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Control of enzyme activity through regulation of three-dimensional structure of proteins: Studies on pyruvate kinase
C O N T R O L OF E N Z Y M E A C T I V I T Y T H R O U G H R E G U L A T I O N OF THREE-DIMENSIONAL STRUCTURE OF P R O T E I N S : S T U D I E S O N PYRUVATE KINASE BENNO HESS and LOTHARBORNMANN Max-Planck-Institut fur Emahrungsphysiologie, Dortmund, Germany INTRODUCTION
THE high molarity of glycolytic enzymes as well as the oligomeric structure of most of the glycolytic enzymes raise the question of the significance of oligomeric structures in general and also of the mechanism of control of oligome~ic structures. Whereas the control of the glycolytic flux and its overall dynamics can be understood as a result of interactions between small molecules and enzymes, the control mechanism of the oligomeric structure of glycolytic enzymes is still obscure. The formation of tetrameric enzyme species clearly is the result of an equilibration between monomoric species and higher states of aggregation favoring the latter state. However, since it became known that dissociation-association equilibria as well as interconvertibility play a significant role within the overall pathway of glycogenolysis and glycolysis, the question of the nature of the control mechanism of oligomeric structures must be asked. Indeed, in glycolysis the oligomeric properties of phosphofructokinaso and pyruvate kinase have been studied by Hofmann and his collaborators and significant dissociation-association equilibria have been described (1). Here it should be stressed that a prerequisite for a mechanism of aggregation is the formation of a fitted tertiary structure of each monomeric unit. Indeed, when a peptido chain is formed by ribosomal synthesis its first function is to fold itself to form a three-dimensional highly specific structure. Whereas conventional points of view indicate that native proteins are the thermodynamically most stable structures, evidence and arguments have recently been presented showing the operation of kinetic constraints in the folding and reassembly of native proteins. The folding of relatively small peptide chains might proceed straightforward in the range of msec in an aqueous milieu supported by suitable mono- or divalent cations and anions, but the folding of large chains with molecular weights up to 50,000 and more 235
236
BENNO HESS
and
LOTHAR BORNMANN
is a complex process. Indeed, if no kinetic constraints were to operate in the folding, and a random search mechanism of folding was to occur, the time of folding would be indefinitely long and incompatible with what is observed in living systems [for summary see (2-4)]. Recent investigation of the acquisition of an active oligomeric structure of pyrnvate kinase of yeast revealed a specific control mechanism effeeting the formation of an active tetrameric enzyme (4, 5) the results of which will be reported in this paper.
MATERIAL AND METHODS
Pyruvate kinase from S. carlsbergensis (ATCC 24699) was prepared as described (6) with a specific activity of 200-250 units/rag under standard assay conditions (7). Methods of crystallization are given elsewhere (8); furthermore, the techniques of analysis of the denaturation and renaturation are described in (5).
RESULTS AND DISCUSSION
Properties and Function of Pyruvate Kinase of Yeast Pyruvate kinase of yeast is a tetrameric enzyme of a molecular weight of 190,000 (9), which dissociates into subunits of identical molecular weight of approximately 48,000-49,000. This result is supported by a chemical study (10), which demonstrates a similarity in the primary structure on the basis of the products obtained by tryptic digestion, the method of cyanogen bromide cleavage as well as the endgroup analysis, indicating an N-acetylated terminus and valine as the C-terminal amino acid. In the course of this study we found that during the treatment with trichloroacctic acid or heat of the native enzyme 1 mole of valine per mole of monomeric enzyme was released, indicating a non-covalently bound valine as component of the native enzyme (5). Since no exchange of radioactive valine incubated with the native enzyme for 3 days at 6 ° occurs, it appears that the non-covalently bound valine is indeed a tightly interacting part of the native protein. The quaternary structure dimensions of the enzyme have been obtained by X-ray small angle scattering of the amorphous enzyme preparation as well as by application of the electron microscopy and optical diffraction technique to the crystals of the enzyme. An optically filtered image of the enzyme crystal is shown in Figure 1, where the white protein bands display a sausagelike form, where two sausages are arranged in one group (9-11). Units of this array are in the order of 93:126:35.2 A, corresponding to an elliptic cylinder resp. a square with a deviation of an angle of 18°. A compari-
FIG. 1. Optically filtered image of pyruvate kinase crystals obtained by optical folding of an electron microscope photograph. Primary magnification 100,000. [Courtesy, J. Molec. Biol. (from 11).]
J A E R 1 3 , f . p 236
FIG. 2. Scanning electron micrograph (scale 10/~m) of crystalline pyruvate kinase of yeast after fixation with 3 % glutaraldehyde [according to (9)].
RENATURATION OF PYRUVATE KINASE
237
son of the volume of the native, amorphous enzyme with the crystal shows that the dimensions are very similar for both the amorphous enzyme in solution and the crystalline preparation (I 1). An examination of the crystal habit was carried out by scanning electron microscope, a typical example of which is shown in Figure 2. A detailed analysis revealed that the crystal habit belongs to the triclinic pedial class, agreeing with the results obtained by the method of electron microscopy and optical diffraction (9). The kinetic analysis of the enzyme activity demonstrated a linearity of the specific activity of the enzyme and the enzyme concentration in the range of 2.5 × 10-1o M up to 5 × 10 -6 M, indicating thehighstabilityof thetetrameric enzyme [see (9)]. The catalytic function of the enzyme has been described on the basis of four identical protomers, occurring in the form of the classic Rand T-states of the Monod-Wyman-Changeux-model, including one additional symmetric hybrid state (12). These studies have been supported by an analysis of the number of binding sites for interacting ligands. In accordance with the tetrameric state of the enzyme, four binding sites for fructose-l,6bisphosphate (12) and phosphoenolpyruvate (13) could be detected. Furthermore, two high affinity binding sites for ATP were discovered, presumably indicating the sites of interaction of allosterically inhibiting ATP (13, 14). The studies were complemented by an analysis of the amino acid residues participating in the catalytic function of the enzyme, yielding as essential residues histidine (15), lysine (16) and SH-groups (17) as part of the active site.
Denaturation and Renaturation of the Enzyme On incubation in 6 u guanidinehydrochloride for 1 hr at 25°C the enzyme readily dissociates into monomers (7), resulting in a complete disappearance of the enzyme activity as well as quenching of protein fluorescence and circular diehroism signals. When the dissociated enzyme is incubated in a renaturation medium containing phosphoenolpyruvate, glutathione, glycerol and valine the enzyme activity reappears with a yield that is dependent on the concentration of L-valine as shown in Figure 3. Here, the plot of the enzyme activity (Vmax) against the molarity of L-valine shows a saturation kinetic with a valine concentration of 17/zM, giving half maximal yield in relation to the amount of the native enzyme initially present in terms of specific activity. In the course of renaturation experiments it was tested whether valine is reincorporated in the enzyme. Indeed, in the presence of radioactive valine during renaturation a 70~o incorporation of L-valine was found, and a non-covalent bondage was indicated. A detailed analysis of the properties of the renatured enzyme demonstrated identical properties compared to those of the native enzyme as summarized in Table 1. In order to analyze the mechanism of the renaturation the kinetics of the
238
BENNO HESS and LOTHAR BORNMANN
[,.,/,,,,]
-2.56 x 103
.x..X....~ " g ' ' "
x./'~
._
._>
g
-1.28 x
K~s= 17/u M
lO~/ff I
tx
I
I
x'
,t
3'0
6'o
9'O~uM [valine]
Fro. 3. L-Valine concentration dependency of the activity of renatured pyruvate kinase: (×) experimental points; ( ) computer fit. [Courtesy, Proc. Natl Acad. Sci. (from 5).]
TABLE 1. COMPARISONOF RENATUREDAND NATIVEPYRUVATEKINASE* Properties Molecular weight Disk electrophoresis i.P. Content of noncovalently bound valine Fluorometry Activation by fructose-l,6bis-phosphate (2 mM) With ADP (1 mM) + phosphoenolpyruvate (0.25 mM) With ADP (I raM) + phosphoenolpyruvate (0.5 mM) pH maximum of Vm,, Activation energy of enzyme activity (V~,) Trypsin degradation
Renatured 195,000 (no dimers) 1 single band; same mobility as in native enzyme 6.05
Native 195,000 1 single band 6.08
0.7 mol/mol of subunit Excitation maximum 285 nm; emission maximum 320 nm
1 mol/mol of subunit Same
5.64-fold
5.55-fold
2.80-fold 6.0
2.64-fold 6.0
13 kcal × mo1-1 15 % inactivation in 30 min
13 kcal × mo1-1 Same
* For reference, see (5). [Courtesy, Proc. Natl Acad. Sci. (from 5).]
239
RENATURATION OF PYRUVATE KINASE
renaturation process was followed in a time-study of the reappearance of the enzyme activity and described by the derivative equation: -- d(Vmax-
vt), ~ k I - [valine] • [ M * ]
dt where valine >), [M*] and kl × [valine] = kl', [M*] being the "unfolded" monomeric reaction partner of valine. As shown in Figure 4, in a semilogarithmic plot of the reaction progress the kinetics follow a pseudo first order reaction course with a rate constant of kl' of 0.05 min- 1 at 25 ° independent of the enzyme concentration. In the temperature range between 5 and 25 ° the activation energy of the renaturation process as expressed by the pseudo first order rate constant was found to be 36 kcal/mole. These studies were complemented by the analysis of the stability of the enzyme at higher temperature since it could be expected that L-valine also increases the stability of the enzyme toward heat denaturation. It could be shown that the heat inactivation curve is shifted to higher temperature when the experiment is carried out in the presence of L-valinc (5). In the course of mechanistic studies it was tested whether the amino acid counteracts the dissociating action of guanidinehydrochloride. Whereas no competition has been evaluated a concentration range was found in which, even in the presence of 0.36 mM guanidinehydrochloride, valine induces the renaturation of the enzyme, as shown in the progress curve of pyruvate formation with time catalyzed by the renatured enzyme in the presence and absence of L-valine (Fig. 5). This experiment also demonstrates that L-valinc is obligatory for renaturation under these conditions. None of the other components of the renaturation medium alone induces the formation of the active enzyme.
g
0.0"
E !
>E o~
g= o.os [.,in'] - 1.0"
""-'~'~'~._
25 ° C
v
- 2.0
l,
6
0
I() I'2 14 16
1'0 t I'l~in'l
FIG. 4. Scmilogarithmi¢ plot of th¢ rcnaturation progress curve in the presence of L-valin¢ and magnesium ions. [Courtesy, Proc. Natl ~lead. ScL (from 5).1 A.F.R.--I
240
BENNO HESS and LOTHARBORNMANN m_.
[pyruvot,] e I~with tram ratine
2.0 ¸
/ 1.0
/ without va[ino
n~?n~
~5
10
,/ 9
15 m i n u t e s
FIG. 5. Progress curve of the pyruvate kinase reaction in the presence and absence of L-valine. Yeast pyruvate kinase in 0.36 Mguanidine hydrochloride. [Courtesy, Proc. Natl Acad. Sci. (from 5).] The stereochemical requirements for the renaturation of the enzyme were tested with a series of analogs. A typical example is the demonstration of the specificity of the L-configuration of the amino acid compared to its D-form as shown in Figure 6. As demonstrated in Table 2, some analogs in which the hydrophobic cluster was varied had minor renaturation activity as compared TABLE 2.
RE~SOC~ON AND ~ N A ~ R A T I O N OF DISSOCIATED PYRUVATE K ~ E OF S. carlsbergensis IN THE PRESENCE OF VALINE AND ANALOGS
Compound*
Activity in ~o
L-Valine Norvaline y-Hydroxyvaline Cysteine D-Valine Isovaleric acid Isobutylamine
100 64 64 37
* Concentration l raM. [Courtesy, Proc. Natl Acad. ScL (from 5).]
to L-valine. All natural amino acids not mentioned in the table were ineffective at concentrations of 1 mM. F r o m these experiments the high specificity of the hydrophobic cluster as well as the acid and base group of the L-valine structure is obvious. In an analysis of a variety of ligands possibly being involved in the control of formation of the tertiary structure of the enzyme it was found that the
241
RENATURATION OF PYRUVATE KINASE Renaturation yield in % L - Valine
50
40 30
/
20
10 D - Valine
# _ o - o-- o--o--o--o--o--o--'v--o 0.5
1.0 mM
[Voline]
FIG. 6. Renaturation yield expressed in percent activity as a function of the concentration of L- and D-valine. [Courtesy, Proc. Natl Acad. ScL (from 5).]
renaturation of enzyme activity could also be induced by magnesium or manganese ions in the absence of L-valine with a yield of 60~o and a pseudo first order rate constant for the activation of 0.0234 min-1 at 25 ° in the presence of magnesium ions (10 mM). The renaturation yield could be increased up to 75Y/o when magnesium ions and valine were tested simultaneously. Here it was found that the presence of both ligands produce a pseudo first order rate constant equal to the sum of the rate constants observed when one of them was tested separately. Thus, for full renaturation both ligands seem to be oligatory, adding up in effectivity when tested simultaneously. The mechanism of this cooperation is still obscure; however, it could be visualized that a folding process could possibly start from two different sites along the peptide, resulting in the formation of the same final product. Our experiments indicate that L-valine as well as magnesium or manganese ions initiate the renaturation of the denatured enzyme with a high yield and a specific requirement of their structures. The mechanism of this renaturation is obviously most complex because of the large number of possible conformation states which could occur with an amino acid sequence of at least 450 units. A process of tertiary structure formation can be visualized as: (1) A folding process in which a disordered monomerie polypeptide chain yields a specific ordered native conformation involving a nucleation step and subsequently a growth event. The formation of a nucleation centre implies a limited number of amino acid residues. During the growth helical and nonhelical regions, pleated sheets and interchain regions are generated. For
242
BENNO HESS a n d LOTHAR BORNMANN
large chains with molecular weights up to 50,000 more than one nucleation centre must be considered. (2) In case of oligomeric enzymes an association of rightly folded monomers toward a higher state of aggregation follows. Here it should be remembered that an association of folded monomers does not imply that the active centre of the monomers is also in the "rightly" folded form. Our kinetic analysis relying on activity measurements yielded a pseudo first order rate constant, indicating that L-valine influences the folding of the monomerie form and does not interact with a dimeric or tetrameric species. It should be stressed that the activity measurements must be complemented by other experimental means to define the functional state of the enzyme during the folding process; however, presently we favor this explanation, not only because of the pseudo first order reaction course being independent of the protein concentration but also because of the stoichiometry of valine in the native and renatured enzyme and the extremely tight binding, indicating that the enzyme is deeply buried in each monomeric structure. Also the high heat of activation of the renaturation process points to a complicated process and not a similar binding event. Thus, we do not feel that a preconditioning mechanism as suggested for phosphofructokinase (18) can be implied in the present case as far as the estimable data allow. Recently, studies on small molecules indicate that the process of folding is inconsistent with thermodynamic determinism of protein structure but rather is determined by kinetic constraints (19, 20). Indeed, the physiological pathway of folding seems to be strictly determined as a one-way street. At the present state the chemical identification of nucleation centres or intermediary states of folding pathways is at its very beginning, although very recently some results have been reported indicating the occurrence of intermediates (20, 21). In case of the larger enzymes occurring in bioenergetic pathways, such as glycolysis (22-24), the rate of regain of enzyme activity from denatured proteins has been found to be a function of the presence of substrates or eofactors in addition to environmental conditions, indicating that kinetic constraints play a significant role in the formation of active enzyme conformations. Considering the high complexity of a folding process of large polypeptides (see 2, 3) the question must be asked whether a possible similarity of the folding pathway depending on a given structural size and enzymic function might not exist resulting from evolutionary development. Indeed, such a similarity of the general topology of the tertiary chain structure in a class of large enzymes has been discovered (25, 26). A number of reasons suggest that similarities of tertiary structure topology of enzymes might result from the evolution of common folding pathways which are kinetically and energetically favored, implying a minimum of energy demand as well as a time
RENATURATION OF PYRUVATE KINASE
243
requirement which is compatible with the high turnover of protein synthesis and degradation in cellular life (27). With respect to physiological conditions the general question must be asked whether results obtained in the study of renaturation simulate what is occurring in nature. At the present state it seems to be difficult to prove whether the folding pathway is what happens in the living cell. However, since the product of renaturation compares well with the natural product it seems justifiable to assume that at least the latter state of the folding pathway, as well as the association steps, is identical to the physiological situation. Also, it should be stressed that the role of ribosomal activity and structure in the folding process has not been elucidated yet. In case of the valine function and the renaturation of pyruvate kinase it seems to be difficult to establish its physiological role since valine affects the renaturation process at concentrations which are at least two orders of magnitude lower compared to the level necessary to induce ribosomal activity. Thus, it might well be that valine as a feed-forward-effect kinetically controls the immediate folding of newly synthesized peptide chains of pyruvate kinase generated by ribosomal activity. A similar mechanism could be expected in case of other large enzyme molecules. For the function of NAD in case of glyceraldehyde-3-phosphate dehydrogenase (23) as well as pyridoxal phosphate in case of phosphorylase (24) as well as other cofactors (see 22) such a mechanism could be considered. Furthermore, it should be stressed that a control of folding by small molecules of intermediary metabolism might be decisive in avoiding the appearance of abortive enzyme conformations which might build up in living cells as inactive monomers and even higher states of aggregation as indicated elsewhere (28, 29).
SUMMARY
A novel function of an amino acid, namely L-valine, in the renaturation process of pyruvate kinase of yeast is described. The amino acid induces the renaturation with a Ko.5 of 17/zM and a pseudo first order rate constant of 0.019 min - I at 25 ° with respect to the monomerie species. This and other results indicate that L-valine influences the folding of the monomeric form from a disordered state to its native conformation, being followed by a spontaneous and rapid reassociation under formation of the tetramerie enzyme. This action of L-valine is highly specific and only shared to a lesser degree by the analogs y-hydroxyvaline and norvaline. All natural amino acids as well as D-valine are inactive in this function. The analysis of the structural configuration necessary to induce renaturation demonstrates that both charge groups as well as the hydrophobic cluster of the L-configuration specifically interacts with a site of the peptide chain. In a discussion of available data and the present study it is concluded that the folding process
244
BENNO HESS
and LOTHAR
BORNMANN
yielding the secondary and tertiary structures of proteins is strongly determined by kinetic constraints. This function adds a new dimension to the control network of metabolic pathways, in which control of enzyme conformation is linked to ubiquitous low-molecular molecules of intermediary metabolism being common controlling agents of enzyme activities and affinities. REFERENCES 1. E. HOFMANN, B. I. KURGANOV,W. SCHELLENBERGER,J. SCHULZ, G. SPARMANN, K.=W. WENZELand G. ZIMMERMANN,Association-dissociation behavior of erythrocyte phosphofructokinase and tumor pyruvate kinase, in this volume, pp. 247-277. 2. D. B. WETLAUFERand S. R~STOW, Acquisition of three-dimensional structure of proteins, Ann. Rev. Biochem. 42, 135-138 (1973). 3. D. B. WETLAUFER,Nucleation, rapid folding and globular interchain regions in proteins, Proc. Natl Acad. ScL, USA 70, 697-701 (1973). 4. B. HESSand L. BORNMANN,Renaturation mechanism of pyruvate kinase of S. carlsbergensls, in Metabolic Interconversion of Enzymes (E. FISHER,ed.), Springer-Verlag, Berlin, Heidelberg, New York, pp. 361-367 (1974). 5. L. BORNMANN,B. HESSand H. ZIMMERMANN-TELSCHOW,Mechanism of renaturation of pyruvate kinase of S. carlsbergensis: Activation by L-valine and magnesium and manganese ions, Proe. Natl Acad. ScL, USA 71, 1525-1529 (1974). 6. P. R6SCHLAUand B. HESS, Affinity chromatography of yeast pyruvate kinase with cibacronblau bound to Sephadex G-200, Hoppe-Seyler's Z. PhysioL Chem. 353, 441--443 (1972). 7. H. BISCHOFBERGER,B. HESS, P. R6SCHLAU, H.-J. WIEKER and H. ZIMMERMANNTELSCHOW, Amino-acid composition and subunit structure of yeast pyruvate kinase, Hoppe-Seyler's Z. PhysioL Chem. 351, 401-408 (1970). 8. P. ROSCHLAUand B. HESS, Purification and crystallization of yeast pyruvate kinase, Hoppe-Seyler's Z. PhysioL Chem. 353, 435-440 (1972). 9. B. HESS and J. SOSSrNKA,Pyruvate kinase of yeast. Properties and crystals, Naturwissenschaften 61, 122-124 (1974). 10. L. BORNMANNand B. HESs, The subunit structure of yeast pyruvate kinase, Europ. J. Biochem. 47, 1-4 (1974). 11. J. SOSSINKAand B. HEss, Electron microscopy of pyruvate kinase crystals from S. carlsbergensis, J. MoL BioL 83, 285-287 (1974). 12. K.-J. JOHANNESand B. HESS,Allosteric kinetics of pyruvate kinase of S. carlsbergensis, J. MoL BioL 76, 181-205 (1973). 13. K. IMAMURAand B. HESs, unpublished results. 14. R. HAECKEL,B. HESS, W. LAUTERBORNand K. H. WOSTER,Purification and allosteric properties of yeast pyruvate kinase, Hoppe-Seyler's Z. PhysioL Chem. 349, 699-714 (1968). 15. L. BORNMANNand B. HESS, Modification of histidine residues in yeast pyruvate kinase by diethylpyrocarbonate, Hoppe-Seyler's Z. PhysioL Chem. 355, 1073-1076 (1974). 16. P. R6SCHLAUand B. HESS, Modification of yeast pyruvate kinase by 2,4,6-trinitrobenzenesulphonic acid, Hoppe-Seyler's Z. PhysioL Chem. 353, 944-948 (1972). 17. H.-J. WEKERand B. HESS, Function of thiol groups in yeast pyruvate kinase, HoppeSeyler's Z. Physiol. Chem. 353, 1877-1893 (1972). 18. J. B. ALPERS, H. PAULUSand G. A. BAZYLEWlCZ,ATP-Catalyzed preconditioning of phosphofructokinase, Proc. Natl Acad. Sci. USA 68, 2937-2940 (1971). 19. T. E. CREIGHTON, The single-disulphide intermediates in the refolding of reduced pancreatic trypsin inhibitor, J. Mol. Biol. 87, 603-624 (1974). 20. D. WETLAUFER,E. KWOK,W. L. ANDERSONand E. R. JOHNSON,Kinetic determinism of lysozyme folding at high temperatures, Biochem. Biophys. Res. Commun. 56, 380-385 (1974).
RENATURATIONOF PYRUVATE KINASE
245
21. T. E. CREIGHTON,Intermediates in the refolding of reduced pancreatic trypsin inhibitor, J. Mol. BioL 87, 579--602 (1974). 22. J. W. TEIPELand D. E. KOSHLAND,JR., Kinetic aspects of conformational changes in proteins. I. Rate of regain of enzyme activity from denatured proteins, Biochemistry 10, 792-798 (1971). 23. W. C. DEAL,Metabolic control and structure of glyeolytic enzymes. IV. Nieotinamideadenine dinucleotide dependent in vitro reversal of dissociation and possible in vivo control of yeast glyceraldehyde 3-phosphate dehydrogenase synthesis, Biochem. 8, 2795-2805 (1969). 24. E. FISCHERand E. G. KgEBS, Relationship of structure to function of muscle phosphorylase, Federation Proc. 25, 1511-1520 (1966). 25. M. G. ROSSMAN~,D. MORASand K. W. OLSEN,Chemical and biological evolution of a nucleotide-bindingprotein, Nature 250, 194-199 (1974). 26. G. E. SCHULZand A. H. SCHIRMER,Topological comparison of adenylate kinase with other proteins, Nature 250, 142-144 (1974). 27. B. HESS, Remarks on the acquisition of active quaternary structure of enzymes, pp. 270-280 in Energy, Biosynthesis and Regulation in Molecular Biology (RICHTER, ed.), Walter de Gruyter Verlag, Berlin-New York (1974). 28. J. W. TEIPELand D. E. KOSHLAND,JR., Kinetic aspects of conformational changes in proteins. II. Structural changes in renaturation of denatured proteins, Biochemistry 10, 798-805 (1971). 29. B. HEss and P. BISCrIOrBERGeg,unpublished observation.
THE high molarity of glycolytic enzymes as well as the oligomeric structure of most of the glycolytic enzymes raise the question of the significance of oligomeric structures in general and also of the mechanism of control of oligome~ic structures. Whereas the control of the glycolytic flux and its overall dynamics can be understood as a result of interactions between small molecules and enzymes, the control mechanism of the oligomeric structure of glycolytic enzymes is still obscure. The formation of tetrameric enzyme species clearly is the result of an equilibration between monomoric species and higher states of aggregation favoring the latter state. However, since it became known that dissociation-association equilibria as well as interconvertibility play a significant role within the overall pathway of glycogenolysis and glycolysis, the question of the nature of the control mechanism of oligomeric structures must be asked. Indeed, in glycolysis the oligomeric properties of phosphofructokinaso and pyruvate kinase have been studied by Hofmann and his collaborators and significant dissociation-association equilibria have been described (1). Here it should be stressed that a prerequisite for a mechanism of aggregation is the formation of a fitted tertiary structure of each monomeric unit. Indeed, when a peptido chain is formed by ribosomal synthesis its first function is to fold itself to form a three-dimensional highly specific structure. Whereas conventional points of view indicate that native proteins are the thermodynamically most stable structures, evidence and arguments have recently been presented showing the operation of kinetic constraints in the folding and reassembly of native proteins. The folding of relatively small peptide chains might proceed straightforward in the range of msec in an aqueous milieu supported by suitable mono- or divalent cations and anions, but the folding of large chains with molecular weights up to 50,000 and more 235
236
BENNO HESS
and
LOTHAR BORNMANN
is a complex process. Indeed, if no kinetic constraints were to operate in the folding, and a random search mechanism of folding was to occur, the time of folding would be indefinitely long and incompatible with what is observed in living systems [for summary see (2-4)]. Recent investigation of the acquisition of an active oligomeric structure of pyrnvate kinase of yeast revealed a specific control mechanism effeeting the formation of an active tetrameric enzyme (4, 5) the results of which will be reported in this paper.
MATERIAL AND METHODS
Pyruvate kinase from S. carlsbergensis (ATCC 24699) was prepared as described (6) with a specific activity of 200-250 units/rag under standard assay conditions (7). Methods of crystallization are given elsewhere (8); furthermore, the techniques of analysis of the denaturation and renaturation are described in (5).
RESULTS AND DISCUSSION
Properties and Function of Pyruvate Kinase of Yeast Pyruvate kinase of yeast is a tetrameric enzyme of a molecular weight of 190,000 (9), which dissociates into subunits of identical molecular weight of approximately 48,000-49,000. This result is supported by a chemical study (10), which demonstrates a similarity in the primary structure on the basis of the products obtained by tryptic digestion, the method of cyanogen bromide cleavage as well as the endgroup analysis, indicating an N-acetylated terminus and valine as the C-terminal amino acid. In the course of this study we found that during the treatment with trichloroacctic acid or heat of the native enzyme 1 mole of valine per mole of monomeric enzyme was released, indicating a non-covalently bound valine as component of the native enzyme (5). Since no exchange of radioactive valine incubated with the native enzyme for 3 days at 6 ° occurs, it appears that the non-covalently bound valine is indeed a tightly interacting part of the native protein. The quaternary structure dimensions of the enzyme have been obtained by X-ray small angle scattering of the amorphous enzyme preparation as well as by application of the electron microscopy and optical diffraction technique to the crystals of the enzyme. An optically filtered image of the enzyme crystal is shown in Figure 1, where the white protein bands display a sausagelike form, where two sausages are arranged in one group (9-11). Units of this array are in the order of 93:126:35.2 A, corresponding to an elliptic cylinder resp. a square with a deviation of an angle of 18°. A compari-
FIG. 1. Optically filtered image of pyruvate kinase crystals obtained by optical folding of an electron microscope photograph. Primary magnification 100,000. [Courtesy, J. Molec. Biol. (from 11).]
J A E R 1 3 , f . p 236
FIG. 2. Scanning electron micrograph (scale 10/~m) of crystalline pyruvate kinase of yeast after fixation with 3 % glutaraldehyde [according to (9)].
RENATURATION OF PYRUVATE KINASE
237
son of the volume of the native, amorphous enzyme with the crystal shows that the dimensions are very similar for both the amorphous enzyme in solution and the crystalline preparation (I 1). An examination of the crystal habit was carried out by scanning electron microscope, a typical example of which is shown in Figure 2. A detailed analysis revealed that the crystal habit belongs to the triclinic pedial class, agreeing with the results obtained by the method of electron microscopy and optical diffraction (9). The kinetic analysis of the enzyme activity demonstrated a linearity of the specific activity of the enzyme and the enzyme concentration in the range of 2.5 × 10-1o M up to 5 × 10 -6 M, indicating thehighstabilityof thetetrameric enzyme [see (9)]. The catalytic function of the enzyme has been described on the basis of four identical protomers, occurring in the form of the classic Rand T-states of the Monod-Wyman-Changeux-model, including one additional symmetric hybrid state (12). These studies have been supported by an analysis of the number of binding sites for interacting ligands. In accordance with the tetrameric state of the enzyme, four binding sites for fructose-l,6bisphosphate (12) and phosphoenolpyruvate (13) could be detected. Furthermore, two high affinity binding sites for ATP were discovered, presumably indicating the sites of interaction of allosterically inhibiting ATP (13, 14). The studies were complemented by an analysis of the amino acid residues participating in the catalytic function of the enzyme, yielding as essential residues histidine (15), lysine (16) and SH-groups (17) as part of the active site.
Denaturation and Renaturation of the Enzyme On incubation in 6 u guanidinehydrochloride for 1 hr at 25°C the enzyme readily dissociates into monomers (7), resulting in a complete disappearance of the enzyme activity as well as quenching of protein fluorescence and circular diehroism signals. When the dissociated enzyme is incubated in a renaturation medium containing phosphoenolpyruvate, glutathione, glycerol and valine the enzyme activity reappears with a yield that is dependent on the concentration of L-valine as shown in Figure 3. Here, the plot of the enzyme activity (Vmax) against the molarity of L-valine shows a saturation kinetic with a valine concentration of 17/zM, giving half maximal yield in relation to the amount of the native enzyme initially present in terms of specific activity. In the course of renaturation experiments it was tested whether valine is reincorporated in the enzyme. Indeed, in the presence of radioactive valine during renaturation a 70~o incorporation of L-valine was found, and a non-covalent bondage was indicated. A detailed analysis of the properties of the renatured enzyme demonstrated identical properties compared to those of the native enzyme as summarized in Table 1. In order to analyze the mechanism of the renaturation the kinetics of the
238
BENNO HESS and LOTHAR BORNMANN
[,.,/,,,,]
-2.56 x 103
.x..X....~ " g ' ' "
x./'~
._
._>
g
-1.28 x
K~s= 17/u M
lO~/ff I
tx
I
I
x'
,t
3'0
6'o
9'O~uM [valine]
Fro. 3. L-Valine concentration dependency of the activity of renatured pyruvate kinase: (×) experimental points; ( ) computer fit. [Courtesy, Proc. Natl Acad. Sci. (from 5).]
TABLE 1. COMPARISONOF RENATUREDAND NATIVEPYRUVATEKINASE* Properties Molecular weight Disk electrophoresis i.P. Content of noncovalently bound valine Fluorometry Activation by fructose-l,6bis-phosphate (2 mM) With ADP (1 mM) + phosphoenolpyruvate (0.25 mM) With ADP (I raM) + phosphoenolpyruvate (0.5 mM) pH maximum of Vm,, Activation energy of enzyme activity (V~,) Trypsin degradation
Renatured 195,000 (no dimers) 1 single band; same mobility as in native enzyme 6.05
Native 195,000 1 single band 6.08
0.7 mol/mol of subunit Excitation maximum 285 nm; emission maximum 320 nm
1 mol/mol of subunit Same
5.64-fold
5.55-fold
2.80-fold 6.0
2.64-fold 6.0
13 kcal × mo1-1 15 % inactivation in 30 min
13 kcal × mo1-1 Same
* For reference, see (5). [Courtesy, Proc. Natl Acad. Sci. (from 5).]
239
RENATURATION OF PYRUVATE KINASE
renaturation process was followed in a time-study of the reappearance of the enzyme activity and described by the derivative equation: -- d(Vmax-
vt), ~ k I - [valine] • [ M * ]
dt where valine >), [M*] and kl × [valine] = kl', [M*] being the "unfolded" monomeric reaction partner of valine. As shown in Figure 4, in a semilogarithmic plot of the reaction progress the kinetics follow a pseudo first order reaction course with a rate constant of kl' of 0.05 min- 1 at 25 ° independent of the enzyme concentration. In the temperature range between 5 and 25 ° the activation energy of the renaturation process as expressed by the pseudo first order rate constant was found to be 36 kcal/mole. These studies were complemented by the analysis of the stability of the enzyme at higher temperature since it could be expected that L-valine also increases the stability of the enzyme toward heat denaturation. It could be shown that the heat inactivation curve is shifted to higher temperature when the experiment is carried out in the presence of L-valinc (5). In the course of mechanistic studies it was tested whether the amino acid counteracts the dissociating action of guanidinehydrochloride. Whereas no competition has been evaluated a concentration range was found in which, even in the presence of 0.36 mM guanidinehydrochloride, valine induces the renaturation of the enzyme, as shown in the progress curve of pyruvate formation with time catalyzed by the renatured enzyme in the presence and absence of L-valine (Fig. 5). This experiment also demonstrates that L-valinc is obligatory for renaturation under these conditions. None of the other components of the renaturation medium alone induces the formation of the active enzyme.
g
0.0"
E !
>E o~
g= o.os [.,in'] - 1.0"
""-'~'~'~._
25 ° C
v
- 2.0
l,
6
0
I() I'2 14 16
1'0 t I'l~in'l
FIG. 4. Scmilogarithmi¢ plot of th¢ rcnaturation progress curve in the presence of L-valin¢ and magnesium ions. [Courtesy, Proc. Natl ~lead. ScL (from 5).1 A.F.R.--I
240
BENNO HESS and LOTHARBORNMANN m_.
[pyruvot,] e I~with tram ratine
2.0 ¸
/ 1.0
/ without va[ino
n~?n~
~5
10
,/ 9
15 m i n u t e s
FIG. 5. Progress curve of the pyruvate kinase reaction in the presence and absence of L-valine. Yeast pyruvate kinase in 0.36 Mguanidine hydrochloride. [Courtesy, Proc. Natl Acad. Sci. (from 5).] The stereochemical requirements for the renaturation of the enzyme were tested with a series of analogs. A typical example is the demonstration of the specificity of the L-configuration of the amino acid compared to its D-form as shown in Figure 6. As demonstrated in Table 2, some analogs in which the hydrophobic cluster was varied had minor renaturation activity as compared TABLE 2.
RE~SOC~ON AND ~ N A ~ R A T I O N OF DISSOCIATED PYRUVATE K ~ E OF S. carlsbergensis IN THE PRESENCE OF VALINE AND ANALOGS
Compound*
Activity in ~o
L-Valine Norvaline y-Hydroxyvaline Cysteine D-Valine Isovaleric acid Isobutylamine
100 64 64 37
* Concentration l raM. [Courtesy, Proc. Natl Acad. ScL (from 5).]
to L-valine. All natural amino acids not mentioned in the table were ineffective at concentrations of 1 mM. F r o m these experiments the high specificity of the hydrophobic cluster as well as the acid and base group of the L-valine structure is obvious. In an analysis of a variety of ligands possibly being involved in the control of formation of the tertiary structure of the enzyme it was found that the
241
RENATURATION OF PYRUVATE KINASE Renaturation yield in % L - Valine
50
40 30
/
20
10 D - Valine
# _ o - o-- o--o--o--o--o--o--'v--o 0.5
1.0 mM
[Voline]
FIG. 6. Renaturation yield expressed in percent activity as a function of the concentration of L- and D-valine. [Courtesy, Proc. Natl Acad. ScL (from 5).]
renaturation of enzyme activity could also be induced by magnesium or manganese ions in the absence of L-valine with a yield of 60~o and a pseudo first order rate constant for the activation of 0.0234 min-1 at 25 ° in the presence of magnesium ions (10 mM). The renaturation yield could be increased up to 75Y/o when magnesium ions and valine were tested simultaneously. Here it was found that the presence of both ligands produce a pseudo first order rate constant equal to the sum of the rate constants observed when one of them was tested separately. Thus, for full renaturation both ligands seem to be oligatory, adding up in effectivity when tested simultaneously. The mechanism of this cooperation is still obscure; however, it could be visualized that a folding process could possibly start from two different sites along the peptide, resulting in the formation of the same final product. Our experiments indicate that L-valine as well as magnesium or manganese ions initiate the renaturation of the denatured enzyme with a high yield and a specific requirement of their structures. The mechanism of this renaturation is obviously most complex because of the large number of possible conformation states which could occur with an amino acid sequence of at least 450 units. A process of tertiary structure formation can be visualized as: (1) A folding process in which a disordered monomerie polypeptide chain yields a specific ordered native conformation involving a nucleation step and subsequently a growth event. The formation of a nucleation centre implies a limited number of amino acid residues. During the growth helical and nonhelical regions, pleated sheets and interchain regions are generated. For
242
BENNO HESS a n d LOTHAR BORNMANN
large chains with molecular weights up to 50,000 more than one nucleation centre must be considered. (2) In case of oligomeric enzymes an association of rightly folded monomers toward a higher state of aggregation follows. Here it should be remembered that an association of folded monomers does not imply that the active centre of the monomers is also in the "rightly" folded form. Our kinetic analysis relying on activity measurements yielded a pseudo first order rate constant, indicating that L-valine influences the folding of the monomerie form and does not interact with a dimeric or tetrameric species. It should be stressed that the activity measurements must be complemented by other experimental means to define the functional state of the enzyme during the folding process; however, presently we favor this explanation, not only because of the pseudo first order reaction course being independent of the protein concentration but also because of the stoichiometry of valine in the native and renatured enzyme and the extremely tight binding, indicating that the enzyme is deeply buried in each monomeric structure. Also the high heat of activation of the renaturation process points to a complicated process and not a similar binding event. Thus, we do not feel that a preconditioning mechanism as suggested for phosphofructokinase (18) can be implied in the present case as far as the estimable data allow. Recently, studies on small molecules indicate that the process of folding is inconsistent with thermodynamic determinism of protein structure but rather is determined by kinetic constraints (19, 20). Indeed, the physiological pathway of folding seems to be strictly determined as a one-way street. At the present state the chemical identification of nucleation centres or intermediary states of folding pathways is at its very beginning, although very recently some results have been reported indicating the occurrence of intermediates (20, 21). In case of the larger enzymes occurring in bioenergetic pathways, such as glycolysis (22-24), the rate of regain of enzyme activity from denatured proteins has been found to be a function of the presence of substrates or eofactors in addition to environmental conditions, indicating that kinetic constraints play a significant role in the formation of active enzyme conformations. Considering the high complexity of a folding process of large polypeptides (see 2, 3) the question must be asked whether a possible similarity of the folding pathway depending on a given structural size and enzymic function might not exist resulting from evolutionary development. Indeed, such a similarity of the general topology of the tertiary chain structure in a class of large enzymes has been discovered (25, 26). A number of reasons suggest that similarities of tertiary structure topology of enzymes might result from the evolution of common folding pathways which are kinetically and energetically favored, implying a minimum of energy demand as well as a time
RENATURATION OF PYRUVATE KINASE
243
requirement which is compatible with the high turnover of protein synthesis and degradation in cellular life (27). With respect to physiological conditions the general question must be asked whether results obtained in the study of renaturation simulate what is occurring in nature. At the present state it seems to be difficult to prove whether the folding pathway is what happens in the living cell. However, since the product of renaturation compares well with the natural product it seems justifiable to assume that at least the latter state of the folding pathway, as well as the association steps, is identical to the physiological situation. Also, it should be stressed that the role of ribosomal activity and structure in the folding process has not been elucidated yet. In case of the valine function and the renaturation of pyruvate kinase it seems to be difficult to establish its physiological role since valine affects the renaturation process at concentrations which are at least two orders of magnitude lower compared to the level necessary to induce ribosomal activity. Thus, it might well be that valine as a feed-forward-effect kinetically controls the immediate folding of newly synthesized peptide chains of pyruvate kinase generated by ribosomal activity. A similar mechanism could be expected in case of other large enzyme molecules. For the function of NAD in case of glyceraldehyde-3-phosphate dehydrogenase (23) as well as pyridoxal phosphate in case of phosphorylase (24) as well as other cofactors (see 22) such a mechanism could be considered. Furthermore, it should be stressed that a control of folding by small molecules of intermediary metabolism might be decisive in avoiding the appearance of abortive enzyme conformations which might build up in living cells as inactive monomers and even higher states of aggregation as indicated elsewhere (28, 29).
SUMMARY
A novel function of an amino acid, namely L-valine, in the renaturation process of pyruvate kinase of yeast is described. The amino acid induces the renaturation with a Ko.5 of 17/zM and a pseudo first order rate constant of 0.019 min - I at 25 ° with respect to the monomerie species. This and other results indicate that L-valine influences the folding of the monomeric form from a disordered state to its native conformation, being followed by a spontaneous and rapid reassociation under formation of the tetramerie enzyme. This action of L-valine is highly specific and only shared to a lesser degree by the analogs y-hydroxyvaline and norvaline. All natural amino acids as well as D-valine are inactive in this function. The analysis of the structural configuration necessary to induce renaturation demonstrates that both charge groups as well as the hydrophobic cluster of the L-configuration specifically interacts with a site of the peptide chain. In a discussion of available data and the present study it is concluded that the folding process
244
BENNO HESS
and LOTHAR
BORNMANN
yielding the secondary and tertiary structures of proteins is strongly determined by kinetic constraints. This function adds a new dimension to the control network of metabolic pathways, in which control of enzyme conformation is linked to ubiquitous low-molecular molecules of intermediary metabolism being common controlling agents of enzyme activities and affinities. REFERENCES 1. E. HOFMANN, B. I. KURGANOV,W. SCHELLENBERGER,J. SCHULZ, G. SPARMANN, K.=W. WENZELand G. ZIMMERMANN,Association-dissociation behavior of erythrocyte phosphofructokinase and tumor pyruvate kinase, in this volume, pp. 247-277. 2. D. B. WETLAUFERand S. R~STOW, Acquisition of three-dimensional structure of proteins, Ann. Rev. Biochem. 42, 135-138 (1973). 3. D. B. WETLAUFER,Nucleation, rapid folding and globular interchain regions in proteins, Proc. Natl Acad. ScL, USA 70, 697-701 (1973). 4. B. HESSand L. BORNMANN,Renaturation mechanism of pyruvate kinase of S. carlsbergensls, in Metabolic Interconversion of Enzymes (E. FISHER,ed.), Springer-Verlag, Berlin, Heidelberg, New York, pp. 361-367 (1974). 5. L. BORNMANN,B. HESSand H. ZIMMERMANN-TELSCHOW,Mechanism of renaturation of pyruvate kinase of S. carlsbergensis: Activation by L-valine and magnesium and manganese ions, Proe. Natl Acad. ScL, USA 71, 1525-1529 (1974). 6. P. R6SCHLAUand B. HESS, Affinity chromatography of yeast pyruvate kinase with cibacronblau bound to Sephadex G-200, Hoppe-Seyler's Z. PhysioL Chem. 353, 441--443 (1972). 7. H. BISCHOFBERGER,B. HESS, P. R6SCHLAU, H.-J. WIEKER and H. ZIMMERMANNTELSCHOW, Amino-acid composition and subunit structure of yeast pyruvate kinase, Hoppe-Seyler's Z. PhysioL Chem. 351, 401-408 (1970). 8. P. ROSCHLAUand B. HESS, Purification and crystallization of yeast pyruvate kinase, Hoppe-Seyler's Z. PhysioL Chem. 353, 435-440 (1972). 9. B. HESS and J. SOSSrNKA,Pyruvate kinase of yeast. Properties and crystals, Naturwissenschaften 61, 122-124 (1974). 10. L. BORNMANNand B. HESs, The subunit structure of yeast pyruvate kinase, Europ. J. Biochem. 47, 1-4 (1974). 11. J. SOSSINKAand B. HEss, Electron microscopy of pyruvate kinase crystals from S. carlsbergensis, J. MoL BioL 83, 285-287 (1974). 12. K.-J. JOHANNESand B. HESS,Allosteric kinetics of pyruvate kinase of S. carlsbergensis, J. MoL BioL 76, 181-205 (1973). 13. K. IMAMURAand B. HESs, unpublished results. 14. R. HAECKEL,B. HESS, W. LAUTERBORNand K. H. WOSTER,Purification and allosteric properties of yeast pyruvate kinase, Hoppe-Seyler's Z. PhysioL Chem. 349, 699-714 (1968). 15. L. BORNMANNand B. HESS, Modification of histidine residues in yeast pyruvate kinase by diethylpyrocarbonate, Hoppe-Seyler's Z. PhysioL Chem. 355, 1073-1076 (1974). 16. P. R6SCHLAUand B. HESS, Modification of yeast pyruvate kinase by 2,4,6-trinitrobenzenesulphonic acid, Hoppe-Seyler's Z. PhysioL Chem. 353, 944-948 (1972). 17. H.-J. WEKERand B. HESS, Function of thiol groups in yeast pyruvate kinase, HoppeSeyler's Z. Physiol. Chem. 353, 1877-1893 (1972). 18. J. B. ALPERS, H. PAULUSand G. A. BAZYLEWlCZ,ATP-Catalyzed preconditioning of phosphofructokinase, Proc. Natl Acad. Sci. USA 68, 2937-2940 (1971). 19. T. E. CREIGHTON, The single-disulphide intermediates in the refolding of reduced pancreatic trypsin inhibitor, J. Mol. Biol. 87, 603-624 (1974). 20. D. WETLAUFER,E. KWOK,W. L. ANDERSONand E. R. JOHNSON,Kinetic determinism of lysozyme folding at high temperatures, Biochem. Biophys. Res. Commun. 56, 380-385 (1974).
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21. T. E. CREIGHTON,Intermediates in the refolding of reduced pancreatic trypsin inhibitor, J. Mol. BioL 87, 579--602 (1974). 22. J. W. TEIPELand D. E. KOSHLAND,JR., Kinetic aspects of conformational changes in proteins. I. Rate of regain of enzyme activity from denatured proteins, Biochemistry 10, 792-798 (1971). 23. W. C. DEAL,Metabolic control and structure of glyeolytic enzymes. IV. Nieotinamideadenine dinucleotide dependent in vitro reversal of dissociation and possible in vivo control of yeast glyceraldehyde 3-phosphate dehydrogenase synthesis, Biochem. 8, 2795-2805 (1969). 24. E. FISCHERand E. G. KgEBS, Relationship of structure to function of muscle phosphorylase, Federation Proc. 25, 1511-1520 (1966). 25. M. G. ROSSMAN~,D. MORASand K. W. OLSEN,Chemical and biological evolution of a nucleotide-bindingprotein, Nature 250, 194-199 (1974). 26. G. E. SCHULZand A. H. SCHIRMER,Topological comparison of adenylate kinase with other proteins, Nature 250, 142-144 (1974). 27. B. HESS, Remarks on the acquisition of active quaternary structure of enzymes, pp. 270-280 in Energy, Biosynthesis and Regulation in Molecular Biology (RICHTER, ed.), Walter de Gruyter Verlag, Berlin-New York (1974). 28. J. W. TEIPELand D. E. KOSHLAND,JR., Kinetic aspects of conformational changes in proteins. II. Structural changes in renaturation of denatured proteins, Biochemistry 10, 798-805 (1971). 29. B. HEss and P. BISCrIOrBERGeg,unpublished observation.