inactive derivatives: Implications for active sites shared between polypeptide chains of aspartate transcarbamoylase

Transcription

1 Proc. Nati. Acad. Sci. USA Vol. 82, pp , January 1985 Biochemistry Regeneration of active enzyme by formation of hybrids from inactive derivatives: Implications for active sites shared between polypeptide chains of aspartate transcarbamoylase (in vitro complementation/interchain hybrids/conformational correction) ELLEN A. ROBEY AND H. K. SCHACHMAN* Departments of Biochemistry and Molecular Biology, Wendell M. Stanley Hall, University of California, Berkeley, CA Contributed by H. K. Schachman, September 24, 1984 ABSTRACT Crystallographic studies of Escherichia coli aspartate transcarbamoylase (aspartate carbamoyltransferase, EC ) in conjunction with chemical modification experiments have led to the suggestion that the active sites of the enzyme are at the interfaces between adjacent polypeptide chains of the catalytic trimers and involve joint participation of amino acid residues from the adjoining chains. However, the precise locations of the active sites and of the residues involved in catalysis are not known. To test the hypothesis that the active sites are shared between chains, we constructed hybrid trimers in which two chains were modified at one presumed active site residue and the third chain was altered at a different active site residue. One parental trimer was a reduced pyridoxal phosphate derivative in which lysine-84 was modified and the other was a mutant protein in which tyrosine-165 was converted to serine by site-directed mutagenesis. Incubating mixtures of these two virtually inactive derivatives under conditions promoting interchain exchange led to a large increase in enzyme activity corresponding approximately to the formation of one active site per trimer. The purified hybrid trimers, containing either two pyridoxylated and one mutant chain or vice versa, had 23% and 28%, respectively, the activity of native wild-type catalytic trimers, compared to 5% and 3% for the parental trimers. The most likely explanation for this large increase in activity is the formation of one "native" active site in each of the hybrid trimers. The results constitute strong evidence for shared active sites in aspartate transcarbamoylase. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. For the majority of enzymes, the amino acid residues constituting an active site are contained within a single polypeptide chain. It is also possible, however, for the active sites to be located at the interfaces between polypeptide chains in oligomeric enzymes and for catalytic activity to be dependent upon the joint participation of amino acids from adjacent chains. With some enzymes such as glutathione reductase (1), glucose-6-phosphate isomerase (2), and aspartate aminotransferase (3), crystallographic studies of the complexes with substrates, coenzymes, or inhibitors have indicated that amino acids from adjoining chains comprise the individual active sites. Similarly, x-ray diffraction studies in conjunction with chemical modification experiments on aspartate transcarbamoylase (ATCase; aspartate carbamoyltransferase; carbamoylphosphate:l-aspartate carbamoyltransferase, EC ) from Escherichia coli have led to the suggestion that the active sites in ATCase are at the interfaces between adjacent catalytic polypeptide chains (4, 5). This paper presents an alternative approach aimed at determining whether the catalytic activity of ATCase requires the joint participation of amino acid residues from adjoining chains. The method is based on measurements of the enzyme activity of different hybrids formed from virtually inactive derivatives of the catalytic subunits. ATCase is composed of six catalytic (c) and six regulatory (r) chains organized in the holoenzyme as two catalytic (C) trimers and three regulatory (R) dimers. Treatment of the enzyme with mercurials causes dissociation of ATCase into discrete subunits that are readily separated (6). The C trimer, although it lacks the regulatory properties of intact ATCase, is fully active and exhibits Michaelian kinetics (6). The crystal structures of unliganded ATCase and of the enzyme with the bound allosteric inhibitor, CTP, have been determined to 2.6- and 2.8-A resolution, respectively (5, 7). Several amino acid residues of the c chains thought to be at the active sites have been identified through studies of chemically modified or mutationally altered enzyme (8-12). However, a clear picture of the active site has not emerged. On the basis of the location of the presumed active site residues, the smaller distance between these residues from adjacent chains as compared to their separation within a single chain, and their location relative to the phosphate binding site (16), Monaco et al. (4) and Honzatko et al. (5) have suggested that the active sites of ATCase are shared between c chains in each C trimer. C trimers are readily dissociated into unfolded polypeptide chains or folded, inactive monomers, and reconstitution studies have shown that the regeneration of enzyme activity is coincident with the formation of trimers (13, 14). Although the inactivity of monomers could be attributed to the absence of the required shared active site, it is also possible that all the residues comprising an active site are present in one monomer but that trimer formation is required to stabilize the active conformation. To test whether the active sites are shared, we constructed hybrid C trimers from two different derivatives, each of which is essentially inactive. This was accomplished by using a pyridoxylated derivative in which lysine-84 is modified (8) and a mutant produced by site-directed mutagenesis in which tyrosine-165 is replaced by serine (15). The finding that the purified hybrid trimers formed from virtually inactive parents have enzyme activities corresponding closely to one active site per trimer constitutes strong evidence for shared active sites between adjacent polypeptide chains in ATCase. Abbreviations: ATCase, aspartate transcarbamoylase; c, catalytic polypeptide chain; r, regulatory polypeptide chain; C, catalytic trimer; R, regulatory dimer; THPA, 3,4,5,6-tetrahydrophthalic anhydride; CMMM, a mutant form of C in which each tyrosine-165 is replaced by serine; Cppp, a pyridoxylated derivative of C in which each c chain is modified at lysine-84 by reaction with pyridoxal 5'- phosphate followed by reduction of the Schiff base with NaBH4. Subscripts are used to designate the types of polypeptide chains in various trimers-e.g., CMMP is a hybrid composed of two mutant and one pyridoxylated polypeptide chain and CMPP is a hybrid trimer containing one mutant and two pyridoxylated chains. *To whom reprint requests should be addressed. 361

2 362 Biochemistry: Robey and Schachman Proc. NatL Acad Sci. USA 82 (1985) Active sites within monomers "unshared" Unmodified trimer 'a derivatives I Hybrid formation +(9 Active sites at monomer interfaces shared" Unmodified trimer derivatives I Hybrid formation FIG. 2. Structure of C trimer within intact unliganded ATCase. The a-carbon trace of each chain is shown as light lines with the side chains of lysine-84 and tyrosine-165 in each chain represented by dark lines. Circles designate the binding sites for phosphate ligands (16). The view is from the center of the ATCase molecule, looking down the 3-fold axis [based on the coordinates from Ke et al. (7)]. One-third active FIG. 1. Schematic view of active sites in C trimers and use of hybrids to test alternative models. Large circles correspond to individual c chains, and o and A represent unmodified active-site residues. The corresponding modified residues that cause inactivation are designated by * and A. An active site requires adjoining and A. The model at the top ("unshared") corresponds to a trimer in which each active site is within a c chain; in the lower model ("shared") the active sites are at the interfaces between chains. One inactive derivative used for the construction of the hybrids involves the modification leading to n, whereas the other has the alteration forming *. EXPERIMENTAL APPROACH The rationale for the construction of hybrid trimers to test the hypothesis that active sites require the participation of amino acid residues from adjacent polypeptide chains is illustrated in Fig. 1. Two possible arrangements of the activesite residues and A in a C trimer are depicted in schematic form. If the active sites are within each monomer, as in the upper model ("unshared"), modification of each protein to give * or A will lead to inactive derivatives. The two hybrids formed from these modified proteins will still be inactive because none of the chains will contain an unmodified active site. If, however, the active sites are located at the interfaces between chains as in the lower model ("shared") and the modifications affect residues on opposite sides of the active sites, then enzyme activity would be restored by the formation of interchain hybrids. In this case both parental derivatives containing the modified residues * or A are inactive, but the construction of the two hybrids results in the formation of one active site per trimer containing C and A at the interface. Thus, the formation of hybrids containing onethird the activity of an unmodified C trimer would provide support for the hypothesis of shared active sites. Fig. 2 shows an a-carbon trace of the three c chains within one C trimer in ATCase (7). Also shown in each chain are lysine-84 and tyrosine-165, two residues which, when modified chemically or by site-specific mutagenesis, lead to a marked reduction in catalytic activity (8-10, 15). The three circles designate the binding sites for phosphate ligands (16) which are competitive inhibitors of carbamoylphosphate binding (17). Thus, the functionally important residues lysine-84 and tyrosine-165 approach the presumed carbamoylphosphate binding site from different polypeptide chains. It should be noted that the distance from the e-amino group of lysine-84 in one chain across the interface to the hydroxyl group of tyrosine-165 in the adjacent chain is still considerable (21.4 A) as compared to their separation within a single chain (28.6 A). This structural information is based on crystallographic studies of ATCase in the absence of active-site ligands and corresponds to the low-activity conformation. No detailed structure of isolated C trimers or the high-activity conformation of ATCase is available. MATERIALS AND METHODS Wild-type ATCase was prepared according to the method of Gerhart and Holoubek (18) from the E. coli strain TR4363 containing the plasmid ppyrb3 (19), which contains the genes coding for the c and r chains of ATCase. Purification of the mutant ATCase766 has been described elsewhere (15) and isolation of the C trimers of the mutant (CMMM) and wild-type ATCase was performed by the procedure of Yang et al. (20). The polypeptide chains in C trimers were chemically modified at lysine-84 to give Cp~p by treatment of the protein with pyridoxal-5'-phosphate and NaBH4 as described by Greenwell et al. (8). ATCase activity was determined by measuring the change in absorbance (21) at 210 nm due to the formation of carbamoylaspartate from carbamoylphosphate (4 mm) and aspartate (10 mm) in 20 mm phosphate at ph 7.0 and 30'C. The assay of Davies et al. (22) with [14C]carbamoylphosphate was used for the determination of the apparent Km for aspartate. Radioactive assays were performed at 30'C in 50 mm imidazole/acetate buffer, ph 7.0, containing 2 mm 2-mercaptoethanol, 0.2 mm EDTA, and 4 mm carbamoylphosphate. Data were fit to the Michaelis- Menten equation.

3 Biochemistry: Robey and Schachman The restoration of enzyme activity from mixtures of inactive derivatives was measured by incubating equal amounts (1.7 mg/ml) of CMMM and Cppp at 40C and ph 8.3 in 50 mm Tris/acetate buffer containing 10 mm sodium pyrophosphate, 2 mm 2-mercaptoethanol, and 0.2 mm EDTA. Aliquots were withdrawn at specific times and diluted about 1:1000 directly into the assay mixture for measurement of enzyme activity. Pyrophosphate has been found to increase the rate of interchain exchange in mixtures of C trimers (unpublished results). A control experiment was done in the same manner except for the omission of the pyrophosphate. Individual interchain hybrids, CMMP and CMPP, were prepared and purified by the procedure of Gibbons and Schachman (23), using the reversible acylating agent 3,4,5,6-tetrahydrophthalic anhydride (THPA) to introduce charged groups and to facilitate separation of the different species in the hybrid set. Cp~p was treated with various amounts of THPA (23, 24) and electrophoretic analysis on cellulose polyacetate strips in a Microzone electrophoresis cell (Beckman-Spinco) was used to determine the optimal degree of acylation. The hybrid set was formed by two different methods. In one procedure, 10 mg of CMMM and 10 mg of tetrahydrophthaloylated Cppp were mixed at 0C and ph 7.5 in 40 ml of 1.25 M NaSCN/40 mm potassium phosphate buffer/2 mm 2-mercaptoethanol/0.2 mm EDTA. Because 1.25 M NaSCN causes the dissociation of C trimers into folded monomers (13), this technique was effective for producing the four-membered hybrid set. After 30 min, the solution was dialyzed against the same buffer without NaSCN to permit the monomers to reassociate into trimers. In the second procedure, equal amounts (1.5 mg/ml) of CMMM and Cppp were incubated together for 72 hr at 4 C and ph 8.3 in 50 mm Tris Cl/20 mm sodium pyrophosphate/2 mm 2-mercaptoethanol/0.2 mm EDTA. The four species were separated by ion-exchange chromatography on DEAE-Sephadex A-50 by eluting the proteins with a gradient of M KCI in 50 mm Tris Cl, ph 7.5/2.0 mm 2-mercaptoethanol/0.2 mm EDTA. After separation of the proteins, the tetrahydrophthaloyl groups were removed (23) by dialyzing the solutions for 2 days at 20 C at ph 6.1. RESULTS Restoration of Enzyme Activity in Mixtures of Derivatives Under Conditions Promoting Interchain Exchange. The possibility of regenerating enzyme activity from virtually inactive derivatives was tested with mixtures of CMMM, in which each tyrosine-165 was replaced by serine (15), and Cppp, in which lysine-84 in each chain was pyridoxylated (8, 25). CMMM had a specific activity of 0.55,umol of carbamoylaspartate formed per hr per,ug of protein at 30 C and the specific activity of Cp~p when assayed under comparable conditions with 10 mm aspartate was 0.89 (,umol/hr)/,g. These activities correspond, respectively, to 2.9% and 4.7% that of native, wild-type C trimers [specific activity 19 (timol/hr)/,4g]. As seen in Fig. 3, mixtures of equal amounts of these two derivatives incubated for 55 hr at ph 8.3 in 50 mm Tris/acetate buffer containing 2.0 mm 2-mercaptoethanol and 0.2 mm EDTA had a constant low specific enzyme activity of 0.6 (,mol/hr)/,ug, a value close to the average for the two proteins. When, however, these same proteins were incubated in the same buffer containing 10 mm sodium pyrophosphate, which promotes interchain exchange (unpublished results), there was more than a 6-fold increase in enzyme activity to about 3.8 (,umol/hr)/,ug. This large increase in enzyme activity is consistent with the view that an unmodified active site is formed in each of the hybrid molecules, CMMP and CMPP, as illustrated in the shared-site model in Fig. 1. It is of interest that the observed >, Proc. NatL. Acad. Sci. USA 82 (1985) Ti me (hours) FIG. 3. Regeneration of enzyme activity from inactive derivatives. Mixtures of CMMM and COPp, each at 1.7 mg/ml, were incubated at 40C in 50 mm Tris/acetate buffer (ph 8.3) containing 2 mm 2- mercaptoethanol and 0.2 mm EDTA with (e) or without (v) 10 mm sodium pyrophosphate, which promotes interchain exchange. At the times indicated, aliquots were removed and diluted approximately 1:1000 into the assay mixture (4 mm carbamoylphosphate and 10 mm aspartate) for measurement of enzyme activity (Amol carbamoylaspartate formed per hr per 1ug of protein). specific activity, 3.8 (jzmol/hr)/,ug, is close to the value, 4.75 (gmol/hr)/,ug, calculated for a binomial distribution of species formed by random interchange of chains from inactive parental trimers. This estimate is based on the assumption that each of the hybrid trimers contains one active site.t Specific Activity of Individual Hybrid Trimers. The results in Fig. 3, though indicating that interchain exchange between CMMM and Cp~p led to active hybrids, do not provide quantitative data regarding the specific activity of the hybrids because the measured activities represent contributions from all four members of the hybrid set. Therefore, each species was isolated so that its specific activity could be measured directly. This was accomplished by the procedure described in Materials and Methods and is illustrated by the electrophoretic patterns in Fig. 4. Both COPp, which had been treated with THPA to alter its net charge, and CMMM exhibited single bands [the band for COPp was substantially broader than that for CMMM because of the heterogeneity introduced by the various degrees of acylation of the pyridoxylated molecules (23)]. Incubating a mixture of these proteins for 72 hr in a buffer containing 20 mm sodium pyrophosphate produced a hybrid set containing four species. As seen in the electrophoresis pattern (Fig. 4), two of the components had mobilities corresponding to the parental trimers and the other two, designated CMMP and CMPP, had intermediate mobilities. All four species were isolated by ion-exchange chromatography, and each produced a single band on electrophoresis (Fig. 4). Measurements of the specific activities of the four species after deacylation (cf. Materials and Methods) are summarized in Table 1. The activities of CMMM and COPp isolated from the hybrid set, corresponding, respectively, to 2.9% and 4.7% that of wild-type C trimers, were the same as those measured for the parental trimers before the hybridization experiment. Thus, the acylation and deacylation procedures and the other manipulations had no significant effect on their activities. As seen in Table 1, the specific activities of the two hybrids, CMMP and CMPP, were considerably greater than those of either of the parental trimers. The specific actit should be noted that the parental trimers were not totally inactive; a small amount of the measured enzyme activity after interchain exchange is due to this residual activity.

4 364 Biochemistry: Robey and Schachman I I: I W-V. origin CMMM I CPPP C SMMm CMPP CpPP i J Parent t ri ners Hybrid set Fractionate d hybrid set FIG. 4. Electrophoretic analysis of hybrid set formed from the two modified derivatives. The patterns for the parental trimers CMMM and Cp1p are shown at the top. COPp was was acylated with THPA to increase the net negative charge and to facilitate resolution of the four individual species in the hybrid set as described in Materials and Methods. The individual species after separation by ionexchange chromatography are shown by the lower four patterns, corresponding to CMMM, CMMP, CMPP, and COPp. tivities of the two hybrids were 28% and 23% that of the wild-type C trimer, values which approach the 33% level expected from the shared-site model if, in each hybrid, one active site were formed at the interface between the two different chains.: If the enzyme activity of the hybrids is due to the formation of a "native" active site, the Km for the reconstructed active site in the hybrids might be expected to be similar to that of unmodified C trimers. Enzyme assays at various aspartate concentrations (and 4 mm carbamoylphosphate) yielded a value of 7 mm for the apparent Km of both CMMP and CMPP as contrasted to 13 mm for wild-type C trimers, 160 mm for CMMM, and 5 mm for Cppp. The value for the hybrids is designated as an apparent Km because of the complications stemming from the residual activity of the mutant and pyridoxylated chains. Since the activity contributed by these chains is small compared to that of the reconstructed active sites, the value for the apparent Km is a good approximation of the actual Km. DISCUSSION As seen in Fig. 3, there is a striking generation of enzyme activity when two different, virtually inactive enzyme derivatives are incubated together under conditions that promote interchain exchange. Moreover, the amount of activity restored is close to that expected for the random formation of hybrid trimers containing one active site per trimer. This high activity is demonstrated more clearly (Table 1) in the quantitative studies on the purified hybrids CMMP and CMPP, which have specific activities of 5.4 and 4.3 (gmol/hr)/pug, respectively; these values are only slightly lower than that expected for a trimer containing one active site [6.3 (,Umol/hr)/,ug]. The most plausible interpretation for the activity of the hybrids is the formation of one unmodified ("native") active site per trimer according to the scheme for the shared-site model in Fig. 1. It is also possible, however, that the contanalogous hybrids were formed from COPp and C trimers that were modified by nitration (9) of tyrosine-165 and tyrosine-240; these parental derivatives had, respectively, 5.5% and 14% the activity of native, wild-type C trimers. The purified hybrids, containing two pyridoxylated and one nitrated chain or one pyridoxylated and two nitrated chains, were more active than the parental trimers, with the former having 16% and the latter, 17% the enzyme activity of native C trimers. The increase in activity is not as striking as that observed with the hybrids CMMP and CMPP. Proc. NatL Acad Sci. USA 82 (1985) Table 1. Specific activity of hybrids* Specific Activity relative activity, to unmodified Type of trimer (kumol/hr)/jug wild-type C, %o Native wild-type CMMM CPPP CMMP t CMPP t *The hybrids CMMP and CMPP and the parental trimers CMMM and CPpp were isolated from the hybrid set formed from CMMM and Cppp as described in Materials and Methods. tlf the enzyme activity of each c chain in the hybrids were the same as its activity in the parental trimer, the activities of the hybrids CMMP and CMPP would be 3.5% and 4.1% that of the unmodified wild-type C trimer. struction of the hybrids leads to partial reactivation of two or three of the sites in the trimer through a mechanism involving conformational correction. This mechanism for the regeneration of activity in the hybrids assumes that the modified amino acids are not critical for catalysis and that the loss of activity in the. derivatives is due to a conformational defect that can be corrected by interaction with a conformationally correct c chain. It is unlikely that this mechanism is operating for the hybrids in this study, because no reactivation of either pyridoxylated or mutant chains occurs when they are incorporated into hybrids with native c chains. It has been shown previously that hybrids containing one native and two pyridoxylated chains or two native and one pyridoxylated chain have specific activities equal, respectively, to 35% and 58% that of native C trimers (25). In addition, incubation of CMMM and wild-type C trimers under conditions promoting interchain exchange (as in Fig. 3) caused no change in activity (data not shown). Although both chemical-modification and site-specific mutagenesis experiments as well as x-ray crystallographic studies indicate that lysine-84 and tyrosine-165 are near the active site, the precise role of these residues in catalysis is not known. It is possible that the loss of activity in derivatives in which these residues are modified is due to a local conformational change that affects the nearby active site. In the case of Cppp, in which a lysine e-amino group in each c chain is converted to a negatively charged pyridoxamine phosphate group, there may be a charge-repulsion effect in the region where anionic substrates bind. If tyrosine-165 or lysine-84 is not directly involved in substrate binding or catalysis, the restoration of activity in the hybrid trimers could still be the result of forming an unmodified active site region at an interface. There are many examples from genetic studies of mutants in which two inactive gene products interact in vivo to form a functional enzyme. This phenomenon, known as interallelic complementation (26-28), has been observed with various mutants of ATCase (12, 29) and it is likely that several different mechanisms are involved. The in vitro reactivation experiments described here represent an extremely efficient example of interallelic complementation. Also, the approach employed here to determine whether shared active sites exist at the interfaces between pairs of catalytic chains in ATCase should have wide applicability to other oligomeric enzymes. We thank Richard B. Honzatko for providing the coordinates of unliganded ATCase that were used for drawing Fig. 2. We are indebted to Ying R. Yang for valuable suggestions, advice, and helpful discussions during the course of the work. This research was supported by National Institute of General Medical Sciences Research Grant GM12159 and by National Science Foundation Research

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