking reagents tested, glutaraldehyde was the most efficient. Diimidates of different chain length, such as dimethyl suberimidate, dimethyl pimelimidate, dimethyl adipimidate, and diethyl pyrocarbonate, only slightly modified the kinetic behavior of dCMPase; the modified protein remained sensitive to the effect of allosteric ligand binding and, in the most favorable cases, 20 to 30% of the modified enzyme was cross-linked in the hexameric form.~2 In contrast, on cross-linking using glutaraldehyde the shape of the enzyme molecule was locked either in the R or the T form, depending on the experimental conditions used. Cross-Linking of dCMPase in the Activated R Conformation The chemical stabilization of the activated R form of dCMPase was carried out in the presence of the allosteric ligand dCTP in order to induce the R conformation, and in the presence of the competitive inhibitor dAMP to protect the enzyme from inactivation, dCMPase was prepared by using a modified version of the method previously described. 6 The enzyme activity was measured at 38° with a thermostated Cary 118 spectrophotometer using dCMP or CH3dCMP as substrates. 7 Cuvettes of 10 or 1 mm light paths were used, depending on the substrate concentration employed. The dCMPase frozen in the R form was prepared in the following way: 1 mg of enzyme was dissolved at 4 ° in 50 mM sodium phosphate buffer, pH 7.5, containing 1 mM mercaptoethanol, 1 mM MgCI2, 50/xM dCTP, 1 mM dAMP, and 20% glycerol. After dissolution of the enzyme, glutaraldehyde (25% Merck, Darmstadt) was added to the solution (0.25% in a final volume of 5 ml) and the mixture was allowed to incubate at 22°. The process of enzyme freezing was followed by studying desensitization of the modified enzyme toward the effector dTTP. Figure 1 shows the time-dependent desensitization of dCMPase. At the indicated times, microliter portions were withdrawn from the reaction mixture to determine the enzyme activity at 38° in the presence and in the absence of 20/zM dTTP. When the determined activities were identical in the two cases (after reaction for 40-50 min), the mixture was cooled in an ice-bath, diluted 10 times with cold quartz-distilled water, and 0.4 ml of a suspen~2 M. Rossi, C. A. Raia, R. Nucci, C. Vaccaro, S. Sepe, and R. Rella, in "Macromolecules in the Functioning Cells'" (A. A. Bayer, ed.), Book 2, p. 52. Nauka, Moscow, 1982.
580
APPLICATION OF IMMOBILIZED ENZYMES/CELLS
152]
60
40j
/
--
20
40 Time {mini
v
60
FIG. 1. Time-dependent desensitization of dCMPase toward the allosteric inhibitor dTTP studied by treatment of the enzyme with glutaraldehyde in the presence of the allosteric activator dCTP. Of 25% glutaraldehyde 2/.d was added to the incubation mixture (0.2 ml) containing 10/zg of dCMPase, 50 mM sodium phosphate (pH 7.5), 20% glycerol, 1 mM MgClz, 50/zM dCTP, and 1 mM dAMP. At the times indicated, portions were taken out for enZyme activity measurements (38°) both in the presence and absence of dTTP. (©) Enzyme activity; (A) enzymatic activity measured in the presence of 10/xM dTTP and expressed as a percentage of the activity measured in the absence of dTTP. From Nucci e t al. I°
sion containing 2% (w/v) alumina gel C7 (Schuchardt) was added to the e n z y m e mixture. After magnetic stirring for 15 min, the suspension was w a s h e d twice with 20 ml portions of I0 m M Tris-HC1 (pH 7.5), 7 m M m e r c a p t o e t h a n o l , 1 m M ethylenediaminetetraacetic acid (EDTA), and 20% glycerol in order to eliminate the excess glutaraldehyde and nucleotides. P r e p a r e d g l u t a r a l d e h y d e - d C M P a s e was eluted from the alumina gel precipitate with 0.5 ml of 0.1 M sodium p h o s p h a t e buffer (pH 7.5) containing 50% glycerol. Properties of d C M P a s e Cross-Linked in the Activated R Conformation The specific d C M P a s e activity expressed in terms of micromoles (with s p e c t r o p h o t o m e t r i c assay) d C M P deaminated per minute and milligrams of protein at 38 ° is 720. T h e average specific activity of 5 preparations of g l u t a r a l d e h y d e - d C M P a s e was 400 -+ 30 /~mol/min and mg of protein. E x p e r i m e n t s carried out using deoxy[5AH]cytidine 5'-triphosphate demonstrated that the d C T P remaining bound to the e n z y m e was less than 0.1
[52]
FREEZING OF d C M P DEAMINASE CONFORMERS
581
nmol/nmol of enzyme molecules.l° The modified enzyme showed a slight cooperativity with respect to the substrate and the n value determined using Hill plots was 1.28. In addition, glutaraldehyde-dCMPase exhibited an apparent Kmfor dCMP (0.18 mM) similar to that of the native enzyme in the presence of dCTP (0.15 mM) and was no longer sensitive to the effect of low concentrations of dTTP. Electrophoretic analysis of glutaraldehyde-dCMPase did not show formation of enzyme aggregates; 90% of the enzyme migrated as the hexamer on analysis of the enzyme preparation by SDS electrophoresis.~° Cross-Linking of dCMPase in the Inhibited T Conformation dCMPase was allowed to react with glutaraldehyde in the absence of ligands and in the presence of the competitive inhibitor dGMP or the allosteric inhibitor dTTP, or both. The reason for adding dGMP was to protect the catalytic sites of the enzyme, whereas that of dTTP was to induce the inhibited T form of the enzyme, dCMPase stabilized in the T conformation was prepared as follows: 2 mg of enzyme was dissolved at 4° in 20 mM sodium phosphate buffer, pH 7.5, containing 2 mM MgC12, 0.1 mM dTTP, 1 mM dGMP, and 20% glycerol. After this step, glutaraldehyde (25% Merck, Darmstadt) was added to the solution (0.25% in a final volume of 10 ml). The mixture was allowed to incubate for 40 min at 22° and was then rapidly cooled in an ice-bath. The cooled mixture was diluted 10 times with cold quartz-distilled water and then 0.6 ml of a suspension containing 2% (w/v) alumina gel was added to the enzyme mixture. After magnetic stirring for 15 min, the suspension was centrifuged, and the supernatant discarded. The alumina gel precipitate was washed twice with 20 ml portions of 10 mM Tris-HCl (pH 7.5), 7 mM mercaptoethanol, 1 mM EDTA, and 20% glycerol to eliminate the excess glutaraldehyde and added nucleotides. The obtained glutaraldehydedCMPase preparation was eluted from the alumina gel precipitate with 0.5 ml of 0.1 M sodium phosphate buffer (pH 7.5) in 50% glycerol. On stabilization of the T form, it was found that dCMPase activity could not be assayed, since dCMP is not a substrate of the enzyme in the dTTP-induced conformation, and therefore it was not easy to analyze the results of the cross-linking procedure. This problem was circumvented by taking advantage of a peculiar kinetic effect observed when a derivative of dCMP mercurated in the 5 position of the pyrimidine ring is used as a dCMPase substrate in the presence of mercaptoethanol. The formed analog, d C M P - H g - S - C H 2 - C H 2 - O H , is a good substrate for the enzyme in the dTTP-induced conformation. This observation permitted us to follow the process of glutaraldehyde cross-linking of the enzyme in the dTTP-
582
APPLICATION OF IMMOBILIZED ENZYMES/CELLS
IS21
induced form and to study the effect of different ligands on cross-linking of the enzyme. H During the reaction with glutaraldehyde, dCMPase activity was measured by using either dCMP or d C M P - H g - S - C H 2 - C H 2 - O H as substrates. The assay using dCMP as a substrate was performed in the presence of dCTP since the enzyme-dTTP complex was not active toward dCMP, and dCTP could shift the equilibrium toward the R form only for enzyme molecules that had not been cross-linked. If the glutaraldehyde treatment had locked the enzyme in the T form, we should observe a decrease in the activity with the time of the reaction when using dCMP as a substrate. However, the activity when using d C M P - H g - S - C H z - C H z OH as a substrate should remain constant because the equilibrium can no longer be shifted toward the R form. Glutaraldehyde rapidly inactivated the enzyme activity toward dCMP (curves at the bottom of the left corner of Fig. 2) and protection by dGMP or dTTP, or both, was inefficient. Upon reaction with glutaraldehyde, however, the activity using d C M P - H g - S - C H z - C H 2 - O H as a substrate increased in all cases about 3-fold and declined at different rates as a function of time. The best protection was observed in the presence of both
30C
ot
20C
10C
3b
is
minules
FIG. 2. Effect of dGMP (1 mM), dTFP (0. ! mM), and dGMP + dTTP (1 + 0.1 raM) on the modification of dCMPase by glutaraldehyde treatment of the enzyme. Each incubation mixture (1 ml) contained 0.02 M sodium phosphate buffer (pH 7.5), 2 mM igCl2, 20% glycerol, 0.20 mg of dCMPase, and nucleotides. The reaction took place at 22°. After measurements of the activity at 0 time (100%), 0.25% glutaraldehyde was added and, at the indicated times, portions were withdrawn and assayed by using either dCMP (curves at bottom of left corner) or d C M P - H g - S - C H 2 - C H 2 - O H as substrates. (D) No addition; (D) 0.1 mM dTTP; (O) 1 mM dGMP; (A) 0.1 mM dTTP + I mM dGMP. From Raia e t al. tt
[52]
FREEZING OF d C M P DEAMINASE CONFORMERS
583
the competitive and the allosteric inhibitors. In all the cases reported in Fig. 2, in addition to enzymatic measurements, samples were withdrawn at different time intervals and were analyzed by SDS gel electrophoresis to measure the size distribution of formed cross-linked species. Figure 3 ~3 shows the kinetics of the formation of dimer, trimer, tetramer, and pentamer + hexamer in the presence of glutaraldehyde as well as the effect on the cross-linking pattern of the isosteric ligand dGMP and of the allosteric inhibitor dTTP. In the absence of complexing ligands -85% of the enzyme was cross-linked as a hexamer after reaction for about 20 min, and it showed very little or no activity either toward the natural substrate or the mercurated analog (Fig. 3a). On the other hand, in the presence of both dGMP and dTTP (Fig. 3b), about 95% of the enzyme molecules were cross-linked as hexamers after reaction for 40 min; this enzyme preparation showed almost no activity using dCMP as a substrate whereas the activity using d C M P - H g - S - C H 2 - C H 2 - O H as a substrate increased to about twice the starting value. An intermediate situation was observed when dGMP or dTTP was present in the cross-linking solutions alone. These data wee taken as evidence that the enzyme under these conditions is stabilized in the T conformation. Properties of dCMPase Cross-Linked in the Inhibited T Conformation In Table I the specific activity of the native enzyme and that of the T form-stabilized enzyme is compared by using dCMP and d C M P - H g - S CH2-CH2-OH as substrates. Also, the effects of dCTP and dTTP on the enzyme activity are listed in Table I. The specific activity of native dCMPase using a saturating concentration of dCMP as a substrate was 820 tzmol/min and mg of protein and that of the enzyme stabilized in the T form was as low as I/zmol/min and mg of protein. In both cases dCTP and dTTP had little or no effect. On the other hand, when using the mercury substrate it was found that the modified enzyme was activated and inhibited by dTTP and dCTP, respectively. However, both ligands inhibited the activity of the enzyme locked in the T conformation in a noncompetitive manner. The assays were carried out at ligand concentrations 10- to 100-fold higher than those effective in modulating the activity of the native enzyme when using dCMP as a substrate. The modified enzyme showed little substrate cooperativity with an n value of 1.5 and the presence of allosteric ligands in the assays yielded the same n value. The kinetic properties of the modified enzyme differed compared with those obtained for native enzyme in studies using d C M P - H g - S - C H 2 13 K. Weber and M. Osborn J. Biol. Chem. 244, 4406 (1969).
584
APPLICATION OF IMMOBILIZED ENZYMES/CELLS
[52]
100
.__.-..--.--..--.-1
>=
10
20
30
I
L,O
minutes
100
~6 50
E I0
20 minutes
30
~'0
FIG. 3. Time-dependent production of oligomeric species by glutaraldehyde treatment of dCMPase in the absence of effector (a) and in the presence of 0.1 mM dTTP + 1 mM dGMP (b). The incubation conditions were the same as those described in the legends to Fig. 2. At the indicated times, portions of 100/~1 (20/zg of protein) were withdrawn and added to 10/~1 of a mixture containing 10% sodium dodecyl sulfate and 10% 2-mercaptoethanol in 0.I M sodium phosphate buffer (pH 7.0). After 5 rain of time of reaction at 90° the samples were analyzed by SDS electrophoresis in 5% polyacrylamide gels according to the method of Weber and Osborn. ~3The relative amounts of monomer (O), dimer (a), trimer (1), tetramer (I-q), and pentamer + hexamer (11) were determined by means of densitometric analysis. From Raia e t al. 11
[52]
FREEZING OF d C M P DEAMINASE CONFORMERS
585
TABLE 1 SPECIFIC ACTIVITY OF NATIVE dCMPase AND GLUTARALDEHYDE-TREATED dCMPase USING dCMP AND d C M P - H g - S - C H 2 - C H ~ - O H AS SUBSTRATES"
dCMP-Hg-S-CH2-CH2-OH
Enzyme system
/zmol dCMP per min and mg of protein
/xmol
dCMPase dCMPase + dCTP (1 /xM) dCMPase + dTTP (5 txM) Glutaraldehyde dCMPase (T form) Glutaraldehyde dCMPase (T form) +
820 820 820 1 4
23 10 71 43 35
1
25
per min and mg of protein
dCTP (20/xM)
Glutaraldehyde dCMPase (T form + dTTP (50/xM) a From Raia et al. 11
CH2-OH as a substrate in the presence of dTTP. This is not surprising since about 30 lysyl residues per molecule had been modified and at least five cross-linking bridges had been formed between subunits. The formed cross-links presumably froze the modified enzyme in the T conformation in such a way that allosteric ligands could no longer shift the equilibrium between the two enzyme forms. In fact, the modified enzyme was still able to bind dCTP or dTTP, but both of them behaved as noncompetitive inhibitors, probably only inducing local conformational changes. It was not possible to stabilize the protein structure containing no ligands complexed with enzyme because, in the absence of substrate or competitive inhibitors, glutaraldehyde rapidly reacted with a reactive amino group localized in the catalytic sites and inactivated the enzyme. In this context, it is worth noting that dCMPase has been entrapped in glutaraldehyde-albumin membranes in both the presence and absence of isosteric and allosteric ligands. 14 The kinetic behavior of the enzyme immobilized in such artificial membranes is markedly different from that of free enzyme. In fact, the enzyme loses its cooperative action toward the substrate and the observed effects on the kinetics of the enzyme in the presence of allosteric ligands could in this case also be attributed to freezing of the enzyme in either of its conformations. However, physical and diffusional resistance to translocation of substrate in the membrane must also be taken into account on interpretation of the obtained kinetic data. 14 G. Iorio, E. Drioli, R. Molinari, and M. Rossi, A n n . N . Y . A c a d . Sci. 369, 235 (1981).