Proc. Nat. Acad. Sci. USA
Vol. 72, No. 11, pp. 4298-4302, November 1975 Biochemistry
Quaternary constraint in hybrid of aspartate transcarbamylase containing wild-type and mutant catalytic subunits (allosteric enzyme/inter-subunit hybrid/inactive mutant/subunit interactions/aspartate carbamoyltransferase)
IAN GIBBONS*, J. E. FLATGAARDt, AND H. K. SCHACHMANt Department of Molecular Biology and The Virus Laboratory, University of California, Berkeley, Calif. 94720
Contributed by H. K. Schachimn, August 15,71975
ABSTRACT Unusual quaternary constraint in the regulatory enzyme, aspartate transcarbamylase (aspartate carbamoyltransferase or carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) from Escherichia coli, was demonstrated with a hybrid composed of one inactive "catalytic" subunit from a mutant strain and one active catalytic subunit and three regulatory subunits from the wild-type strain. The hybrid had a high affinity for three molecules of the bi-substrate analog, N(phosphonacetyl)-L-aspartate, compared to the six strong binding sites in the wild-type enzyme and none in the mutant. However, the Vow of the hybrid was only about 25% that of the wild-type enzyme. In addition, the hybrid exhibited a very low apparent affinity for the substrate, aspartate [Michaelis constant (Ki) about 90 mM], as compared to the wild-type enzyme (apparent Km of 7 mM). No homotropic effect was observed for the hybrid in the absence of nucleotides as contrasted to the cooperativity of the wildtype enzyme; also, large changes in the VMow of the hybrid were caused by the addition of the nucleotide effectors, CTP and ATP, which do not affect the V.. of the wild-type, but influence only the cooperativity and the apparent Km. Although the hybrid undergoes a ligand-promoted conformational change analogous to that of the wild-type enzyme, this transition required a 20-fold higher concentration o the substrate analog, succinate. It appears that the "paralysis" of the wild-type catalytic subunit in the hybrid can be attributed to subunit interactions which constrain the molecule in a lowaffinity state.
Aspartate transcarbamylase (ATCase) (aspartate carbamoyltransferase or carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) contributes to the regulation of pyrimidine biosynthesis in Escherichia colh in two ways (1-3). On the one hand, the enzyme activity changes in a sigmoidal fashion with increasing concentration of the substrate, aspartate; on the other hand, the enzyme is subject to inhibition by CTP and activation by ATP (4). These phenomena, termed homotropic and heterotropic effects, respectively, are considered to be the result of an equilibrium between two distinct conformations of the allosteric enzyme that have different affinities for substrate (5). Inhibition is achieved by stabilization of the constrained (low-affinity) state, whereas activation favors the relaxed (high-affinity) form. Several types of subunit interactions are involved in stabilizing ATCase as an oligomer (6) composed of six catalytic Abbreviations: ATCase, aspartate transcarbamylase or aspartate carbamoyltransferase; C, catalytic subunit; R, regulatory subunit; N(subscript), native wild-type subunit; M(subscript), mutant subunit; T(subscript), subunit acylated with 3,4,5,6-tetrahydrophthalic anhydride; p(subscript), pyridoxylated subunit; H4Pht-, 3,4,5,6-tetrahydrophthalic; H4Phtoyl-, 3,4,5,6-tetrahydrophthaloyl-; PALA, N-(phosphonacetyl)L-aspartate. *
Present address: Department of Biochemistry, University of Bristol, Bristol BS8 1TD, U.K.
tPresent address: Cetus Corporation, Berkeley, Calif. 94710. To whom requests for reprints should be sent. 4298
(c) and six regulatory (r) polypeptide chains (7-9) which are organized as two catalytic (C) and three regulatory (R) subunits (9-13). In this structure, C2R3, there are six c:c bonding domains linking the c chains in the two C trimers; three r:r bonding domains linking the r chains in the three R dimers; and six c:r bonding domains linking the c and r chains (13, 14). It has been shown that cooperativity and feedback inhibition are exhibited by a hybrid containing one active (native) C and one inactive (chemically modified) C and three native R subunits (15). For these studies, inactivation of the C subunits was achieved by a specific reaction involving reduction of the Schiff base formed between pyridoxyl 5'phosphate and a single lysyl residue of each of the c chains. Apparently the chemical modification does not have a large effect on the subunit interactions and the allosteric transition of the hybrid (15). We present here studies of another ATCase hybrid that contains one active C subunit from the wild-type strain and an inactive C subunit isolated from a mutant strain of E. coli which produces almost completely inactive ATCase-like molecules. The substitution due to a missense mutation and responsible for the inactivation is in the catalytic polypeptide chains, and probably represents the only difference between the mutant and wild-type proteins (J. E. Flatgaard, K. Wall, I. Gibbons, and H. K. Schachman, unpublished). The mutant catalytic subunit (CM) is able to bind the substrate, carbamyl phosphate, but, unlike the wild-type subunit, CN, it shows little affinity for the aspartate analog, succinate. Although CM is similar in these properties to the pyridoxylated subunit, Cp, the inter-subunit ATCase-like hybrids, CNCMR3 and CNCpR3, are markedly different. Even though it contains three fully active catalytic chains, the hybrid CNCMR3 exhibits low enzyme activity and an extremely poor affinity for the substrate, aspartate. This apparent "paralysis" of the active subunit is another manifestation of quaternary constraint in ATCase-like molecules that results presumably from interactions transmitted by the R subunits.
MATERIALS AND METHODS
Wild-type ATCase, CN, and RN were prepared as described earlier (15). The strain which produced the mutant ATCase was constructed (16) by replacing the pyr B and pyr F alleles in the E. coli K12 strain 23-5 (17) with the pyr B- allele from strain YA 231 (18) and the leaky pyr F« allele from strain YA 149 r21 (19), respectively. Further selection was based on its ability to grow on exogenous carbamyl aspartate or orotic acid while failing to grow on minimal medium. This strain produces inactive ATCase-like molecules whose synthesis is derepressed when the exogenous uracil is consumed, as long as orotic acid (or some other intermediate in the pyrimidine pathway between carbamyl aspartate and
Biochemistry:
Proc. Nat. Acad. Sci. USA 72 (1975)
Gibbons et al.
orotidine 5'-phosphate) is present as a source of pyrimidines. The ATCase-like protein was purified by the procedure of Gerhart and Holoubek (19) by using electrophoresis on polyacrylamide gels to monitor the efficacy of the individual steps. Characterization of the mutant protein indicated that it had a molecular weight of about 3.1 X 105 and like the wild-type enzyme was an oligomer of both C and R subunits in a complex designated C2R3. The RM subunits have physical and functional properties similar to those of RN, and reconstituted ATCase-like molecules from CN and RM exhibited the allosteric behavior of the wild-type enzyme. In contrast, the CM subunits, although similar in physical properties to CN, lacked enzyme activity and differed in their interaction with specific ligands. The construction of the strain, purification of the mutant ATCase, and its characterization will be described elsewhere (J. E. Flatgaard, K. Wall, I. Gibbons, and H. K. Schachman, in preparation). The CM subunits were prepared by the method of Kirschner (20); acylation with H4Pht-anhydride was performed as described elsewhere (21). Protein concentrations were determined spectrophotometrically (11). Absorbance measurements in conjunction with refractometric determinations of concentration showed that the extinction coefficients of the mutant ATCase, CM and RM, were equal to those for the wild-type proteins. Enzyme activity was assayed either by the method of Porter et al. (22) or by measuring in a radiometer pH-stat the amount of KOH required to maintain pH 8.0 during the reaction between carbamyl phosphate and aspartate (23, 24). Zone electrophoresis was performed with a Beckman model R-101 Microzone Electrophoresis Cell on Gelman Sepraphore III cellulose acetate strips at 250 V for 20 min. The buffer was 25 mM Tris-HCl at pH 8.0 containing 2 mM EDTA. Polyacrylamide gel electrophoresis in 5% gels was conducted with the buffer system of Jovin et al. (25). Binding of N-(phosphonacetyl)-L-aspartate (PALA) was measured by difference spectra titrations based on the change in absorbance at 286.3 nm (26). The ligand-promoted changes in sedimentation coefficient, Asl§, were measured as described elsewhere (27). RESULTS Preparation of CNCMR3 Because CN and CM are very similar chemically and almost indistinguishable both by chromatography and electrophoresis (J. E. Flatgaard, K. Wall, I. Gibbons, and H. K. Schachman, unpublished), it was not possible to separate CNCMRO from other members of the inter-subunit hybrid set formed by reconstituting ATCase-like molecules from CN, CM, and R. This difficulty was circumvented by using H4Pht-anhydride to acylate about 30% of the amino groups in CM. As shown earlier (15, 21), the H4Phtoyl-groups serve as a reversible "chromatographic handle" through the introduction of charged groups which permit chromatographic fractionation and then can be removed readily by incubating the desired species at pH 6.0. Acylation of CM with 1 mol of H4Pht-anhydride per mol of amino groups gave a derivative, CMT, with an electrophoretic mobility on cellulose acetate strips (Fig. 1) corresponding to an average of 5.5 H4Phtoyl-groups per polypeptide chain (21). Less than 50% of the acylated derivative was converted to CMTCM,TR3 upon the addition of excess RN. However, on mixing CMT with CN prior to the addition of RN, a larger fraction of CMT formed ATCase-like com-
4299
Origin CN
CM,T Hybrid set
a
I
CNCNR3J
CNCM,TR3
tPosition
of
CM,TCM,TR3
FIG. 1. Electrophoretic analysis of the hybrid set. The hybrid set was formed by mixing 11 mg of CN with 20 mg of CM,T and adding 20 mg of R in a final volume of 9 ml (9). Electrophoresis was conducted on cellulose acetate strips as described in Materials and Methods. The patterns, from top to bottom, show CN, CM,T, and the hybrid set. A separate experiment was conducted to
determine the position of CM,TCM,TR3-
plexes§. As shown by the electrophoresis pattern for the
inter-subunit hybrid set in Fig. 1, most of the CMT was incorporated into the hybrid, CNCM,TR3, which was identified by its electrophoretic mobility in comparison with other derivatives (15, 21). Chromatography on DEAE-Sephadex (9) resolved the hybrid set into the individual species, and the H4Phtoyl-groups were removed from CNCM.TR3 by incubation at pH 6.0 and 230 for 36 hr. Physical properties of CNCMR3 The hybrid, CNCMR3, was shown by gel electrophoresis to be free of aggregates, R-deficient ATCase-like molecules (28), and free C and R subunits. Its electrophoretic mobility on cellulose acetate strips and in polyacrylamide gels and its sedimentation coefficient were virtually identical to those for the native wild-type enzyme. The number of substrate binding sites in CNCMR3 was determined by spectrophotometric titration with the bi-substrate analog, PALA, which incorporates structural features of both carbamyl phosphate and aspartate (26). As shown by Jacobson and Stark (29), PALA binds tightly and specifically to the six active sites of wild-type ATCase. In contrast, the mutant ATCase shows no change in the ultraviolet absorption spectrum upon the addition of PALA at a concentration 10 times that required to saturate the wild-type enzyme. With CNCMR3 the amplitude of the difference spectrum at 286.3 nm increased linearly, with the amount of added PALA reaching a plateau at three molecules of PALA per hybrid molecule. Thus, all three wild-type catalytic sites in CNCMR3 were capable of binding PALA with high affinity. Kinetic properties of CNCMR3 As seen in Fig. 2, the hybrid, CNCMR3, had a much lower apparent affinity for the substrate, aspartate, than did the reconstituted, wild-type enzyme, CNCNR3. Moreover, the specific activity of CNCMRS, as indicated by V.., was much less than that expected for a molecule having three active chains as compared to six in CNCNR3. In contrast, the activity of the acylated hybrid, CNCM,TR3, was close to half that of CNCNR3. Each of the hybrids exhibited Michaelisderivatives of the catalytic subunit (9, 15, 21) the reconstitution of ATCase-like complexes has been very efficient, and the distribution of species formed in hybridization experiments was approximately binomial.
§ With other acylated
Proc. Nat. Acad. Sci. USA 72 (1975)
Biochemistry: Gibbons et al.
4300
6
4)
~~~~~CNCII
*O L
0
5
10
20
15
30
25
[Aspartate], mM 1.0
M 0.
0.6
04
CN CMR3\
CL C,,
0 0
2
4
6
8
10
12
Specific activity FIG. 2. Kinetics of the hybrid, CNCMR3, and the reconstituted wild-type enzyme, CNCNR3. Assays were performed at 30° in 50 mM imidazole acetate, pH 7.0, containing 0.2 mM EDTA and 4 mM (saturating) carbamyl phosphate. Specific activity is given as ,tmol of carbamyl aspartate produced per hr/ug of protein. 0, Results for CNCMR3; results for CNCNR3. (a) Specific activity as a function of aspartate concentration (mM); (b) specific activity divided by aspartate concentration versus specific activity. 0,
Menten kinetics when nucleotide effectors were absent. The kinetic data for the hybrids, along with those for related species, are summarized in Table 1. Of particular interest is a
of the hybrids, CNCMR3 and CNCPR3. The latcomposed of three active wild-type chains in one C sub-
comparison
ter,
Table 2. Release of active catalytic subunit upon dissociation of CNCM R3 with mercurial
Table 1. Kinetic properties of hybrids and parental species
Species Wild-type ATCase Mutant ATCase CNCNR3 CNCMR3 CNCM,TR3
CNCpR3
Hill Vmax* Km t coeffi(units/ag) (mM) cient 11 < 0.001
11
7 7
Aspar-
Vmax per
active site t
1.75
1.83
1.75
1.83
-
-
2.7
90
1.00
0.90
5.2
25
1.00
1.73
5.7
12
1.38
1.90
are values obtained by extrapolation of the Eadie plots to correspond to infinite aspartate concentration. No nucleotides were present. A unit is 1 zmol of carbamyl aspartate produced per hr. t Km corresponds to the aspartate concentration at one-half Vmax. t Value for the observed Vmax divided by the number of wild-type catalytic polypeptide chains in the molecule. *
These
unit and three chemically inactivated wild-type c chains in the other subunit, shows the characteristic behavior of wildtype ATCase. The value of Vm,,. per active site in CNCpR% is equal to that of the native enzyme, and the hybrid shows considerable cooperativity (Hill coefficient of 1.38). This hybrid has an apparent Km which is somewhat greater than that of the native enzyme, In contrast, the V per active site in CNCMR3 is about one half that of the wild-type enzyme (and CNCpR3). Also, CNCMR3 exhibits no cooperativity (Hill coefficient of 1.00), and the value of 90 mM for Km is about 13 times that of the native, wild-type enzyme. This "paralysis" of the active catalytic subunit in CNCMR3 is much less in the acylated derivative, as seen by comparing the values of Km and Vm per active site exhibited by CNCM,TR% with those for CNCNR3 and CNCMR3. The striking kinetic properties of CNCMR3 raised the possibility that the wild-type catalytic subunit in the hybrid was damaged in the preparation of the hybrid set and/or in the deacylation procedure. In this regard it should be noted that the values for CNCNR3 were obtained from the sample isolated from the hybrid set which yielded CNCMR3. Proof that the wild-type catalytic subunit in CNCMR3 was not damaged and was potentially fully active is summarized in Table 2. Since the reaction of ATCase with the mercurial, para-hydroxymercuribenzoate, is known to cause the release of free, active C subunits (6), the dissociation of CNCMR3 (and CNCNRS as a control) was followed by measuring the enzyme activity in a pH-stat (23, 24) before and after the addition of the mercurial. As shown in Table 2, the activity (per wild-type catalytic subunit) of the hybrid, CNCMR3, was much less than that of CNCNR3 at both 5 and 20 mM aspartate. After dissociation of the complexes, however, the CN released from the hybrid was as active as that from CNCNR3 at both substrate concentrations. The intact wild-type enzyme is much less active at 5 mM aspartate than its free CN subunits because the enzyme is in the constrained (low-affinity) state at low concentrations. When the aspartate concentration is increased to 20 mM, the wild-type enzyme undergoes the allosteric transition to the relaxed (high-affinity) state, and the activity per CN is almost the same as that of the free CN subunits. In contrast, the hybrid, CNCMRS, is much less active relative to its CN subunit at both concentrations of aspartate.
Specific activity* (units/,ug of CN)
tate concen-
tration
(mM)
Species
Intact
5
CNCMR3
0.3 1.8 2 21
20
CNCNR3
CNCMR3
CNCNR3
Dissociated
10 8.3 22
19
* Activities are expressed per Ag of CN, measured at 300 by the pHstat assay (23, 24). Units are jimol of KOH required to maintain a 2 ml volume (containing 50 mM potassium acetate) at pH 8.0. Carbamyl phosphate was present at 10 mM; aspartate was present at either 5 or 20 mM. After the activity of the intact species had been measured, 100 nmol of para-hydroxymercuribenzoate were added to the assay solution; dissociation of the enzymes was completed in 10 min, as indicated by the linearity of the consumption of alkali with time, and the activity of the released subunits was measured.
Biochemistry:
Proc. Nat. Acad. Sci. USA 72 (1975)
Gibbons et al.
Effect of CTP and ATP on activity of CNCMR3 Although the nucleotide effectors, CTP and ATP, inhibit and activate, respectively, wild-type ATCase at low aspartate concentrations, they do not cause any change in VMa, (4). With CNCMR3, however, the value of VMA, is lowered by CTP and increased by ATP. Plots of the kinetic data according to Eadie (30) gave V1x values of 0.7, 3.5, and 2.7 ,gmol of carbamyl aspartate formed per hr per ,ug of protein in the presence of CTP, ATP, and no effector, respectively. Conformational changes in CNCMR3 Because the enzyme activity of CNCMR3 was less than that expected for an ATCase-like molecule containing three active sites and there was no evidence of cooperativity, it was of interest to determine whether the hybrid would undergo a ligand-promoted conformational change analogous to that of the wild-type enzyme (27). Accordingly, difference sedimentation measurements were performed as a probe of conformational changes (27). The value of As/I caused by saturating carbamyl phosphate (2 mM) was virtually zero for CNCMR3 compared to -0.5% for CNCNR3. Upon the subsequent addition of succinate, however, the hybrid did undergo a conformational change similar to that of the wild-type enzyme. But, as seen in Fig. 3, the concentration of succinate required for the hybrid was much larger (about 20fold) than that for the wild-type enzyme. Even at 12 mM succinate (in the presence of carbamyl phosphate) the change in As/i was not complete for CNCMR%, whereas 0.5 mM succinate was sufficient for the wild-type enzyme. With PALA, however, the value of As/I was -3.8% for the hybrid as compared to -3.1% for CNCNR3. DISCUSSION As shown in Fig. 2 and Table 1, the behavior of the hybrid, CNCMR3, is strikingly different from that of the native, wild-type enzyme. In the absence of nucleotides the hybrid exhibits hyperbolic kinetics as compared to the cooperativity of the wild-type enzyme. Also, the Km of the hybrid is much higher (about 13-fold) and the Vm. per active site is lower than that of the wild-type enzyme; they further differ in that Vmax of the hybrid changes upon the addition of nucleotides, whereas Vm,,, of the wild-type enzyme is not affected. The presence of three functional wild-type active sites in the hybrid was established both by the demonstration of three strong binding sites for PALA and by the release of the full enzyme activity of the CN subunit when the hybrid was dissociated by the addition of the mercurial, para-hydroxymercuribenzoate. The hybrid undergoes a ligand-promoted conformational change similar to that identified as the allosteric transition in the wild-type enzyme, but much higher levels (20-fold) of the ligand, succinate, are required for CNCMR3. This distinctive behavior of CNCMR3 cannot be attributed solely to the loss of three active sites because the analogous hybrid, CNCpR3, is similar in all respects to the native enzyme; however, the two inactive subunits, CM and Cp, are functionally similar in that both bind carbamyl phosphate but fail to bind succinate. The reduced Vmax and cooperativity and the increased Km of CNCpR3, as compared with the native enzyme, can be accounted for in terms of the fewer active sites by assuming that this hybrid, like the native enzyme, undergoes a concerted ligand-promoted conformational change, and that the subunit interactions are not affected by the pyridoxylation of the single lysyl residue in
4301
CL
_
i1 0
2
4
8
6
[Succinate],
lO
12
mM
FIG. 3. Ligand-promoted conformational change in CNCmR3 and CNCNR3. Measurements of the change in the sedimentation coefficient, As/s, were performed as. described elsewhere (27). The buffer was 50 mM potassium phosphate at pH 7.0 containing 0.2 mM EDTA, 2 mM 2-mercaptoethanol, and 2 mM carbamyl phosphate. Glutarate was added to the reference solution to compensate for the viscosity and density contributions of the succinate. O. Results for CNCMR3; *, results for CNCNR3. The protein concentration was 2-4 mg/ml.
each chain of Cp (15). In contrast, we can account for the "S9paralysis" of the wild-type catalytic subunit in CNCMR3 by assuming that the hybrid has a structure analogous to the constrained state of the wild-type enzyme, and that the subunit interactions are such that the hybrid is almost "frozen" in the low-affinity form. This could occur if the polypeptide chains of the mutant subunit have such a stable' tertiary structure that the ligand-promoted conformational change of the whole molecule is less favored thermodynamically than that of the wild-type enzyme. According to this hypothesis, the CN subunit in CNCMR3 cannot undergo the allosteric transition independently. Alternatively, the tertiary structure of the mutant chains may be so altered that the bonding domains between CN and RN are distorted even in the absence of ligands. In turn, the conformation of the RN subunits and the bonds between RN and CN may be changed with the result that the active sites in CNare affectedl. Experiments designed to examine.the c:c and c:r bonding domains formed by the mutant catalytic subunit are necessary in order to distinguish between these alternatives. Although the hybrid, CNCmR3, exhibits heterotropic effects in that it is inhibited by CTP and activated by ATP, it must be noted that these effects are markedly different from those obtained with the wild-type enzyme (4). Whereas the latter is designated a K-class allosteric enzyme because the inhibitor and activator affect only the apparent K. and cooperativity and their effect is overcome at high substrate concentrations, the hybrid has characteristics of both a K- and V-class system (5) for which the principal heterotropic effect is a change in Vma,,. In view of this unusual heterotropic effect and the lack of cooperativity exhibited by the hybrid, it is pertinent to consider the applicability of the two-state model of Monod et al. (5) which accounts so satisfactorily for most of the properties of the wild-type enzyme (31). Usually it is assumed that the putative constrained and reI
Other hypotheses can be proposed to account for the altered Km and Vm,,. of CNCMR3. For example, the size and shape of CM may be sufficiently different from CN to alter the geometry of the complex and to hinder access of ligands.
4302
Proc. Nat. Acad. Sci. USA 72 (1975)
Biochemistry: Gibbons et al.
laxed forms of the enzyme have identical turnover numbers. However, the kinetic behavior of the wild-type enzyme would be unaffected if the constrained state had a lower activity (turnover number) than the relaxed state, inasmuch as the former contributes so little to the overall activity even at low substrate concentrations. If we assume that the constrained state of CNCMR% had a lower activity than the relaxed state, the anomalous kinetic properties of the hybrid could be rationalized on the basis of the two-state model. The equilibrium constant favoring the constrained state of the hybrid would have to be much greater than for the wildtype enzyme, and the ratio of affinities of the two forms of the hybrid for aspartate would have to be less than that for the wild-type enzyme. Although this speculation shows that the properties of CNCMR3 can be accommodated qualitatively within the framework of the two-state model, alternative models must be considered. It is noteworthy that removal of the H4Phtoyl-groups fromx CNCM,TR3 leads to a marked enhancement of the quaternary constraint. Not only did Km increase about 4-fold, but Vmkx also was reduced about 2-fold. A similar effect was observed (15) for the hybrid, CNCPTR& which exhibited no cooperativity and had a much lower Km than the deacylated, allosteric hybrid, CNCpR3. In both cases, it seems that the electrostatic destabilization stemming from the charged acyl groups is greater for the constrained or compact (higher 820w) form of the enzyme than it is for the relaxed or swollen state; hence, the acylated derivatives exhibit less quaternary constraint. We thank J. C. Gerhart for innumerable and invaluable suggestions and G. R. Stark for the PALA. This investigation was supported by NIH Research Grant GM 12159 from the National Institute of General Medical Sciences, by Training Grant CA 05028 from the National Cancer Institute, and by National Science Foundation Grant GB 32812X. J.E.F. was the recipient of NIH Postdoctoral Fellowship GM 46941 from the National Institute of General Medical Sciences.
1. O'Donovan, G. A. & Neuhard, J. (1970) Bacteriol. Rev. 34, 278-43. 2. Gerhart, J. C. (1970) Curr. Top. Cell. Regul. 2,275-325. 3. Jacobson, G. R. & Stark, G. R. (1973) in The Enzymes, ed. Boyer, P. D. (Academic Press, New York), Vol. 9, pp. 225308.
4. Gerhart, J. C. & Pardee, A. B. (1962) J. Btol. Chem. 237, 891-896. 5. Monod, J., Wyman, J. & Changeux, J.-P. (1965) J. Mol. Biol. 12,88-118. 6. Gerhart, J. C. & Schachman, H. K. (1965) Biochemistry 4, 1054-1062. 7. Weber, K. (1968) Nature 218, 1116-1119. 8. Wiley, D. C. & Lipscomb, W. N. (1968) Nature 218, 11191121. 9. Meighen, E. A., Pigiet, V. & Schachman, H. K. (1970) Proc. Nat. Acad. Sci. USA 65,234-241. 10. Nagel, G. M., Schachman, H. K. & Gerhart, J. C. (1972) Fed. Proc. 31, 423 Abstr. 11. Nagel, G. M. & Schachman, H. K. (1975) Biochemistry 14, 3195-3203. 12. Rosenbusch, J. P. & Weber, K. (1971) J. Biol. Chem. 246, 1644-1657. 13. Cohlberg, J. A., Pigiet, V. P., Jr. & Schachman, H. K. (1972) Biochemistry 11, 3396-3411. 14. Schachman, H. K. (1974) Hanvey Lect. 68, 67-113. 15. Gibbons, I., Yang, Y. R. & Schachman, H. K. (1974) Proc. Nat. Aced. Sci. USA 71, 4452-4456. 16. Flatgaard, J. E., Gerhart, J. C. & Schachman, H. K. (1974) Fed. Proc. 33, No. J, 1533 Abstr. 17. Yu, M. T., Kaney, A. R. & Atwood, K. C. (1965) J. Bacteriol. 90, 1150-1152. 18. Beckwith, J. R., Pardee, A. B., Austrian, R. & Jacob, F. (1962) J. Mol. Blol. 5,618-634. 19. Gerhart, J. C. & Holoubek, H. (1967) J. Biol. Chem. 242, 2886-2892. 20. Kirschner, M. W. (1971) in Ph.D. Dissertation, University of California, Berkeley, pp. 176-181. 21. Gibbons, I. & Schachman, H. K. (1975) Biochemistry, in press. 22. Porter, R. W., Modebe, M. 0. & Stark, G. R. (1969) J. Biol. Chem. 244,1846-1859. 23. Gerhart, J. C. (1962) in Ph.D. Dissertation, University of California, Berkeley, p. 17. 24. Mort, J. S. & Chan, W. W.-C. (1975) J. Biol. Chem. 250,
65360. 25. Jovin, T., Chrambach, A. & Naughton, M. A. (1964) Anal.
Biochem. 9,351-369. 26. Collins, K. D. & Stark, G. R. (1971) J. Biol. Chem. 246,
6599-6605. 27. Gerhart, J. C. & Schachman, H. K. (1968) Biochemistry 7, 538-552. 28. Yang, Y. R., Syvanen, J. M., Nagel, G. M. & Schachman, H. K. (1974) Proc. Nat. Acad. Sd. USA 71, 918-922. 29. Jacobson, G. R. & Stark, G. R. (1973) J. Biol. Chem. 248, 8003-8014. 30. Eadie, G. S. (1942) J. BRiol. Chem. 146,85-93. 31. Changeux, J.-P. & Rubin, M. M. (1968) Biochemistry 7,553561.
Vol. 72, No. 11, pp. 4298-4302, November 1975 Biochemistry
Quaternary constraint in hybrid of aspartate transcarbamylase containing wild-type and mutant catalytic subunits (allosteric enzyme/inter-subunit hybrid/inactive mutant/subunit interactions/aspartate carbamoyltransferase)
IAN GIBBONS*, J. E. FLATGAARDt, AND H. K. SCHACHMANt Department of Molecular Biology and The Virus Laboratory, University of California, Berkeley, Calif. 94720
Contributed by H. K. Schachimn, August 15,71975
ABSTRACT Unusual quaternary constraint in the regulatory enzyme, aspartate transcarbamylase (aspartate carbamoyltransferase or carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) from Escherichia coli, was demonstrated with a hybrid composed of one inactive "catalytic" subunit from a mutant strain and one active catalytic subunit and three regulatory subunits from the wild-type strain. The hybrid had a high affinity for three molecules of the bi-substrate analog, N(phosphonacetyl)-L-aspartate, compared to the six strong binding sites in the wild-type enzyme and none in the mutant. However, the Vow of the hybrid was only about 25% that of the wild-type enzyme. In addition, the hybrid exhibited a very low apparent affinity for the substrate, aspartate [Michaelis constant (Ki) about 90 mM], as compared to the wild-type enzyme (apparent Km of 7 mM). No homotropic effect was observed for the hybrid in the absence of nucleotides as contrasted to the cooperativity of the wildtype enzyme; also, large changes in the VMow of the hybrid were caused by the addition of the nucleotide effectors, CTP and ATP, which do not affect the V.. of the wild-type, but influence only the cooperativity and the apparent Km. Although the hybrid undergoes a ligand-promoted conformational change analogous to that of the wild-type enzyme, this transition required a 20-fold higher concentration o the substrate analog, succinate. It appears that the "paralysis" of the wild-type catalytic subunit in the hybrid can be attributed to subunit interactions which constrain the molecule in a lowaffinity state.
Aspartate transcarbamylase (ATCase) (aspartate carbamoyltransferase or carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) contributes to the regulation of pyrimidine biosynthesis in Escherichia colh in two ways (1-3). On the one hand, the enzyme activity changes in a sigmoidal fashion with increasing concentration of the substrate, aspartate; on the other hand, the enzyme is subject to inhibition by CTP and activation by ATP (4). These phenomena, termed homotropic and heterotropic effects, respectively, are considered to be the result of an equilibrium between two distinct conformations of the allosteric enzyme that have different affinities for substrate (5). Inhibition is achieved by stabilization of the constrained (low-affinity) state, whereas activation favors the relaxed (high-affinity) form. Several types of subunit interactions are involved in stabilizing ATCase as an oligomer (6) composed of six catalytic Abbreviations: ATCase, aspartate transcarbamylase or aspartate carbamoyltransferase; C, catalytic subunit; R, regulatory subunit; N(subscript), native wild-type subunit; M(subscript), mutant subunit; T(subscript), subunit acylated with 3,4,5,6-tetrahydrophthalic anhydride; p(subscript), pyridoxylated subunit; H4Pht-, 3,4,5,6-tetrahydrophthalic; H4Phtoyl-, 3,4,5,6-tetrahydrophthaloyl-; PALA, N-(phosphonacetyl)L-aspartate. *
Present address: Department of Biochemistry, University of Bristol, Bristol BS8 1TD, U.K.
tPresent address: Cetus Corporation, Berkeley, Calif. 94710. To whom requests for reprints should be sent. 4298
(c) and six regulatory (r) polypeptide chains (7-9) which are organized as two catalytic (C) and three regulatory (R) subunits (9-13). In this structure, C2R3, there are six c:c bonding domains linking the c chains in the two C trimers; three r:r bonding domains linking the r chains in the three R dimers; and six c:r bonding domains linking the c and r chains (13, 14). It has been shown that cooperativity and feedback inhibition are exhibited by a hybrid containing one active (native) C and one inactive (chemically modified) C and three native R subunits (15). For these studies, inactivation of the C subunits was achieved by a specific reaction involving reduction of the Schiff base formed between pyridoxyl 5'phosphate and a single lysyl residue of each of the c chains. Apparently the chemical modification does not have a large effect on the subunit interactions and the allosteric transition of the hybrid (15). We present here studies of another ATCase hybrid that contains one active C subunit from the wild-type strain and an inactive C subunit isolated from a mutant strain of E. coli which produces almost completely inactive ATCase-like molecules. The substitution due to a missense mutation and responsible for the inactivation is in the catalytic polypeptide chains, and probably represents the only difference between the mutant and wild-type proteins (J. E. Flatgaard, K. Wall, I. Gibbons, and H. K. Schachman, unpublished). The mutant catalytic subunit (CM) is able to bind the substrate, carbamyl phosphate, but, unlike the wild-type subunit, CN, it shows little affinity for the aspartate analog, succinate. Although CM is similar in these properties to the pyridoxylated subunit, Cp, the inter-subunit ATCase-like hybrids, CNCMR3 and CNCpR3, are markedly different. Even though it contains three fully active catalytic chains, the hybrid CNCMR3 exhibits low enzyme activity and an extremely poor affinity for the substrate, aspartate. This apparent "paralysis" of the active subunit is another manifestation of quaternary constraint in ATCase-like molecules that results presumably from interactions transmitted by the R subunits.
MATERIALS AND METHODS
Wild-type ATCase, CN, and RN were prepared as described earlier (15). The strain which produced the mutant ATCase was constructed (16) by replacing the pyr B and pyr F alleles in the E. coli K12 strain 23-5 (17) with the pyr B- allele from strain YA 231 (18) and the leaky pyr F« allele from strain YA 149 r21 (19), respectively. Further selection was based on its ability to grow on exogenous carbamyl aspartate or orotic acid while failing to grow on minimal medium. This strain produces inactive ATCase-like molecules whose synthesis is derepressed when the exogenous uracil is consumed, as long as orotic acid (or some other intermediate in the pyrimidine pathway between carbamyl aspartate and
Biochemistry:
Proc. Nat. Acad. Sci. USA 72 (1975)
Gibbons et al.
orotidine 5'-phosphate) is present as a source of pyrimidines. The ATCase-like protein was purified by the procedure of Gerhart and Holoubek (19) by using electrophoresis on polyacrylamide gels to monitor the efficacy of the individual steps. Characterization of the mutant protein indicated that it had a molecular weight of about 3.1 X 105 and like the wild-type enzyme was an oligomer of both C and R subunits in a complex designated C2R3. The RM subunits have physical and functional properties similar to those of RN, and reconstituted ATCase-like molecules from CN and RM exhibited the allosteric behavior of the wild-type enzyme. In contrast, the CM subunits, although similar in physical properties to CN, lacked enzyme activity and differed in their interaction with specific ligands. The construction of the strain, purification of the mutant ATCase, and its characterization will be described elsewhere (J. E. Flatgaard, K. Wall, I. Gibbons, and H. K. Schachman, in preparation). The CM subunits were prepared by the method of Kirschner (20); acylation with H4Pht-anhydride was performed as described elsewhere (21). Protein concentrations were determined spectrophotometrically (11). Absorbance measurements in conjunction with refractometric determinations of concentration showed that the extinction coefficients of the mutant ATCase, CM and RM, were equal to those for the wild-type proteins. Enzyme activity was assayed either by the method of Porter et al. (22) or by measuring in a radiometer pH-stat the amount of KOH required to maintain pH 8.0 during the reaction between carbamyl phosphate and aspartate (23, 24). Zone electrophoresis was performed with a Beckman model R-101 Microzone Electrophoresis Cell on Gelman Sepraphore III cellulose acetate strips at 250 V for 20 min. The buffer was 25 mM Tris-HCl at pH 8.0 containing 2 mM EDTA. Polyacrylamide gel electrophoresis in 5% gels was conducted with the buffer system of Jovin et al. (25). Binding of N-(phosphonacetyl)-L-aspartate (PALA) was measured by difference spectra titrations based on the change in absorbance at 286.3 nm (26). The ligand-promoted changes in sedimentation coefficient, Asl§, were measured as described elsewhere (27). RESULTS Preparation of CNCMR3 Because CN and CM are very similar chemically and almost indistinguishable both by chromatography and electrophoresis (J. E. Flatgaard, K. Wall, I. Gibbons, and H. K. Schachman, unpublished), it was not possible to separate CNCMRO from other members of the inter-subunit hybrid set formed by reconstituting ATCase-like molecules from CN, CM, and R. This difficulty was circumvented by using H4Pht-anhydride to acylate about 30% of the amino groups in CM. As shown earlier (15, 21), the H4Phtoyl-groups serve as a reversible "chromatographic handle" through the introduction of charged groups which permit chromatographic fractionation and then can be removed readily by incubating the desired species at pH 6.0. Acylation of CM with 1 mol of H4Pht-anhydride per mol of amino groups gave a derivative, CMT, with an electrophoretic mobility on cellulose acetate strips (Fig. 1) corresponding to an average of 5.5 H4Phtoyl-groups per polypeptide chain (21). Less than 50% of the acylated derivative was converted to CMTCM,TR3 upon the addition of excess RN. However, on mixing CMT with CN prior to the addition of RN, a larger fraction of CMT formed ATCase-like com-
4299
Origin CN
CM,T Hybrid set
a
I
CNCNR3J
CNCM,TR3
tPosition
of
CM,TCM,TR3
FIG. 1. Electrophoretic analysis of the hybrid set. The hybrid set was formed by mixing 11 mg of CN with 20 mg of CM,T and adding 20 mg of R in a final volume of 9 ml (9). Electrophoresis was conducted on cellulose acetate strips as described in Materials and Methods. The patterns, from top to bottom, show CN, CM,T, and the hybrid set. A separate experiment was conducted to
determine the position of CM,TCM,TR3-
plexes§. As shown by the electrophoresis pattern for the
inter-subunit hybrid set in Fig. 1, most of the CMT was incorporated into the hybrid, CNCM,TR3, which was identified by its electrophoretic mobility in comparison with other derivatives (15, 21). Chromatography on DEAE-Sephadex (9) resolved the hybrid set into the individual species, and the H4Phtoyl-groups were removed from CNCM.TR3 by incubation at pH 6.0 and 230 for 36 hr. Physical properties of CNCMR3 The hybrid, CNCMR3, was shown by gel electrophoresis to be free of aggregates, R-deficient ATCase-like molecules (28), and free C and R subunits. Its electrophoretic mobility on cellulose acetate strips and in polyacrylamide gels and its sedimentation coefficient were virtually identical to those for the native wild-type enzyme. The number of substrate binding sites in CNCMR3 was determined by spectrophotometric titration with the bi-substrate analog, PALA, which incorporates structural features of both carbamyl phosphate and aspartate (26). As shown by Jacobson and Stark (29), PALA binds tightly and specifically to the six active sites of wild-type ATCase. In contrast, the mutant ATCase shows no change in the ultraviolet absorption spectrum upon the addition of PALA at a concentration 10 times that required to saturate the wild-type enzyme. With CNCMR3 the amplitude of the difference spectrum at 286.3 nm increased linearly, with the amount of added PALA reaching a plateau at three molecules of PALA per hybrid molecule. Thus, all three wild-type catalytic sites in CNCMR3 were capable of binding PALA with high affinity. Kinetic properties of CNCMR3 As seen in Fig. 2, the hybrid, CNCMR3, had a much lower apparent affinity for the substrate, aspartate, than did the reconstituted, wild-type enzyme, CNCNR3. Moreover, the specific activity of CNCMRS, as indicated by V.., was much less than that expected for a molecule having three active chains as compared to six in CNCNR3. In contrast, the activity of the acylated hybrid, CNCM,TR3, was close to half that of CNCNR3. Each of the hybrids exhibited Michaelisderivatives of the catalytic subunit (9, 15, 21) the reconstitution of ATCase-like complexes has been very efficient, and the distribution of species formed in hybridization experiments was approximately binomial.
§ With other acylated
Proc. Nat. Acad. Sci. USA 72 (1975)
Biochemistry: Gibbons et al.
4300
6
4)
~~~~~CNCII
*O L
0
5
10
20
15
30
25
[Aspartate], mM 1.0
M 0.
0.6
04
CN CMR3\
CL C,,
0 0
2
4
6
8
10
12
Specific activity FIG. 2. Kinetics of the hybrid, CNCMR3, and the reconstituted wild-type enzyme, CNCNR3. Assays were performed at 30° in 50 mM imidazole acetate, pH 7.0, containing 0.2 mM EDTA and 4 mM (saturating) carbamyl phosphate. Specific activity is given as ,tmol of carbamyl aspartate produced per hr/ug of protein. 0, Results for CNCMR3; results for CNCNR3. (a) Specific activity as a function of aspartate concentration (mM); (b) specific activity divided by aspartate concentration versus specific activity. 0,
Menten kinetics when nucleotide effectors were absent. The kinetic data for the hybrids, along with those for related species, are summarized in Table 1. Of particular interest is a
of the hybrids, CNCMR3 and CNCPR3. The latcomposed of three active wild-type chains in one C sub-
comparison
ter,
Table 2. Release of active catalytic subunit upon dissociation of CNCM R3 with mercurial
Table 1. Kinetic properties of hybrids and parental species
Species Wild-type ATCase Mutant ATCase CNCNR3 CNCMR3 CNCM,TR3
CNCpR3
Hill Vmax* Km t coeffi(units/ag) (mM) cient 11 < 0.001
11
7 7
Aspar-
Vmax per
active site t
1.75
1.83
1.75
1.83
-
-
2.7
90
1.00
0.90
5.2
25
1.00
1.73
5.7
12
1.38
1.90
are values obtained by extrapolation of the Eadie plots to correspond to infinite aspartate concentration. No nucleotides were present. A unit is 1 zmol of carbamyl aspartate produced per hr. t Km corresponds to the aspartate concentration at one-half Vmax. t Value for the observed Vmax divided by the number of wild-type catalytic polypeptide chains in the molecule. *
These
unit and three chemically inactivated wild-type c chains in the other subunit, shows the characteristic behavior of wildtype ATCase. The value of Vm,,. per active site in CNCpR% is equal to that of the native enzyme, and the hybrid shows considerable cooperativity (Hill coefficient of 1.38). This hybrid has an apparent Km which is somewhat greater than that of the native enzyme, In contrast, the V per active site in CNCMR3 is about one half that of the wild-type enzyme (and CNCpR3). Also, CNCMR3 exhibits no cooperativity (Hill coefficient of 1.00), and the value of 90 mM for Km is about 13 times that of the native, wild-type enzyme. This "paralysis" of the active catalytic subunit in CNCMR3 is much less in the acylated derivative, as seen by comparing the values of Km and Vm per active site exhibited by CNCM,TR% with those for CNCNR3 and CNCMR3. The striking kinetic properties of CNCMR3 raised the possibility that the wild-type catalytic subunit in the hybrid was damaged in the preparation of the hybrid set and/or in the deacylation procedure. In this regard it should be noted that the values for CNCNR3 were obtained from the sample isolated from the hybrid set which yielded CNCMR3. Proof that the wild-type catalytic subunit in CNCMR3 was not damaged and was potentially fully active is summarized in Table 2. Since the reaction of ATCase with the mercurial, para-hydroxymercuribenzoate, is known to cause the release of free, active C subunits (6), the dissociation of CNCMR3 (and CNCNRS as a control) was followed by measuring the enzyme activity in a pH-stat (23, 24) before and after the addition of the mercurial. As shown in Table 2, the activity (per wild-type catalytic subunit) of the hybrid, CNCMR3, was much less than that of CNCNR3 at both 5 and 20 mM aspartate. After dissociation of the complexes, however, the CN released from the hybrid was as active as that from CNCNR3 at both substrate concentrations. The intact wild-type enzyme is much less active at 5 mM aspartate than its free CN subunits because the enzyme is in the constrained (low-affinity) state at low concentrations. When the aspartate concentration is increased to 20 mM, the wild-type enzyme undergoes the allosteric transition to the relaxed (high-affinity) state, and the activity per CN is almost the same as that of the free CN subunits. In contrast, the hybrid, CNCMRS, is much less active relative to its CN subunit at both concentrations of aspartate.
Specific activity* (units/,ug of CN)
tate concen-
tration
(mM)
Species
Intact
5
CNCMR3
0.3 1.8 2 21
20
CNCNR3
CNCMR3
CNCNR3
Dissociated
10 8.3 22
19
* Activities are expressed per Ag of CN, measured at 300 by the pHstat assay (23, 24). Units are jimol of KOH required to maintain a 2 ml volume (containing 50 mM potassium acetate) at pH 8.0. Carbamyl phosphate was present at 10 mM; aspartate was present at either 5 or 20 mM. After the activity of the intact species had been measured, 100 nmol of para-hydroxymercuribenzoate were added to the assay solution; dissociation of the enzymes was completed in 10 min, as indicated by the linearity of the consumption of alkali with time, and the activity of the released subunits was measured.
Biochemistry:
Proc. Nat. Acad. Sci. USA 72 (1975)
Gibbons et al.
Effect of CTP and ATP on activity of CNCMR3 Although the nucleotide effectors, CTP and ATP, inhibit and activate, respectively, wild-type ATCase at low aspartate concentrations, they do not cause any change in VMa, (4). With CNCMR3, however, the value of VMA, is lowered by CTP and increased by ATP. Plots of the kinetic data according to Eadie (30) gave V1x values of 0.7, 3.5, and 2.7 ,gmol of carbamyl aspartate formed per hr per ,ug of protein in the presence of CTP, ATP, and no effector, respectively. Conformational changes in CNCMR3 Because the enzyme activity of CNCMR3 was less than that expected for an ATCase-like molecule containing three active sites and there was no evidence of cooperativity, it was of interest to determine whether the hybrid would undergo a ligand-promoted conformational change analogous to that of the wild-type enzyme (27). Accordingly, difference sedimentation measurements were performed as a probe of conformational changes (27). The value of As/I caused by saturating carbamyl phosphate (2 mM) was virtually zero for CNCMR3 compared to -0.5% for CNCNR3. Upon the subsequent addition of succinate, however, the hybrid did undergo a conformational change similar to that of the wild-type enzyme. But, as seen in Fig. 3, the concentration of succinate required for the hybrid was much larger (about 20fold) than that for the wild-type enzyme. Even at 12 mM succinate (in the presence of carbamyl phosphate) the change in As/i was not complete for CNCMR%, whereas 0.5 mM succinate was sufficient for the wild-type enzyme. With PALA, however, the value of As/I was -3.8% for the hybrid as compared to -3.1% for CNCNR3. DISCUSSION As shown in Fig. 2 and Table 1, the behavior of the hybrid, CNCMR3, is strikingly different from that of the native, wild-type enzyme. In the absence of nucleotides the hybrid exhibits hyperbolic kinetics as compared to the cooperativity of the wild-type enzyme. Also, the Km of the hybrid is much higher (about 13-fold) and the Vm. per active site is lower than that of the wild-type enzyme; they further differ in that Vmax of the hybrid changes upon the addition of nucleotides, whereas Vm,,, of the wild-type enzyme is not affected. The presence of three functional wild-type active sites in the hybrid was established both by the demonstration of three strong binding sites for PALA and by the release of the full enzyme activity of the CN subunit when the hybrid was dissociated by the addition of the mercurial, para-hydroxymercuribenzoate. The hybrid undergoes a ligand-promoted conformational change similar to that identified as the allosteric transition in the wild-type enzyme, but much higher levels (20-fold) of the ligand, succinate, are required for CNCMR3. This distinctive behavior of CNCMR3 cannot be attributed solely to the loss of three active sites because the analogous hybrid, CNCpR3, is similar in all respects to the native enzyme; however, the two inactive subunits, CM and Cp, are functionally similar in that both bind carbamyl phosphate but fail to bind succinate. The reduced Vmax and cooperativity and the increased Km of CNCpR3, as compared with the native enzyme, can be accounted for in terms of the fewer active sites by assuming that this hybrid, like the native enzyme, undergoes a concerted ligand-promoted conformational change, and that the subunit interactions are not affected by the pyridoxylation of the single lysyl residue in
4301
CL
_
i1 0
2
4
8
6
[Succinate],
lO
12
mM
FIG. 3. Ligand-promoted conformational change in CNCmR3 and CNCNR3. Measurements of the change in the sedimentation coefficient, As/s, were performed as. described elsewhere (27). The buffer was 50 mM potassium phosphate at pH 7.0 containing 0.2 mM EDTA, 2 mM 2-mercaptoethanol, and 2 mM carbamyl phosphate. Glutarate was added to the reference solution to compensate for the viscosity and density contributions of the succinate. O. Results for CNCMR3; *, results for CNCNR3. The protein concentration was 2-4 mg/ml.
each chain of Cp (15). In contrast, we can account for the "S9paralysis" of the wild-type catalytic subunit in CNCMR3 by assuming that the hybrid has a structure analogous to the constrained state of the wild-type enzyme, and that the subunit interactions are such that the hybrid is almost "frozen" in the low-affinity form. This could occur if the polypeptide chains of the mutant subunit have such a stable' tertiary structure that the ligand-promoted conformational change of the whole molecule is less favored thermodynamically than that of the wild-type enzyme. According to this hypothesis, the CN subunit in CNCMR3 cannot undergo the allosteric transition independently. Alternatively, the tertiary structure of the mutant chains may be so altered that the bonding domains between CN and RN are distorted even in the absence of ligands. In turn, the conformation of the RN subunits and the bonds between RN and CN may be changed with the result that the active sites in CNare affectedl. Experiments designed to examine.the c:c and c:r bonding domains formed by the mutant catalytic subunit are necessary in order to distinguish between these alternatives. Although the hybrid, CNCmR3, exhibits heterotropic effects in that it is inhibited by CTP and activated by ATP, it must be noted that these effects are markedly different from those obtained with the wild-type enzyme (4). Whereas the latter is designated a K-class allosteric enzyme because the inhibitor and activator affect only the apparent K. and cooperativity and their effect is overcome at high substrate concentrations, the hybrid has characteristics of both a K- and V-class system (5) for which the principal heterotropic effect is a change in Vma,,. In view of this unusual heterotropic effect and the lack of cooperativity exhibited by the hybrid, it is pertinent to consider the applicability of the two-state model of Monod et al. (5) which accounts so satisfactorily for most of the properties of the wild-type enzyme (31). Usually it is assumed that the putative constrained and reI
Other hypotheses can be proposed to account for the altered Km and Vm,,. of CNCMR3. For example, the size and shape of CM may be sufficiently different from CN to alter the geometry of the complex and to hinder access of ligands.
4302
Proc. Nat. Acad. Sci. USA 72 (1975)
Biochemistry: Gibbons et al.
laxed forms of the enzyme have identical turnover numbers. However, the kinetic behavior of the wild-type enzyme would be unaffected if the constrained state had a lower activity (turnover number) than the relaxed state, inasmuch as the former contributes so little to the overall activity even at low substrate concentrations. If we assume that the constrained state of CNCMR% had a lower activity than the relaxed state, the anomalous kinetic properties of the hybrid could be rationalized on the basis of the two-state model. The equilibrium constant favoring the constrained state of the hybrid would have to be much greater than for the wildtype enzyme, and the ratio of affinities of the two forms of the hybrid for aspartate would have to be less than that for the wild-type enzyme. Although this speculation shows that the properties of CNCMR3 can be accommodated qualitatively within the framework of the two-state model, alternative models must be considered. It is noteworthy that removal of the H4Phtoyl-groups fromx CNCM,TR3 leads to a marked enhancement of the quaternary constraint. Not only did Km increase about 4-fold, but Vmkx also was reduced about 2-fold. A similar effect was observed (15) for the hybrid, CNCPTR& which exhibited no cooperativity and had a much lower Km than the deacylated, allosteric hybrid, CNCpR3. In both cases, it seems that the electrostatic destabilization stemming from the charged acyl groups is greater for the constrained or compact (higher 820w) form of the enzyme than it is for the relaxed or swollen state; hence, the acylated derivatives exhibit less quaternary constraint. We thank J. C. Gerhart for innumerable and invaluable suggestions and G. R. Stark for the PALA. This investigation was supported by NIH Research Grant GM 12159 from the National Institute of General Medical Sciences, by Training Grant CA 05028 from the National Cancer Institute, and by National Science Foundation Grant GB 32812X. J.E.F. was the recipient of NIH Postdoctoral Fellowship GM 46941 from the National Institute of General Medical Sciences.
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4. Gerhart, J. C. & Pardee, A. B. (1962) J. Btol. Chem. 237, 891-896. 5. Monod, J., Wyman, J. & Changeux, J.-P. (1965) J. Mol. Biol. 12,88-118. 6. Gerhart, J. C. & Schachman, H. K. (1965) Biochemistry 4, 1054-1062. 7. Weber, K. (1968) Nature 218, 1116-1119. 8. Wiley, D. C. & Lipscomb, W. N. (1968) Nature 218, 11191121. 9. Meighen, E. A., Pigiet, V. & Schachman, H. K. (1970) Proc. Nat. Acad. Sci. USA 65,234-241. 10. Nagel, G. M., Schachman, H. K. & Gerhart, J. C. (1972) Fed. Proc. 31, 423 Abstr. 11. Nagel, G. M. & Schachman, H. K. (1975) Biochemistry 14, 3195-3203. 12. Rosenbusch, J. P. & Weber, K. (1971) J. Biol. Chem. 246, 1644-1657. 13. Cohlberg, J. A., Pigiet, V. P., Jr. & Schachman, H. K. (1972) Biochemistry 11, 3396-3411. 14. Schachman, H. K. (1974) Hanvey Lect. 68, 67-113. 15. Gibbons, I., Yang, Y. R. & Schachman, H. K. (1974) Proc. Nat. Aced. Sci. USA 71, 4452-4456. 16. Flatgaard, J. E., Gerhart, J. C. & Schachman, H. K. (1974) Fed. Proc. 33, No. J, 1533 Abstr. 17. Yu, M. T., Kaney, A. R. & Atwood, K. C. (1965) J. Bacteriol. 90, 1150-1152. 18. Beckwith, J. R., Pardee, A. B., Austrian, R. & Jacob, F. (1962) J. Mol. Blol. 5,618-634. 19. Gerhart, J. C. & Holoubek, H. (1967) J. Biol. Chem. 242, 2886-2892. 20. Kirschner, M. W. (1971) in Ph.D. Dissertation, University of California, Berkeley, pp. 176-181. 21. Gibbons, I. & Schachman, H. K. (1975) Biochemistry, in press. 22. Porter, R. W., Modebe, M. 0. & Stark, G. R. (1969) J. Biol. Chem. 244,1846-1859. 23. Gerhart, J. C. (1962) in Ph.D. Dissertation, University of California, Berkeley, p. 17. 24. Mort, J. S. & Chan, W. W.-C. (1975) J. Biol. Chem. 250,
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