Catalytic Polypeptide Chains Research Articles

Assembly and regulation of acetylcholinesterase at the vertebrate neuromuscular junction

The collagen-tailed form of acetylcholinesterase (ColQ-AChE) is the major if not unique form of the enzyme associated with the neuromuscular junction (NMJ). This enzyme form consists of catalytic and non-catalytic subunits encoded by separate genes, assembled as three enzymatic tetramers attached to the three-stranded collagen-like tail (ColQ). This synaptic form of the enzyme is tightly attached to the basal lamina associated with the glycosaminoglycan perlecan. Fasciculin-2 is a snake toxin that binds tightly to AChE. Localization of junctional AChE on frozen sections of muscle with fluorescent Fasciculin-2 shows that the labeled toxin dissociates with a half-life of about 36 h. The fluorescent toxin can subsequently be taken up by the muscle fibers by endocytosis giving the appearance of enzyme recycling. Newly synthesized AChE molecules undergo a lengthy series of processing events before final transport to the cell surface and association with the synaptic basal lamina. Following co-translational glycosylation the catalytic subunit polypeptide chain interacts with several molecular chaperones, glycosidases and glycosyltransferases to produce a catalytically active enzyme that can subsequently bind to one of two non-catalytic subunits. These molecular chaperones can be rate limiting steps in the assembly process. Treatment of muscle cells with a synthetic peptide containing the PRAD attachment sequence and a KDEL retention signal results in a large increase in assembled and exportable AChE, providing an additional level of post-translational control. Finally, we have found that Pumilio2, a member of the PUF family of RNA-binding proteins, is highly concentrated at the vertebrate neuromuscular junction where it plays an important role in regulating AChE translation through binding to a highly conserved NANOS response element in the 3′-UTR. Together, these studies define several new levels of AChE regulation in electrically excitable cells.

In vivo assembly of aspartate transcarbamoylase from fragmented and circularly permuted catalytic polypeptide chains.

Previous studies on Escherichia coli aspartate transcarbamoylase (ATCase) demonstrated that active, stable enzyme was formed in vivo from complementing polypeptides of the catalytic (c) chain encoded by gene fragments derived from the pyrBI operon. However, the enzyme lacked the allosteric properties characteristic of wild-type ATCase. In order to determine whether the loss of homotropic and heterotropic properties was attributable to the location of the interruption in the polypeptide chain rather than to the lack of continuity, we constructed a series of fragmented genes so that the breaks in the polypeptide chains would be dispersed in different domains and diverse regions of the structure. Also, analogous molecules containing circularly permuted c chains with altered termini were constructed for comparison with the ATCase molecules containing fragmented c chains. Studies were performed on four sets of ATCase molecules containing cleaved c chains at positions between residues 98 and 99, 121 and 122, 180 and 181, and 221 and 222; the corresponding circularly permuted chains had N termini at positions 99, 122, 181, and 222. All of the ATCase molecules containing fragmented or circularly permuted c chains exhibited the homotropic and heterotropic properties characteristic of the wild-type enzyme. Hill coefficients (n(H:)) and changes in them upon the addition of ATP and CTP were similar to those observed with wild-type ATCase. In addition, the conformational changes revealed by the decrease in sedimentation coefficient upon the addition of a bisubstrate analog were virtually identical to that for the wild-type enzyme. Differential scanning calorimetry showed that neither the breakage of the polypeptide chains nor the newly formed covalent bond between the termini in the wild-type enzyme had a significant impact on the thermal stability of the assembled dodecamers. The studies demonstrate that continuity of the polypeptide chain within structural domains is not essential for the assembly, activity, and allosteric properties of ATCase.

Purification and characterization of the first bacterial laccase in the rhizospheric bacterium Azospirillum lipoferum

Azospirillum lipoferum is a soil bacterium known for its ability to colonize roots and to promote plant growth. Recently, a laccase-like polyphenol oxidase activity has been reported in a strain isolated from the rhizosphere of rice. Purification to apparent homogeneity of A. lipoferum laccase was achieved after cell disruption by sonication, protein solubilization by Triton X-100, acetone and protamine precipitation, hydroxyapatite column chromatography in the presence of urea (5 M) and NaCl (2 M), and finally by conventional hydroxyapatite column chromatography. After this final step, laccase was in an inactive form but could be reactivated by adding sodium dodecyl sulfate (SDS). Based on gel electrophoresis results, we suggest that native A. lipoferum laccase is composed of a catalytic polypeptide chain with low molecular mass (16.3 kDa) and one or two regulatory/structural chains with high molecular mass (81.5 kDa). The purified enzyme was found to be thermostable up to 70°C for 10 min, had an optimal pH of 6.0, and was inhibited by tropolone, a known inhibitor of metal-containing enzymes. Although the natural substrate of this enzyme is unknown, its biochemical characterization may facilitate further investigations on the ecological role of this laccase in the process of root colonization by A. lipoferum.

In vivo formation of allosteric aspartate transcarbamoylase containing circularly permuted catalytic polypeptide chains: implications for protein folding and assembly.

Because the N- and C-terminal amino acids of the catalytic (c) polypeptide chains of Escherichia coli aspartate transcarbamoylase (ATCase) are in close proximity to each other, it has been possible to form in vivo five different active ATCase variants in which the terminal regions of the wild-type c chains are linked in a continuous polypeptide chain and new termini are introduced elsewhere in either of the two structural domains of the c chain. These circularly permuted (cp) chains were produced by constructing tandem pyrB genes, which encode the c chain of ATCase, followed by application of PCR. Chains expressed in this way assemble efficiently in vivo to form active, stable ATCase variants. Three such variants have been purified and shown to have the kinetic and physical properties characteristic of wild-type ATCase composed of two catalytic (C) trimers and three regulatory (R) dimers. The values of Vmax for cpATCase122, cpATCase222, and cpATCase281 ranged from 16-21 mumol carbamoylaspartate per microgram per h, compared with 15 for wild-type ATCase, and the values for K0.5 for the variants were 4-17 mM aspartate, whereas wild-type ATCase exhibited a value of 6 mM. Hill coefficients for the three variants varied from 1.8 to 2.1, compared with 1.4 for the wild-type enzyme. As observed with wild-type ATCase, ATP activated the variants containing the circularly permuted chains, as shown by the lowering of K0.5 for aspartate and a decrease in the Hill coefficient (nH). In contrast, CTP caused both an increase in K0.5 and nH for the variants, just as observed with wild-type ATCase. Thus, the enzyme containing the permuted chains with widely diverse N- and C-termini exhibited the homotropic and heterotropic effects characteristic of wild-type ATCase. The decrease in the sedimentation coefficient of the variants caused by the binding of the bisubstrate ligand N-(phosphonacetyl)-L-aspartate (PALA) was also virtually identical to that obtained with wild-type ATCase, thereby indicating that these altered ATCase molecules undergo the analogous ligand-promoted allosteric transition from the taut (T) state to the relaxed (R) conformation. These ATCase molecules with new N- and C-termini widely dispersed throughout the c chains are valuable models for studying in vivo and in vitro folding of polypeptide chains.

Structural similarity between ornithine and aspartate transcarbamoylases of Escherichia coli: implications for domain switching.

Each catalytic (c) polypeptide chain of Escherichia coli aspartate transcarbamoylase (ATCase) is composed of two globular domains connected by two interdomain helices. Helix 12, near the C-terminus, extends from the second domain back through the first domain, bringing the two termini close together. This helix is of critical importance for the assembly of a stable enzyme. The trimeric E. coli enzyme ornithine transcarbamoylase (OTCase) is proposed to be similar in tertiary and quaternary structure to the ATCase trimer and has a predicted alpha-helical segment near its C-terminus. In our companion paper, we have shown that this putative helix is essential for OTCase folding and assembly (Murata L, Schachman HK, 1996, Protein Sci 5:709-718). Here, the similarity between OTCase and the ATCase trimer, which are 32% identical in sequence, was tested further by the construction of several chimeras in which various structural elements were switched between the enzymes by genetic techniques. These elements included the two globular domains and regions containing the C-terminal helices. In contrast to results reported previously (Houghton J, O'Donovan G, Wild J, 1989, Nature 338:172-174), none of the chimeric proteins exhibited in vivo activity and all were insoluble when overexpressed. Attempts to make hybrid trimers composed of c chains from ATCase and OTCase were also unsuccessful. These results underscore the complexities of specific intrachain and interchain side-chain interactions required to maintain tertiary and quaternary structures in these enzymes.

Aspartate transcarbamoylase containing circularly permuted catalytic polypeptide chains.

Based on the demonstration that active enzyme is formed in vitro and in vivo from polypeptide fragments of the catalytic chains of aspartate transcarbamoylase (ATCase; EC 2.1.3.2) and the evidence that NH2 and COOH termini of wild-type chains are in close proximity, we constructed altered genes to determine whether circularly permuted catalytic chains could fold and assemble into active catalytic trimers. Two slightly different genetic constructs led to the expression in good yield of circularly permuted catalytic chains, which associated in vivo into active trimers. They, in turn, combined in vitro with wild-type regulatory dimers to form ATCase-like molecules. Both polypeptide chains began at residue 235 in a different domain from the NH2 terminus of wild type and had an overlapping sequence of eight residues at the COOH terminus. One had a six-amino-acid linker, and the other had a deletion of four residues. Enzymes containing rearranged chains were similar to their wild-type counterparts in physical properties. Whereas values of Vmax were close to those of wild-type trimers and ATCase, the Km values were more than 10-fold greater. Also the allosteric properties characteristic of wild-type ATCase were lacking in the enzymes containing permuted chains. Denaturation of trimers by urea was reversible, and recovery of activity in both rate and yield was comparable to that of wild-type trimers. The experiments demonstrate that folding of chains into clearly defined domains and the assembly of active, thermodynamically stable oligomers are not dependent on the positions of NH2 and COOH termini; the folded structures are a consequence of the final sequence and not the order of biosynthetic addition of amino acids.

In vivo formation of active aspartate transcarbamoylase from complementing fragments of the catalytic polypeptide chains.

Despite the complexity of Escherichia coli aspartate transcarbamoylase (ATCase), composed of 12 polypeptide chains organized as two catalytic (C) trimers and three regulatory (R) dimers, it is possible to form active stable enzyme in vivo even with fragmented catalytic (c) chains. Based on the observation that chymotryptic digestion of the C trimers yields an active protein that can be dissociated into fragmented chains and then reconstituted in high yield, genetically engineered plasmids carrying the genes encoding each of the fragments were constructed. When the N-terminal peptide (residues 1-242) and the C-terminal peptide (residues 235-310) were expressed separately, each incomplete polypeptide chain was found in the insoluble fraction of the individual cell extracts. Mixing the two insoluble pellets in 6.5 M urea, followed by a 10-fold dilution in buffer, led to the formation of active C trimers composed of incomplete polypeptide chains with an 8-amino acid redundancy. When the two partial genes were linked into a single transcriptional unit separated by a 15-nucleotide untranslated region containing a sequence for ribosome binding, the cells produced high yields of active C trimers composed of the incomplete, partially overlapping chains. The resulting protein, purified as C trimers or as holoenzyme formed by the addition of R subunits, has a specific activity (Vmax) only slightly less than that of the wild-type C trimer and ATCase. However, Km for aspartate exhibited by the C trimer composed of fragmented chains is more than 10-fold larger than that of the wild-type trimer. The holoenzyme formed from the C trimer containing the coexpressed peptides is devoid of cooperativity with a Hill coefficient of 1.0, as contrasted to wild-type ATCase for which the Hill coefficient is 1.7. Km for aspartate as well as Kd for the binding of the bisubstrate analog N-(phosphonacetyl)-L-aspartate are significantly higher than the analogous values for wild-type ATCase. Sedimentation velocity experiments indicate that the holoenzyme containing the incomplete chains has a conformation analogous to that of the R state of wild-type ATCase.

Asparate transcarbamoylase: Current opinion in structural biology 1993, 3:960–967

Various approaches have been used to investigate the allosteric transition of aspartate transcarbamoylase. Mutant forms involving amino acid substitutions in both catalytic and regulatory polypeptide chains have yielded valuable insights. Catalytic mechanisms have been examined using isotope effects as probes, and the formation of the enzyme in vitro and in vivo from polypeptide fragments has stimulated interest in the mechanism of interallelic complementation. A zinc-containing polypeptide fragment of the regulatory chain is shown to interact with the catalytic trimer, thereby forming a stable complex with markedly altered catalytic properties.

Negative complementation in aspartate transcarbamylase. Analysis of hybrid enzyme molecules containing different arrangements of polypeptide chains from wild-type and inactive mutant catalytic subunits.

A comprehensive set of hybrid molecules of aspartate transcarbamylase (ATCase) from Escherichia coli has been constructed of wild-type and mutationally altered catalytic chains. The mutant enzymes that were virtually devoid of activity contained a replacement of Gly-128 in the catalytic polypeptide chains by either Asp or Arg. The kinetic properties of these hybrid enzyme-like molecules were analyzed to evaluate the basis for the unusual quaternary constraint demonstrated by an intersubunit hybrid containing one wild-type catalytic subunit, one inactive mutant subunit (containing the Gly to Asp replacement), and three wild-type regulatory subunits. A similar intersubunit hybrid was constructed from the wild-type catalytic subunit and the mutant in which Gly-128 was replaced by Arg, and it too demonstrated a pronounced decrease in activity relative to that expected for a hybrid containing three active sites. Moreover, neither of these hybrid holoenzymes exhibited the cooperativity with respect to aspartate that is characteristic of wild-type ATCase. In contrast, hybrid holoenzymes containing at least one wild-type chain in each catalytic subunit showed cooperativity. Also, hybrid enzymes containing different arrangements of five, four, three, or two wild-type catalytic chains with an appropriate complement of mutant chains had specific activities proportional to the number of wild-type chains in the holoenzymes. Exceptions were observed only in hybrids in which one of the two subunits in the holoenzyme was composed completely of mutant catalytic chains. For these hybrids the negative complementation was manifested as a much lower enzyme activity than expected from the number of wild-type chains in the enzyme and the loss of cooperativity. Thus, the activity and allosteric properties of these hybrids is dependent on the arrangement of catalytic chains in the holoenzyme, in contrast to results obtained for hybrids containing native and chemically modified catalytic chains. Intrasubunit hybrid catalytic trimers containing one or two wild-type chains exhibited one-third and two-thirds the activity of the intact wild-type catalytic subunit, respectively, indicating the dominant negative effect that was seen in intersubunit hybrid holoenzymes is absent within trimers.

Role of a carboxyl-terminal helix in the assembly, interchain interactions, and stability of aspartate transcarbamoylase.

The six individual catalytic polypeptide chains within the two catalytic trimers of Escherichia coli aspartate transcarbamoylase (ATCase; EC 2.1.3.2) are folded into two discrete structural domains interconnected in part by helix 12, which comprises residues 285-305 and is located near the carboxyl terminus of the chain. The essential role of this helix in folding of the chains and their assembly into ATCase was demonstrated by introducing a stop codon at the position corresponding to amino acid 284, 291, or 299. Cells containing these mutations are pyrimidine auxotrophs lacking ATCase-like protein in cell extracts. In contrast, stable active enzyme is formed from chains truncated at position 306 or 307, showing that all 310 amino acids are not required for assembly. Replacements of Gln-288, Asn-291, Arg-296, and Ala-298 were introduced to assess the effect of alterations within helix 12 on protein stability. Stability of the trimers was measured both by differential scanning microcalorimetry and by the rate of exchange of chains at 4 degrees C when mutant trimers were incubated with succinylated wild-type trimers. Melting temperatures of the mutant trimers spanned a range of more than 20 degrees C, with a few higher and others lower than that of wild-type trimers. Large changes in interchain interaction energies were observed for the trimers, but there was no direct correlation between the ease of dissociation of the trimers and their thermal stability. Calorimetry on the mutant holoenzymes revealed alterations in the interactions between trimers and regulatory subunits within the intact enzymes. The striking changes in stability of both trimers and holoenzymes demonstrated that effects of relatively localized amino acid replacements in helix 12 are manifested by indirect global alterations propagated throughout the structure.

Amino acid substitutions which stabilize aspartate transcarbamoylase in the R state disrupt both homotropic and heterotropic effects

We have used site-specific amino acid substitutions to investigate the linkage between the allosteric properties of arpartate transcarbamoylase and the global conformational transition exhibited by the enzyme upon binding active-site ligands. Two mutationally altered enzymes in which an amino acid substitution had been introduced at a single position in the catalytic polypeptide chain (Lys-164----Glu and Glu-239----Lys) and a third species harboring both of these substitutions (Lys-164:Glu-239----Glu:Lys) were constructed. Sedimentation velocity difference studies were performed in order to assess the effects of the amino acid substitutions on the quaternary structure of the holoenzyme in the absence and presence of various active-site ligands, including the bisubstrate analog, N-(phosphonacetyl)-L-aspartate (PALA), which has been shown previously to promote the allosteric transition. In the absence of ligand, two of the mutationally altered enzymes, Lys-164----Glu and Lys-164:Glu-239----Glu:Lys, existed in the R conformation, isomorphous with that of the PALA-liganded wild-type holoenzyme. These enzymes exhibited no conformational change upon binding PALA. The unliganded Glu-239----Lys enzyme had an average sedimentation coefficient intermediate between that of the unliganded and PALA-liganded states of the wild-type enzyme which could be accounted for in terms of a mixture of T- and R-state molecules. This mutant enzyme was converted to the fully swollen conformation upon binding PALA, phosphate or carbamoyl phosphate. The allosteric properties of the mutationally altered species were investigated by PALA-binding studies and by steady-state enzyme kinetics. In each case, the mutationally altered enzymes were devoid of both homotropic and heterotropic effects, supporting the premise that the allosteric properties of the wild-type enzyme are linked to a ligand-promoted change in quaternary structure.

Recent evolutionary divergence of plasma prekallikrein and factor XI

The evolution in mammals of the zymogens of the contact activation system of coagulation (factor XII, prekallikrein and factor XI) has been investigated. The NH2-terminal sequences of human plasma prekallikrein and the heavy and light chains of kallikrein have been determined and compared with those of bovine prekallikrein and of human and bovine factors XII and XI. The human and bovine NH 2-terminal sequences of the light chains (catalytic polypeptide) show striking similarities both among themselves and with those of the catalytic polypeptide chains of other coagulation and digestive proteases, indicating a common origin. Comparison of the NH 2-terminal sequences of human prekallikrein with those of the bovine prekallikrein and human and bovine factors XIa and XIIa indicates a common origin of the heavy chain of kallikrein and factor XIa, different from that of either factor XIIa or other known amino acid sequences. Ancestral sequences for human and bovine prekallikrein and factor XI, deduced by genetic analysis of the minimum number of base changes indicate that the NH 2-terminus of prekallikrein and factor XI have evolved at about the same rate. The estimated time for the gene duplication was about 124 million years ago, a value consistent with the age of the mammals.

Regulation of aspartate transcarbamoylase synthesis in Escherichia coli: analysis of deletion mutations in the promoter region of the pyrBI operon.

The catalytic and regulatory polypeptide chains of Escherichia coli aspartate transcarbamoylase are encoded by the pyrB and pyrI genes, respectively, which constitute a single transcriptional unit in the pyrBI operon. The DNA sequence immediately preceding the first structural gene, pyrB, contains a short open reading frame that could encode a 44-amino acid leader peptide and a (G+C)-rich region of dyad symmetry followed by eight thymidine residues. Synthesis of the enzyme is negatively controlled at the level of transcription depending on the cellular level of UTP, and an attenuation mechanism has been proposed to account for the 70-fold increase in pyrBI expression on pyrimidine starvation. The potential role of the dyad and eight thymidines as an attenuator was tested with a plasmid containing the promoter region of the pyrBI operon upstream of the galK coding sequence. When cells containing this plasmid, pPYRB10, were grown in a medium low in uracil, there was an 83-fold increase in galactokinase activity compared with the same cells grown at high uracil levels. This regulation is similar to that for aspartate transcarbamoylase synthesis in cells depleted of pyrimidines. Deletions constructed in the promoter region of pPYRB10 from the 3' side produced one plasmid that retained normal control of galK expression and five that exhibited greatly reduced regulation. Nucleotide sequence determination showed that the one deletion mutation that was functionally similar to the wild-type plasmid contained the entire region of dyad symmetry, including the eight thymidines. The plasmids with more extensive deletions lacked the region with dyad symmetry and the eight thymidines. One of the deletion mutants that exhibited very low levels of regulation lacks the entire sequence coding for the putative leader peptide up to the major promoter. The results demonstrating the crucial role of a 19-nucleotide sequence (from -33 to -15) support an attenuation model but indicate that other mechanisms also contribute to the regulation of the pyrBI operon.

Monomeric solubilized sarcoplasmic reticulum Ca pump protein: demonstration of Ca binding and dissociation coupled to ATP hydrolysis.

The sarcoplasmic reticulum Ca-pump protein can be solubilized in monomeric form in nonionic detergents with full retention of ATPase activity. It is impossible to prove directly that coupling between ATP hydrolysis and Ca2+ transport is maintained in the soluble state, because separate compartments for Ca2+ uptake and Ca2+ discharge are required to demonstrate this, but here we provide strong indirect evidence that coupling in fact persists, in both the forward and reverse directions of the normal pump cycle. Demonstration of coupling in the forward direction makes use of the fact that the solubilized protein is structurally labile in the absence of bound Ca2+. Loss of activity accompanying ATP hydrolysis in solution is quantitatively consistent with sequential Ca2+ binding and dissociation during the hydrolysis cycle. Coupling in the reverse direction is demonstrated by the dependence of ATP synthesis on Ca2+ concentration. Although substantial differences between solubilized (monomeric) and membrane-bound protein are shown to exist, as previously reported, they do not affect the main conclusion from this work, which is that the molecular machinery for free-energy coupling in active Ca2+ transport is an inherent property of each individual catalytic polypeptide chain of the pump protein.

Assembly of the aspartate transcarbamoylase holoenzyme from transcriptionally independent catalytic and regulatory cistrons.

The cistrons encoding the regulatory and catalytic polypeptides of aspartate transcarbamoylase (EC 2.1.3.2) from Escherichia coli K-12 have been cloned separately on plasmids from different incompatability groups. The catalytic cistron (pyrB) was carried by pACYC184 and expressed from its own promoter, whereas the regulatory cistron was expressed from the lac po of pBH20. The catalytic polypeptide chains assembled into enzymatically active trimers (c3) in vivo when expressed in the absence of regulatory subunits. Similarly, the regulatory polypeptide chains assembled into regulatory dimers (r2) in vivo in the absence of catalytic subunits. When cellular extracts containing regulatory dimers and catalytic trimers synthesized in separate cells were combined in vitro, partial spontaneous holoenzyme assembly occurred. When pyrB and pyrI were expressed from transcriptionally independent cistrons in the same cell, all detectable catalytic polypeptides were incorporated into the functional aspartate transcarbamoylase holoenzyme, 2(c3):3(r2). Thus, it is clear that the in vivo assembly of ATCase holoenzyme is a direct, spontaneous process involving the association of preformed regulatory subunits (r2) and catalytic subunits (c3). This procedure provides a general method for the construction of hybrid aspartate transcarbamoylase in vivo and may be applicable to other oligomeric enzymes constructed from different polypeptides.

The organization and regulation of the pyrBI operon in E. coli includes a rho-independent attenuator sequence.

1. The two polypeptide chains that comprise aspartate carbamoyltransferase in Escherichia coli are encoded by adjacent cistrons expressed in the order, promoter-leader-catalytic cistron-regulatory cistron (p-leader-pyrBI). These two cistrons and their single control region have been cloned as a 2,800 base pair (bp) fragment (The minimal coding requirement for the catalytic and regulatory polypeptides is about 1,350 bp plus control regions). The genes contained by this fragment are subject to normal repression controls and thus possess the intact control regions. 2. By deleting an internal fragment with specific restriction endonucleases, it was possible to construct shortened fragments which no longer produced the regulatory polypeptide. In these cases the expression of the catalytic cistron was normal and subject to repression upon growth in the presence of uracil. Since the pyrB cistron retained transcriptional control, the regulatory polypeptide was not required for expression or control of the catalytic cistron. As expected, the catalytic trimer (Mr = 100,000 daltons) from these deletion mutants had no effector response nor did it exhibit homotropic kinetics for aspartate. The enzyme was identical to the c3 trimer purified from the native holoenzyme by neohydrin dissociation. 3. Insertion of Mu d1(lac Apr) into the structural region of pyrB had a negative effect on the expression of pyrI. This supports the idea that the catalytic and regulatory polypeptide chains of aspartate carbamoyl-transferase are encoded by a single bicistronic operon. Detailed restriction analysis of the cloned pyrBI region has produced a genetic map of restriction sites which is colinear with the published amino acid sequences of the two polypeptides. These maps indicate that the 3'-terminus of the catalytic cistron is adjacent to the 5'-terminus of the regulatory cistron and separated by 10-20 bp. 4. DNA sequence analysis of the 5'-proximal regions of pyrBI revealed that an extensive leader sequence separated the promoter and first structural gene pyrB. This leader of approximately 150 bp contains an attenuator sequence and the translational signals required for the production of a leader polypeptide of 43 amino acids. In this paper we describe the structural organization of pyrBI, and provide a detailed analysis of its regulatory region including its DNA sequence.

Assembly of catalytic subunits of aspartate transcarbamoylase from Escherichia coli

Although extensive studies have been conducted on the assembly of the allosteric enzyme, aspartate transcarbamoylase (ATCase) from isolate, intact catalytic (C) and regulatory (R) subunits, there has been little research on the formation of these subunits from individual catalytic (c) and regulatory (r) polypeptide chains. Such studies would be useful for evaluating the strengths of the interchain bonding domains within the subunits just as earlier experiments provided valuable data regarding interactions between the subunits in ATCase. The intact enzyme comprising two C trimers and three R dimers is designated as C/sub 2/R/sub 3/ or c/sub 6/r/sub 6/.

Communication between catalytic subunits in hybrid aspartate transcarbamoylase molecules: effect of ligand binding to active chains on the conformation of unliganded, inactive chains.

In the regulatory enzyme aspartate transcarbamoylase (aspartate carbamoyltransferase; carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) of Escherichia coli, the six catalytic polypeptide chains are arranged as two distinct catalytic trimers "crosslinked" by three regulatory dimers. Because in allosteric proteins it is assumed that the binding of a ligand to one site promotes a conformational change that affects the subsequent binding to other sites in the oligomeric protein, it was of interest to determine directly whether the effects of ligand binding to chains in one catalytic subunit are "communicated" to unliganded chains in the other subunit. Accordingly, hybrid enzyme molecules were constructed containing sensitive chromophores on the three inactive catalytic chains in one subunit along with an active catalytic subunit and three native regulatory subunits. The derivative exhibited the allosteric properties characteristic of the native enzyme. Communication between the two catalytic subunits was demonstrated by spectral measurements showing that the effects of ligand binding to the three active chains are propagated to the chromophores on the unliganded, inactive chains in the other subunit. Moreover, the change in the tertiary structure of the unliganded catalytic chains is tightly linked to the alteration in the quaternary structure.

Purification and characterization of a mutant aspartate transcarbamoylase lacking enzyme activity.

A mutant form of aspartate transcarbamoylase (ATCase) from Escherichiu coli which lacks catalytic activity has been purified from a strain which produces large quantities of the inactive protein. Analysis of hybrid enzymes composed of subunits from the mutant and wild type enzyme showed that the mutation was in the structural gene for the catalytic subunits. No alteration of the regulatory subunits was detected. The mutant enzyme had the same sedimentation coefficient, 11.7 S, as the wild type enzyme, and the molecular weights of the individual catalytic and regulatory polypeptide chains of the mutant enzyme evaluated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were the same as the wild type enzyme. Amino acid analysis of the catalytic and regulatory subunits showed no differences, within 3% error, between the two forms of the enzyme. Electrophoresis in polyacrylamide gels containing 8 M urea indicated that the catalytic polypeptide chains of the mutant had a charge difference of -1 relative to those of the wild type enzyme. Ligand binding studies were performed by ultraviolet difference spectroscopy. Although the mutant catalytic subunit bound the substrate, carbamoylphosphate, the dissociation constant, 150 pM, was much greater than that for the wild type catalytic subunit, 9 pM. Neither the substrate analog, succinate, nor the bisubstrate analog, N-(phosphonacetyl)+aspartate (PALA), was bound by the mutant protein, whereas both of these ligands are bound with high affinity by the wild type enzyme. Preparations of the mutant enzyme do not contain a regulatory subunit-deficient species which is usually found in preparations of the wild type enzyme. Moreover, this species could not be generated by limited degradation of the mutant ATCase by mercurials nor by reconstitution of mutant enzyme from mixtures of catalytic and regulatory subunits with the former in excess. These results indicate that the

Communication between dissimilar subunits in aspartate transcarbamoylase: Effect of inhibitor and activator on the conformation of the catalytic polypeptide chains

Although local, direct effects of ligand binding to proteins are readily differentiated conceptually from gross, indirect conformational changes in regions of the protein remote from the site of binding, it has been difficult experimentally to distinguish between them. In oligomeric proteins, for example, the binding of ligands to one chain may cause a conformational change in the unliganded chains, but many physical chemical probes are not sufficiently discriminating to demonstrate where the change occurred. Evidence has been lacking as to whether the inhibitor, CTP, or the activator, ATP, in binding to the regulatory chains of the allosteric enzyme aspartate transcarbamoylase (aspartate carbamoyltransferase; carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) causes a conformational change that is propagated throughout the enzyme to the catalytic chains. To demonstrate this "communication" of effects of binding at one site on the conformation of other polypeptide chains, we constructed molecules containing native regulatory subunits and enzymically active nitrated catalytic subunits having one sensitive nitrotyrosyl chromophore per polypeptide chain. These hybrid molecules exhibited the characteristic regulatory properties of the native enzyme. Upon the addition of CTP the population of molecules was shifted toward the constrained or T state, as shown by the change in the sedimentation coefficient and the altered enzyme kinetics. Moreover, there was a decrease in absorbance at 430 nm due to the altered environment of the nitrated catalytic polypeptide chains. In contrast, ATP caused a shift toward the relaxed or R conformation, and the absorbance due to the nitrotyrosyl residues was increased. Different types of experiments indicated that the modified enzyme molecules are in a preexisting equilibrium that is perturbed by CTP or ATP; the resulting conformational changes in the nitrated catalytic subunits are detected by opposite alterations in their absorbance spectrum.