Effects of cAMP-binding site mutations on intradomain cross-communication in the regulatory subunit of cAMP-dependent protein kinase I

Each protomer of the regulatory subunit dimer of cAMP-dependent protein kinase contains two tandem and homologous cAMP-binding domains, A and B, and cooperative cAMP binding to these two sites promotes holoenzyme dissociation. Several amino acid residues in the type I regulatory subunit, predicted to lie in close proximity to each bound cyclic nucleotide based on affinity labeling and model building, were replaced using recombinant techniques. The mutations included replacement of 1) Glu-200, predicted to hydrogen bond to the 2'-OH of cAMP bound to site A, with Asp, 2) Tyr-371, the site of affinity labeling with 8-N3-cAMP in site B, with Trp, and 3) Phe-247, the position in site A that is homologous to Tyr-371 in site B, with Tyr. Each mutation caused an approximate 2-fold increase in both the Ka(cAMP) and Kd(cAMP); however, the off-rates for cAMP and the characteristic pattern of affinity labeling with 8-N3-cAMP differed markedly for each mutant protein. Furthermore, these mutations affect the cAMP binding properties not only of the site containing the mutation, but of the adjacent nonmutated site as well, thus confirming that extensive cross-communication occurs between the two cAMP-binding domains. Photoaffinity labeling of the native R-subunit results in the covalent modification of two residues, Trp-260 and Tyr-371, by 8-N3-cAMP bound to sites A and B, respectively, with a stoichiometry of 1 mol of 8-N3-cAMP incorporated per mol of R-monomer (Bubis, J., and Taylor, S. S. (1987) Biochemistry 26, 3478-3486). A stoichiometry of 1 mol of 8-N3-cAMP incorporated per R-monomer was observed for each mutant regulatory subunit as well, even when 2 mol of 8-N3-cAMP were bound per R-monomer; however, the major sites of covalent modification were altered as follows: R(Y371/W), Trp-371; R(E200/D), Tyr-371, and R(F247/Y), Tyr-371.     

A point mutation abolishes binding of cAMP to site A in the regulatory subunit of cAMP-dependent protein kinase

Each regulatory subunit of cAMP-dependent protein kinase has two tandem cAMP-binding sites, A and B, at the carboxyl terminus. Based on sequence homologies with the cAMP-binding domain of the Escherichia coli catabolite gene activator protein, a model has been constructed for each cAMP-binding domain. Two of the conserved features of each cAMP-binding site are an arginine and a glutamic acid which interact with the negatively charged phosphate and with the 2'-OH on the ribose ring, respectively. In the type I regulatory subunit, this arginine in cAMP binding site A is Arg-209. Recombinant DNA techniques have been used to change this arginine to a lysine. The resulting protein binds cAMP with a high affinity and associates with the catalytic subunit to form holoenzyme. The mutant holoenzyme also is activated by cAMP. However, the mutant R-subunit binds only 1 mol of cAMP/R-monomer. Photoaffinity labeling confirmed that the mutant R-subunit has only one functional cAMP-binding site. In contrast to the native R-subunit which is labeled at Trp-260 and Tyr-371 by 8-N3cAMP, the mutant R-subunit is convalently modified at a single site, Tyr-371, which correlates with a functional cAMP-binding site B. The lack of functional cAMP-binding site A also was confirmed by activating the mutant holoenzyme with analogs of cAMP which have a high specificity for either site A or site B. 8-NH2-methyl cAMP which preferentially binds to site B was similar to cAMP in its ability to activate both mutant and wild type holoenzyme whereas N6-monobutyryl cAMP, a site A-specific analog, was a very poor activator of the mutant holoenzyme. The results support the conclusions that 1) Arg-209 is essential for cAMP binding to site A and 2) cAMP binding to domain A is not essential for dissociation of the mutant holoenzyme.     

Dissecting the domain structure of the regulatory subunit of cAMP-dependent protein kinase I and elucidating the role of MgATP

A truncated regulatory subunit of cAMP-dependent protein kinase I was constructed which contained deletions at both the carboxyl terminus and at the amino terminus. The entire carboxyl-terminal cAMP-binding domain was deleted as well as the first 92 residues up to the hinge region. This monomeric truncated protein still forms a complex with the catalytic subunit, and activation of this complex is mediated by cAMP. The affinity of this mutant holoenzyme for cAMP and its activation by cAMP are nearly identical to holoenzyme formed with a regulatory subunit having only the carboxyl-terminal deletion and very similar to native holoenzyme. The off rate for cAMP from both mutant regulatory subunits, however, is monophasic and very fast relative to the biphasic off rate seen for the native regulatory subunit. The effects of NaCl, urea, and pH on cAMP binding are also very similar for the mutant and native holoenzymes. Like the native type I holoenzyme, both mutant holoenzymes bind ATP with a high affinity. The positive cooperativity seen for MgATP binding to the native holoenzyme, however, is abolished in the double deletion mutant. The Hill coefficient for ATP binding to this mutant holoenzyme is 1.0 in contrast to 1.6 for the native holoenzyme. The Kd (cAMP) is increased by approximately 1 order of magnitude for both mutant forms of the holoenzyme in the presence of MgATP. A similar shift is seen for the native holoenzyme. Further characterization of the MgATP-binding properties of the wild-type holoenzyme indicates that a binary complex containing catalytic subunit and MgATP is required, in particular, for reassociation with the cAMP-bound regulatory subunit. This binary complex is required for rapid dissociation of the bound cAMP and is probably responsible for the observed reduction in cAMP-binding affinity for the type I holoenzyme in the presence of MgATP.     

Correlation of photolabeling with occupancy of cAMP binding sites in the regulatory subunit of cAMP-dependent protein kinase I

Each regulatory subunit of the cAMP-dependent protein kinase contains two in-tandem cAMP binding sites. Photolabeling of holoenzyme I with 8-azidoadenosine 3',5'-monophosphate (8-N3-cAMP) leads to the covalent modification of two residues, Trp-260 and Tyr-371. In order to correlate photolabeling of these two residues with occupancy of each specific cAMP binding site, photolabeling was carried out in the presence of various analogues of cAMP that bind preferentially to one site. Photolabeling of holoenzyme I after dissociation of 60% of 8-N3-[3H]cAMP with an excess of N6-monobutyryl-cAMP nearly abolished the incorporation of 8-N3-cAMP into Trp-260, whereas the modification of Tyr-371 was reduced by 49%. When 8-N3-[32P]cAMP was bound under equilibrium conditions in the presence of various cAMP analogues, N6-monobutyryl-cAMP also selectively abolished incorporation of radioactivity into Trp-260, whereas 8-(methylamino)-cAMP preferentially reduced the covalent modification of Tyr-371. Photolabeling with trace amounts of 8-N3-[32P]cAMP in the presence of saturating amounts of N6-monobutyryl-cAMP led to the covalent modification of only Tyr-371. In addition, photolabeling of Tyr-371 was enhanced synergistically in the presence of N6-monobutyryl-cAMP. MgATP reduced the covalent modification of both Trp-260 and Tyr-371 but showed no selectivity for either site. These studies support a model that correlates photolabeling of Trp-260 with occupancy of cAMP binding site A and photolabeling of Tyr-371 with occupancy of cAMP binding site B.(ABSTRACT TRUNCATED AT 250 WORDS)     

cAMP-dependent protein kinase regulatory subunit type IIbeta: active site mutations define an isoform-specific network for allosteric signaling by cAMP

cAMP-dependent protein kinase (cAPK) contains a regulatory (R) subunit dimer bound to two catalytic (C) subunits. Each R monomer contains two cAMP-binding domains, designated A and B. The sequential binding of two cAMPs releases active C. We describe here the properties of RIIbeta and two mutant RIIbeta subunits, engineered by converting a conserved Arg to Lys in each cAMP-binding domain thereby yielding a protein that contains one intact, high affinity cAMP-binding site and one defective site. Structure and function were characterized by circular dichroism, steady-state fluorescence, surface plasmon resonance and holoenzyme activation assays. The Ka for RIIbeta is 610 nM, which is 10-fold greater than its Kd(cAMP) and significantly higher than for RIalpha and RIIalpha. The Arg mutant proteins demonstrate that the conserved Arg is important for both cAMP binding and organization of each domain and that binding to domain A is required for activation. The Ka of the A domain mutant protein is 21-fold greater than that of wild-type and the Kd(cAMP) is increased 7-fold, confirming that cAMP must bind to the mutated site to initiate activation. The domain B mutant Ka is 2-fold less than its Kd(cAMP), demonstrating that, unlike RIalpha, cAMP can access the A site even when the B site is empty. Removal of the B domain yields a Ka identical to the Kd(cAMP) of full-length RIIbeta, indicating that the B domain inhibits holoenzyme activation for RIIbeta. In RIalpha, removal of the B domain generates a protein that is more difficult to activate than the wild-type protein.     

Role of MgATP in the activation and reassociation of cAMP-dependent protein kinase I: consequences of replacing the essential arginine in cAMP binding site A.

The type I form of cAMP-dependent protein kinase binds MgATP with a high affinity, and binding of MgATP decreases the affinity of the holoenzyme for cAMP [Hofmann et al. (1975) J. Biol. Chem. 250, 7795]. Holoenzyme was formed here with a mutant form of the bovine recombinant type I regulatory subunit where the essential arginine in site A, Arg-209, was replaced with Lys. Although this mutation does not significantly change the high-affinity binding of MgATP to the holoenzyme, it does abolish high-affinity binding of cAMP to site A. In the absence of MgATP, binding of cAMP to site B is sufficient to promote dissociation of the holoenzyme complex and activation of the catalytic subunit [Bubis et al. (1988) J. Biol. Chem. 263, 9668]. In the presence of MgATP however, holoenzyme formed with this mutant regulatory subunit is very resistant to cAMP. The Kd(cAMP) was greater than 1 microM, and the Ka(cAMP) increased 60-fold from 130 nM to 6.5 microM in the presence of MgATP. Thus, MgATP serves as a lock that selectively stabilizes the holoenzyme and inhibits activation. Both site A and site B are shielded from cAMP in the presence of MgATP. These results suggest that Arg-209 may play a role in stabilizing the MgATP.holoenzyme complex in addition to its role in binding the exocyclic oxygens of cAMP when cAMP is bound to the regulatory subunit. The catalytic subunit also reassociates rapidly with this mutant regulatory subunit, and in contrast to the wild-type regulatory subunit, holoenzyme formation does not require MgATP.     

Activation of type I cyclic AMP-dependent protein kinases with defective cyclic AMP-binding sites

Two S49 mouse lymphoma cell variants hemizygous for expression of mutant regulatory (R) subunits of type I cyclic AMP-dependent protein kinase were used to investigate functional consequences of lesions in the putative cAMP-binding sites of R subunit. Kinase activation properties of wild-type and mutant enzymes were compared using cAMP and six site-selective analogs of cAMP. Kinases from both mutant sublines were relatively resistant to cyclic nucleotide-dependent activation, but they were fully activable by at least some effectors. Relative resistances of the mutant kinases varied from about 5-fold for analogs selective for their nonmutated sites to as much as 700-fold for analogs selective for their mutated sites; resistance to cAMP was intermediate. Apparent affinities of wild-type and mutant R subunits for [3H]cAMP were not appreciably different, but competition experiments with site-selective analogs of cAMP suggested that binding of cAMP to mutant R subunits was primarily to their nonmutated sites. Analyses of cooperativity in cyclic nucleotide-dependent activation of mutant kinases, synergism between site I- and site II-selective analogs in activating the mutant enzymes, and dissociation of bound cAMP from mutant R subunits provided additional evidence that the mutations in these strains selectively inactivated single classes of cAMP-binding sites: phenomena attributable in wild-type enzyme to intrachain interactions between sites I and II were always absent or severely diminished in experiments with the mutant enzymes. These results confirm that R subunit sequences implicated in cAMP binding by homology with other cyclic nucleotide-binding proteins actually correspond to functional cAMP-binding sites. Furthermore, occupation of either cAMP-binding site I or II is apparently sufficient for activation of cAMP-dependent protein kinase. The presence of four functional cAMP-binding sites in wild-type kinase enhances the cooperativity and sensitivity of cAMP-mediated activation.     

Site-directed mutagenesis of the cAMP-binding sites of the recombinant type I regulatory subunit of cAMP-dependent protein kinase

The type I regulatory subunit (R-I) of rat brain cAMP-dependent protein kinase was expressed in E. coli and site-directed mutagenesis was used to substitute amino acids in the putative cAMP-binding sites. The wild-type recombinant R-I bound 2 mol of cAMP/mol subunit, while two mutant R-Is with a single amino acid substitution in one of the two intrachain cAMP-binding sites (clone N153:a glutamate for Gly-200, and clone C254:an aspartate for Gly-324) bound 1 mol of cAMP/mol subunit. When these two substitutions were made in one mutant, cAMP did not bind to this mutant, indicating that binding of cAMP to N153 or C254 was to their nonmutated sites. Competition experiments with site-selective analogs and dissociation of bound cAMP from mutant R-Is provided evidence for strong intrachain interactions between the two classes of cAMP-binding sites in R-I.     

Site-specific cyclic nucleotide binding and dissociation of the holoenzyme of cAMP-dependent protein kinase

The photoaffinity reagent 8-azidoadenosine 3':5'-monophosphate (8-N3cAMP) was previously shown to modify a single tyrosine residue on the type II regulatory subunit of cAMP-dependent protein kinase (Kerlavage, A.R., and Taylor, S.S. (1980) J. Biol. Chem, 255, 8483-8488). In the present studies, the binding stoichiometries of type II holoenzyme for cAMP and 8-N3cAMP were determined using Millipore filtration assays in the absence (Assay A) and presence (Assay B) of 2 M NaCl and histone. The binding stoichiometry of holoenzyme for cAMP was 2 mol/mol with Assay A, and 4 mol/mol with assay B. The binding stoichiometry for 8-N3cAMP was 2 mol/mol with Assay B or with Assay A following photolysis of the holoenzyme:8-N3cAMP mixture. In the absence of photolysis, the binding stoichiometry for 8-N3cAMP was 0.4 mol/mol with Assay A. Both 8-N3cAMP and cAMP fully dissociated the holoenzyme. Holoenzyme, labeled with 8-N3[3H]cAMP on a preparative scale, incorporated 1 mol of 8-N3[3H]cAMP/mol of regulatory subunit (RII) monomer. The labeled RII was separated from catalytic subunit, cleaved with cyanogen bromide, and the resultant peptides were separated by high performance liquid chromatography. A single radioactive peptide was observed which had the same NH2 terminal residue and amino acid composition as the peptide obtained when dissociated RII was labeled with 8-N3cAMP.     

Function of threonine-55 in the carbamoyl phosphate binding site of Escherichia coli aspartate transcarbamoylase

Carbamoyl phosphate is held in the active site of Escherichia coli aspartate transcarbamoylase by a variety of interactions with specific side chains of the enzyme. In particular, the carbonyl group of carbamoyl phosphate interacts with Thr-55, Arg-105, and His-134. Site-specific mutagenesis was used to create a mutant version of the enzyme in which Thr-55 was replaced by alanine in order to help define the role of this residue in the catalytic mechanism. The Thr-55----Ala holoenzyme exhibits a 4.7-fold reduction in maximal observed specific activity, no alteration in aspartate cooperativity, and a small reduction in carbamoyl phosphate cooperativity. The mutation also causes 14-fold and 35-fold increases in the carbamoyl phosphate and aspartate concentrations required for half the maximal observed specific activity, respectively. Circular dichroism spectroscopy has shown that saturating carbamoyl phosphate does not induce a conformational change in the Thr-55----Ala holoenzyme as it does for the wild-type holoenzyme. The kinetic properties of the Thr-55----Ala catalytic subunit are altered to a greater extent than the mutant holoenzyme. The mutant catalytic subunit cannot be saturated by either substrate under the experimental conditions. Furthermore, as opposed to the wild-type catalytic subunit, the Thr-55----Ala catalytic subunit shows cooperativity for aspartate and can be activated by N-(phosphonoacetyl)-L-aspartate in the presence of low concentrations of aspartate and high concentrations of carbamoyl phosphate. As deduced by circular dichroism spectroscopy, the conformation of the Thr-55----Ala catalytic subunit in the absence of active-site ligands is distinctly different from the wild-type catalytic subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deletion mutants as probes for localizing regions of subunit interaction in cAMP-dependent protein kinase

The regulatory subunit of cAMP-dependent protein kinase has a well-defined domain structure, and recombinant DNA techniques have been used to define further the functional properties that are associated with each domain. Our initial question was to define the minimal structural unit that is required for forming a stable complex with the catalytic subunit that will still bind and hence be dissociated by cAMP. To answer these questions, the entire second cAMP-binding domain was deleted using oligonucleotide-directed mutagenesis to introduce a premature stop codon at Trp260. This mutation results in the expression of a stable protein with an Mr of 38,000 based on polyacrylamide gel electrophoresis. The resulting mutant protein is a dimer; and like the native R-subunit, the two protomers of the dimer are cross-linked by disulfide bonds at the amino terminus. The mutant R-subunit binds 1 mol of cAMP/monomer based on equilibrium dialysis. The Kd(cAMP) was 25 nM, which is slightly higher than the Kd(cAMP) for the native R-subunit. The removal of the second cAMP domain does not prevent aggregation with the catalytic subunit, and the inactive holoenzyme complex that is formed in the absence of cAMP can still be dissociated and consequently activated by cAMP. In conjunction with previous results based on limited proteolysis, it is concluded that the region extending from Arg94 to Lys259 constitutes a structural unit that will be sufficient to interact with the catalytic subunit in a cAMP-dependent manner.     

Function of arginine-234 and aspartic acid-271 in domain closure, cooperativity, and catalysis in Escherichia coli aspartate transcarbamylase

Two mutant versions of Escherichia coli aspartate transcarbamylase were created by site-specific mutagenesis. Arg-234 of the 240s loop was replaced by serine in order to help deduce the function of the interactions that normally occur between Arg-234 and both Glu-50 and Gln-231 in the R state of the enzyme. The other mutation involved the replacement of Asp-271 by asparagine to further test the functional importance of the Tyr-240-Asp-271 link that has previously been proposed to stabilize the T state of the enzyme [Middleton, S. A., & Kantrowitz, E. R. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5866-5870]. The Arg-234----Ser holoenzyme exhibits no cooperativity, a 24-fold reduction in maximal velocity, normal affinity for carbamyl phosphate, and substantially reduced affinity for aspartate and N-(phosphonoacetyl)-L-aspartate (PALA). Unlike the wild-type enzyme, the heterotropic effectors ATP and CTP are able to influence the activity of the Arg-234----Ser enzyme at saturating aspartate concentrations. The Arg-234----Ser catalytic subunit exhibits a 33-fold reduction in maximal activity, an aspartate Km of 261 mM, compared to 5.7 mM for the wild-type catalytic subunit, and only a small alteration in the Km for carbamyl phosphate. Together these results provide additional evidence that the interdomain bridging interactions between Glu-50 of the carbamyl phosphate domain and both Arg-167 and Arg-234 of the aspartate domain are necessary for the stabilization of the high-activity-high-affinity configuration of the active site of the enzyme. Furthermore, without the interdomain bridging interactions, the holoenzyme no longer exhibits homotropic cooperativity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions between regulatory and catalytic subunits of the Candida albicans cAMP-dependent protein kinase are modulated by autophosphorylation of the regulatory subunit

The cAMP-dependent protein kinase (PKA) from Candida albicans is a tetramer composed of two catalytic subunits (C) and two type II regulatory subunits (R). To evaluate the role of a putative autophosphorylation site of the R subunit (Ser(180)) in the interaction with C, this site was mutated to an Ala residue. Recombinant wild-type and mutant forms of the R subunit were expressed in Escherichia coli and purified. The wild-type recombinant R subunit was fully phosphorylated by the purified C subunit, while the mutant form was not, confirming that Ser(180) is the target for the autophosphorylation reaction. Association and dissociation experiments conducted with both recombinant R subunits and purified C subunit showed that intramolecular phosphorylation of the R subunit led to a decreased affinity for C. This diminished affinity was reflected by an 8-fold increase in the concentration of R subunit needed to reach half-maximal inhibition of the kinase activity and in a 5-fold decrease in the cAMP concentration necessary to obtain half-maximal dissociation of the reconstituted holoenzyme. Dissociation of the mutant holoenzyme by cAMP was not affected by the presence of MgATP. Metabolic labeling of yeast cells with [(32)P]orthophosphate indicated that the R subunit exists as a serine phosphorylated protein. The possible involvement of R subunit autophosphorylation in modulating C. albicans PKA activity in vivo is discussed.     

Mutating protein kinase cAMP-binding sites into cGMP-binding sites. Mechanism of cGMP selectivity.

The cAMP-dependent protein kinase contains two different cAMP-binding sites referred to as the slow and fast sites. Mutation of Ala-334 to a threonine in the slow site of the bovine type I regulatory subunit created a site with marked increase in cGMP affinity without changing cAMP affinity (Shabb, J. B., Ng. L., Corbin, J. D. (1990) J. Biol. Chem. 265, 16031-16034). The corresponding fast site residue (Ala-210) was changed to a threonine by oligonucleotide-directed mutagenesis, and a double mutant containing a threonine in each site was also made. Holoenzymes were formed from native catalytic subunit and each recombinant regulatory subunit. The fast site mutant holoenzyme exhibited an improved cGMP activation constant and an impaired cAMP activation constant. The double mutant cGMP/cAMP selectivity was 200-fold greater than that of wild-type holoenzyme, making it as responsive to cGMP as native cGMP-dependent protein kinase. The increased intrinsic binding energies of mutated sites for cGMP were 2.7-3.0 kcal mol-1, consistent with the presence of an extra hydrogen bond. Cyclic nucleotide analog studies implied that this hydrogen bond was between the threonine hydroxyl and the 2-amino of cGMP. Comparisons of amino acid sequences and cyclic nucleotide specificities suggested that the Ala/Thr difference may also impart cAMP/cGMP binding selectivity to related proteins such as cyclic nucleotide-gated ion channels.     

Site-specific substitutions of the Tyr-165 residue in the catalytic chain of aspartate transcarbamoylase promotes a T-state preference in the holoenzyme

The amino acid residue Tyr-165C of aspartate transcarbamoylase (EC 2.1.3.2) of Escherichia coli has been proposed to be involved in the transition from the T-state to the R-state upon binding of the bisubstrate analogue N-(phosphonacetyl)-L-aspartate. Site-specific mutagenesis has been used to substitute phenylalanine for tyrosine, thus maintaining the aromatic R-group but removing the charged hydroxyl moiety. This mutation dramatically altered the aspartate requirements for the holoenzyme but did not substantially affect the homotropic or heterotropic characteristics of the oligomer. The aspartate requirements for half-maximal saturation increased from 5.5 mM at pH 7.0 for the native holoenzyme to approximately 90 mM in the mutant enzyme. Nonetheless, estimates of the kinetic cooperativity index remained similar (Hill coefficients: Tyr-165C, n = 2.1; Phe-165C, n = 2.5). CTP inhibited both enzymes approximately 70% and ATP activated approximately 40% at the aspartate concentrations required for half-maximal saturation (5 and 90 mM, respectively). The maximal velocity of the mutant holoenzyme is almost identical to that of the wild-type enzyme. The phenylalanine substitution does not affect the stability of the holoenzyme to heat or mercurials, and the Vmax of the catalytic trimer was 444% greater than that of the holoenzyme. Upon dissociation of the wild-type native enzyme into catalytic trimers, the Vmax increased 450%. The Km for aspartate in the separated catalytic trimer is approximately 2-fold higher than for the native catalytic trimer (16.5 versus 8 mM at pH 7.0). It is clear from the data that although Tyr-165C is not directly involved in the active site of the enzyme, it does play a pivotal role in catalytic transitions of the holoenzyme. In addition, the homotropic and heterotropic characteristics of the enzyme do not seem to be altered by the substitution of phenylalanine for Tyr-165C in the E. coli aspartate transcarbamoylase, although other substitutions have been reported (Robey, E. H., and Schachman, H. K. (1984) J. Biol. Chem. 259, 11180-11183) which show more complex effects.     

Peptide-protein interaction markedly alters the functional properties of the catalytic subunit of aspartate transcarbamoylase.

Interaction of a 70-amino acid zinc-binding polypeptide from the regulatory chain of aspartate transcarbamoylase (ATCase) with the catalytic (C) subunit leads to dramatic changes in enzyme activity and affinity for ligand binding at the active sites. The complex between the polypeptide (zinc domain) and wild-type C trimer exhibits hyperbolic kinetics in contrast to the sigmoidal kinetics observed with the intact holoenzyme. Moreover, the Scatchard plot for binding N-(phosphonacetyl)-L-aspartate (PALA) to the complex is linear with a Kd corresponding to that evaluated for the holoenzyme converted to the relaxed (R) state. Additional evidence that the binding of the zinc domain to the C trimer converts it to the R state was attained with a mutant form of ATCase in which Lys 164 in the catalytic chain is replaced by Glu. As shown previously (Newell, J.O. & Schachman, H.K., 1990, Biophys. Chem. 37, 183-196), this mutant holoenzyme, which exists in the R conformation even in the absence of active site ligands, has a 50-fold greater affinity for PALA than the free C subunit. Adding the zinc domain to the C trimer containing the Lys 164-->Glu substitution leads to a 50-fold enhancement in the affinity for the bisubstrate analog yielding a value of Kd equal to that for the holoenzyme. A different mutant ATCase containing the Gln 231 to Ile replacement was shown (Peterson, C.B., Burman, D.L., & Schachman, H.K., 1992, Biochemistry 31, 8508-8515) to be much less active as a holoenzyme than as the free C trimer. For this mutant holoenzyme, the addition of substrates does not cause its conversion to the R state. However, the addition of the zinc domain to the Gln 231-->Ile C trimer leads to a marked increase in enzyme activity, and PALA binding data indicate that the complex resembles the R state of the holoenzyme. This interaction leading to a more active conformation serves as a model of intergenic complementation in which peptide binding to a protein causes a conformational correction at a site remote from the interacting surfaces resulting in activation of the protein. This linkage was also demonstrated by difference spectroscopy using a chromophore covalently bound at the active site, which served as a spectral probe for a local conformational change. The binding of ligands at the active sites was shown also to lead to a strengthening of the interaction between the zinc domain and the C trimer.     

The role of intersubunit interactions for the stabilization of the T state of Escherichia coli aspartate transcarbamoylase

Homotropic cooperativity in Escherichia coli aspartate transcarbamoylase results from the substrate-induced transition from the T to the R state. These two alternate states are stabilized by a series of interdomain and intersubunit interactions. The salt link between Lys-143 of the regulatory chain and Asp-236 of the catalytic chain is only observed in the T state. When Asp-236 is replaced by alanine the resulting enzyme exhibits full activity, enhanced affinity for aspartate, no cooperativity, and no heterotropic interactions. These characteristics are consistent with an enzyme locked in the functional R state. Using small angle x-ray scattering, the structural consequences of the D236A mutant were characterized. The unliganded D236A holoenzyme appears to be in a new structural state that is neither T, R, nor a mixture of T and R states. The structure of the native D236A holoenzyme is similar to that previously reported for another mutant holoenzyme (E239Q) that also lacks intersubunit interactions. A hybrid version of aspartate transcarbamoylase in which one catalytic subunit was wild-type and the other had the D236A mutation was also investigated. The hybrid holoenzyme, with three of the six possible interactions involving Asp-236, exhibited homotropic cooperativity, and heterotropic interactions consistent with an enzyme with both T and R functional states. Small angle x-ray scattering analysis of the unligated hybrid indicated that the enzyme was in a new structural state more similar to the T than to the R state of the wild-type enzyme. These data suggest that three of the six intersubunit interactions involving D236A are sufficient to stabilize a T-like state of the enzyme and allow for an allosteric transition.     

Differential scanning calorimetric studies of E. coli aspartate transcarbamylase. III. The denaturational thermodynamics of the holoenzyme with single-site mutations in the catalytic chain

Aspartate transcarbamylase (EC 2.1.3.2) from E. coli is a multimeric enzyme consisting of two catalytic subunits and three regulatory subunits whose activity is regulated by subunit interactions. Differential scanning calorimetric (DSC) scans of the wild-type enzyme consist of two peaks, each comprised of at least two components, corresponding to denaturation of the catalytic and regulatory subunits within the intact holoenzyme (Vickers et al., J. Biol. Chem. 253 (1978) 8493; Edge et al., Biochemistry 27 (1988) 8081). We have examined the effects of nine single-site mutations in the catalytic chains. Three of the mutations (Asp-100-Gly, Glu-86-Gln, and Arg-269-Gly) are at sites at the C1: C2 interface between c chains within the catalytic subunit. These mutations disrupt salt linkages present in both the T and R states of the molecule (Honzatko et al., J. Mol. Biol. 160 (1982) 219; Krause et al., J. Mol. Biol. 193 (1987) 527). The remainder (Lys-164-Ile, Tyr-165-Phe, Glu-239-Gln, Glu-239-Ala, Tyr-240-Phe and Asp-271-Ser) are at the C1: C4 interface between catalytic subunits and are involved in interactions which stabilize either the T or R state. DSC scans of all of the mutants except Asp-100-Gly and Arg-269-Gly consisted of two peaks. At intermediate concentrations, Asp-100-Gly and Arg-269-Gly had only a single peak near the Tm of the regulatory subunit transition in the holoenzyme, although their denaturational profiles were more complex at high and low protein concentrations. The catalytic subunits of Glu-86-Gln, Lys-164-Ile and Asp-271-Ser appear to be significantly destabilized relative to wild-type protein while Tyr-165-Phe and Tyr-240-Phe appear to be stabilized. Values of delta delta G degree cr, the difference between the subunit interaction energy of wild-type and mutant proteins, evaluated as suggested by Brandts et al. (Biochemistry 28 (1989) 8588) range from -3.7 kcal mol-1 for Glu-86-Gln to 2.4 kcal mol-1 for Tyr-165-Phe.     

Identification of a ternary complex between cAMP and a trimeric form of cAMP-dependent protein kinase

One of the intermediates involved in dissociation and reassociation of the subunits of the type II cAMP-dependent protein kinase has been characterized. This intermediate can be generated when the protein kinase is prepared from the isolated catalytic subunit (C) and the isolated regulatory subunit-[3H]cAMP complex (R2-[3H]cAMP4) by dialysis for 18 h followed by gel filtration. The intermediate, which could be separated from the holoenzyme and the isolated subunits by polyacrylamide gel electrophoresis, had an apparent molecular weight of 149,000, consistent with an R2C form. Following electrophoresis, measurements of R and bound nucleotide indicated that R2C was half-saturated with [3H]cAMP. The bound [3H]cAMP exhibited biphasic dissociation kinetics indicating that both types of cAMP binding sites were occupied. These findings suggested that the intermediate is R2C-cAMP2. This intermediate was not seen when the dialysis time was increased to 5 days, but could be observed when cAMP was added to the holoenzyme or when holoenzyme was mixed with R2cAMP4 and cAMP. The presence of two occupied cAMP binding sites on this intermediate suggests that there is minimal cooperativity between the two members of the regulatory subunit dimer, i.e. one member of the dimer binds 2 molecules of cAMP while the other binds C.     

Consequences of cAMP-binding site mutations on the structural stability of the type I regulatory subunit of cAMP-dependent protein kinase

The regulatory (R) subunit of cAMP-dependent protein kinase (cAPK) is a multidomain protein with two tandem cAMP-binding domains, A and B. The importance of cAMP binding on the stability of the R subunit was probed by intrinsic fluorescence and circular dichroism (CD) in the presence and absence of urea. Several mutants were characterized. The site-specific mutants R(R209K) and R(R333K) had defects in cAMP-binding sites A and B, respectively. R(M329W) had an additional tryptophan in domain B. Delta(260-379)R lacked Trp260 and domain B. The most destabilizing mutation was R209K. Both CD and fluorescence experiments carried out in the presence of urea showed a decrease in cooperativity of the unfolding, which also occurred at lower urea concentrations. Unlike native R, R(R209K) was not stabilized by excess cAMP. Additionally, CD revealed significant alterations in the secondary structure of the R209K mutant. Therefore, Arg209 is important not only as a contact site for cAMP binding but also for the intrinsic structural stability of the full-length protein. Introducing the comparable mutation into domain B, R333K, had a smaller effect on the integrity and stability of domain A. Unfolding was still cooperative; the protein was stabilized by excess cAMP, but the unfolding curve was biphasic. The R(M329W) mutant behaved functionally like the native protein. The Delta(260-379)R deletion mutant was not significantly different from wild-type RIalpha in its stability. Consequently, domain B and the interaction between Trp260 and cAMP bound to site A are not critical requirements for the structural stability of the cAPK regulatory subunit.