Active oligomeric states of pyruvate decarboxylase and their functional characterization.

Homomeric pyruvate decarboxylase (E.C 4.1.1.1) from yeast consists of dimers and tetramers under physiological conditions, a K(d) value of 8.1 microM was determined by analytical ultracentrifugation. Dimers and monomers of the enzyme could be populated by equilibrium denaturation using urea as denaturant at defined concentrations and monitored by a combination of optical (fluorescence and circular dichroism) and hydrodynamic methods (analytical ultracentrifugation). Dimers occur after treatment with 0.5 M urea, monomers with 2.0 M urea independent of the protein concentration. The structured monomers are catalytically inactive. At even higher denaturant concentrations (6 M urea) the monomers unfold. The contact sites of two monomers in forming a dimer as the smallest enzymatically active unit are mainly determined by aromatic amino acids. Their interactions have been quantified both by structure-theoretical calculations on the basis of the X-ray crystallography structure, and experimentally by binding of the fluorescent dye bis-ANS. The contact sites of two dimers in tetramer formation, however, are mainly determined by electrostatic interactions. Homomeric pyruvate decarboxylase (PDC) is activated by its substrate pyruvate. There was no difference in the steady-state activity (specific activity) between dimers and tetramers. The activation kinetics of the two oligomeric states, however, revealed differences in the dissociation constant of the regulatory substrate (K(a)) by one order of magnitude. The tetramer formation is related to structural consequences of the interaction transfer in the activation process causing an improved substrate utilization.     

Changes in catalytic activity and association state of pyruvate carboxylase which are dependent on enzyme concentration

The specific activity of chicken liver pyruvate carboxylase has been shown to decrease with decreasing enzyme concentration, even at 100 microM, which is close to the estimated physiological concentration. The kinetics of the loss of enzyme specific activity following dilution were biphasic. Incubation of dilution-inactivated enzyme with ATP, acetyl CoA, Mg2+ + ATP or, to a lesser degree, with Mg2+ alone resulted in a high degree of reactivation, while no reactivation occurred in the presence of pyruvate. The association state of the enzyme before, during, and after dilution inactivation has been assessed by gel filtration chromatography. These studies indicate that on dilution, there is dissociation of the catalytically active tetrameric enzyme species into inactive dimers. Reactivation of the enzyme resulted in reassociation of enzymic dimers into tetramers. The enzyme was shown to form high molecular weight aggregates at high enzyme concentrations.     

The crystal structure of pyruvate decarboxylase from Kluyveromyces lactis. Implications for the substrate activation mechanism of this enzyme

The crystal structure of pyruvate decarboxylase from Kluyveromyces lactis has been determined to 2.26 A resolution. Like other yeast enzymes, Kluyveromyces lactis pyruvate decarboxylase is subject to allosteric substrate activation. Binding of substrate at a regulatory site induces catalytic activity. This process is accompanied by conformational changes and subunit rearrangements. In the nonactivated form of the corresponding enzyme from Saccharomyces cerevisiae, all active sites are solvent accessible due to the high flexibility of loop regions 106-113 and 292-301. The binding of the activator pyruvamide arrests these loops. Consequently, two of four active sites become closed. In Kluyveromyces lactis pyruvate decarboxylase, this half-side closed tetramer is present even without any activator. However, one of the loops (residues 105-113), which are flexible in nonactivated Saccharomyces cerevisiae pyruvate decarboxylase, remains flexible. Even though the tetramer assemblies of both enzyme species are different in the absence of activating agents, their substrate activation kinetics are similar. This implies an equilibrium between the open and the half-side closed state of yeast pyruvate decarboxylase tetramers. The completely open enzyme state is favoured for Saccharomyces cerevisiae pyruvate decarboxylase, whereas the half-side closed form is predominant for Kluyveromyces lactis pyruvate decarboxylase. Consequently, the structuring of the flexible loop region 105-113 seems to be the crucial step during the substrate activation process of Kluyveromyces lactis pyruvate decarboxylase.     

Control of the reactivation kinetics of homodimeric triosephosphate isomerase from unfolded monomers

Homodimeric triosephosphate isomerases from Trypanosoma cruzi (TcTIM) and Trypanosoma brucei (TbTIM) have markedly similar catalytic properties and 3-D structures; their overall amino acid sequence identity is 68% and 85% in their interface residues. Nonetheless, active dimer formation from guanidinium chloride unfolded monomers is faster and more efficient in TcTIM than in TbTIM. The enzymes thus provide a unique opportunity for exploring the factors that control the formation of active dimers. The kinetics of reactivation at different protein concentrations showed that the process involved three reactions: monomer folding, association of folded monomers, and a transition from inactive to active dimers. The rate constants of the reactions indicated that, at relatively low protein concentrations, the rate-limiting step of reactivation was the association reaction; at high protein concentrations the transition of inactive to active dimers was rate limiting. The rates of the latter two reactions were higher in TcTIM than in TbTIM. Studies with a mutant of TcTIM that had the interface residues of TbTIM showed that the association rate constant was similar to that of TbTIM. However, the rate of the transition from inactive to active dimers was close to that of TcTIM; thus, this transition depends on the noninterfacial portion of the enzymes. When unfolded monomers of TcTIM and TbTIM were allowed to reactivate together, TcTIM, the hybrid, and TbTIM were formed in a proportion of 1:0.9:0.2. This distribution suggests that, in the hybrid, the characteristics of the TcTIM monomers influence the properties of TbTIM monomers.     

Purification and characterization of indolepyruvate decarboxylase. A novel enzyme for indole-3-acetic acid biosynthesis in Enterobacter cloacae.

Indolepyruvate decarboxylase, a key enzyme for indole-3-acetic acid biosynthesis, was found in extracts of Enterobacter cloacae. The enzyme catalyzes the decarboxylation of indole-3-pyruvic acid to yield indole-3-acetaldehyde and carbon dioxide. The enzyme was purified to apparent homogeneity from Escherichia coli cells harboring the genetic locus for this enzyme obtained from E. cloacae. The results of gel filtration experiments showed that indolepyruvate decarboxylase is a tetramer with an M(r) of 240,000. In the absence of thiamine pyrophosphate and Mg2+, the active tetramers dissociate into inactive monomers and dimers. However, the addition of thiamine pyrophosphate and Mg2+ to the inactive monomers and dimers results in the formation of active tetramers. These results indicate that the thiamine pyrophosphate-Mg2+ complex functions in the formation of the tetramer, which is the enzymatically active holoenzyme. The enzyme exhibited decarboxylase activity with indole-3-pyruvic acid and pyruvic acid as substrates, but no decarboxylase activity was apparent with L-tryptophan, indole-3-lactic acid, beta-phenylpyruvic acid, oxalic acid, oxaloacetic acid, and acetoacetic acid. The Km values for indole-3-pyruvic acid and pyruvic acid were 15 microM and 2.5 mM, respectively. These results indicate that indole-3-acetic acid biosynthesis in E. cloacae is mediated by indolepyruvate decarboxylase, which has a high specificity and affinity for indole-3-pyruvic acid.     

Dissociation and reassociation of prolyl 4-hydroxylase subunits after cross-linking of monomers

1. Incubation of prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate : oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2) with H2O2 leads to a decrease of 50% in the specific activity of enzyme tetramers, followed by dissociation into inactive dimers in which the monomers are covalently cross-linked by S-S bridge formation. 2. Incubation of the enzyme with K3Fe(CN)6 leads to a comparable decrease in activity of enzyme tetramers. Addition of urea leads to dissociation into inactive dimers with similarly cross-linked monomers. 3. Removal of the dissociating agent leads to reassociation of cross-linked dimers to tetramers and to about 50% reactivation. The enzyme is further reactivated by preincubation with dithiothreitol. 4. Dissociation of the enzyme with dithiothreitol, urea or LiCl, or at low pH (4.15) produces inactive monomers, which could not be reassociated.     

Single-Molecule characterization of oligomerization kinetics and equilibria of the tumor suppressor p53

The state of oligomerization of the tumor suppressor p53 is an important factor in its various biological functions. It has a well-defined tetramerization domain, and the protein exists as monomers, dimers and tetramers in equilibrium. The dissociation constants between oligomeric forms are so low that they are at the limits of measurement by conventional methods in vitro. Here, we have used the high sensitivity of single-molecule methods to measure the equilibria and kinetics of oligomerization of full-length p53 and its isolated tetramerization domain, p53tet, at physiological temperature, pH and ionic strength using fluorescence correlation spectroscopy (FCS) in vitro. The dissociation constant at 37 °C for tetramers dissociating into dimers for full-length p53 was 50 ± 7 nM, and the corresponding value for dimers into monomers was 0.55 ± 0.08 nM. The half-lives for the two processes were 20 and 50 min, respectively. The equivalent quantities for p53tet were 150 ± 10 nM, 1.0 ± 0.14 nM, 2.5 ± 0.4 min and 13 ± 2 min. The data suggest that unligated p53 in unstressed cells should be predominantly dimeric. Single-molecule FCS is a useful procedure for measuring dissociation equilibria, kinetics and aggregation at extreme sensitivity.     

Synchrotron radiation solution X-ray scattering study of the pH dependence of the quaternary structure of yeast pyruvate decarboxylase.

The pH dependence of the quaternary structure of pyruvate decarboxylase from yeast was studied in the range 6.2 less than pH less than 8.4. There is an equilibrium with a midpoint around pH 7.5 between tetramers and dimers, and the catalytic activity of the enzyme depends on the volume fraction of tetramer. This equilibrium may provide an additional regulating mechanism besides substrate activation since accumulation of pyruvate would lead to a reduction in pH and hence an increase of the concentration of the catalytically active tetramer. Radiation damage during the X-ray scattering experiments results in a shift of this equilibrium and in the formation of octamers. These effects could be circumvented and analyzed using experimental and data processing methods which can be readily applied to other radiation-sensitive systems. The low-resolution shapes of the dimers and tetramers were determined from the scattering curves using spherical harmonics. The results indicate that a conformational change must occur in the dimers upon formation of the tetramers, in agreement with earlier circular dichroism measurements.     

Studies on dilution inactivation of sheep liver pyruvate carboxylase

When sheep liver pyruvate carboxylase was diluted below 4 EU/ml, it underwent inactivation involving two kinetically distinct processes, i.e., a rapid initial burst followed by a slower second phase. The catalytic activity of the diluted enzyme eventually approached zero, suggesting the occurrence of an irreversible process. Analysis of the quaternary structure of the enzyme by gel filtration chromatography and electron microscopy showed that most of the enzyme molecules occur as tetramers at high enzyme concentrations. However, dilution of the enzyme below 4 EU/ml led to the appearance of dimers and monomers which were essentially inactive under the conditions of the assay system used. The presence of acetyl-CoA during dilution prevented inactivation from occurring and preserved the tetrameric structure. When added after dilution, acetyl-CoA prevented further inactivation from occurring but did not reactivate the enzyme. However, acetyl-CoA did cause a relatively rapid reassociation of the inactive monomers and dimers to form inactive tetramers.     

The oligomeric distribution of SecYEG is altered by SecA and translocation ligands

The multimeric membrane protein complex translocase mediates the transport of preproteins across and integration of membrane proteins into the inner membrane of Escherichia coli. The translocase consists of the peripheral membrane-associated ATPase SecA and the heterotrimeric channel-forming complex consisting of SecY, SecE and SecG. We have investigated the quaternary structure of the SecYEG complex in proteoliposomes. Fluorescence resonance energy transfer demonstrates that SecYEG forms oligomers when embedded in the membrane. Freeze-fracture techniques were used to examine the oligomeric composition under non-translocating and translocating conditions. Our data show that membrane-embedded SecYEG exists in a concentration-dependent equilibrium between monomers, dimers and tetramers, and that dynamic exchange of subunits between oligomers can occur. Remarkably, the formation of dimers and tetramers in the lipid environment is stimulated significantly by membrane insertion of SecA and by the interaction with translocation ligands SecA, preprotein and ATP, suggesting that the active translocation channel consists of multiple SecYEG complexes.     

Nativelike intermediate on the unfolding pathway of pig kidney fructose-1,6-bisphosphatase

The unfolding and dissociation of the tetrameric enzyme fructose-1,6-bisphosphatase from pig kidney by guanidine hydrochloride have been investigated at equilibrium by monitoring enzyme activity, ANS binding, intrinsic (tyrosine) protein fluorescence, exposure of thiol groups, fluorescence of extrinsic probes (AEDANS, MIANS), and size-exclusion chromatography. The unfolding is a multistate process involving as the first intermediate a catalytically inactive tetramer. The evidence that indicates the existence of this intermediate is as follows: (1) the loss of enzymatic activity and the concomitant increase of ANS binding, at low concentrations of Gdn.HCl (midpoint at 0.75 M), are both protein concentration independent, and (2) the enzyme remains in a tetrameric state at 0.9 M Gdn.HCl as shown by size-exclusion chromatography. At slightly higher Gdn.HCl concentrations the inactive tetramer dissociates to a compact dimer which is prone to aggregate. Further evidence for dissociation of tetramers to dimers and of dimers to monomers comes from the concentration dependence of AEDANS-labeled enzyme anisotropy data. Above 2.3 M Gdn.HCl the change of AEDANS anisotropy is concentration independent, indicative of monomer unfolding, which also is detected by a red shift of MIANS-labeled enzyme emission. At Gdn.HCl concentrations higher than 3.0 M, the protein elutes from the size-exclusion column as a single peak, with a retention volume smaller than that of the native protein, corresponding to the completely unfolded monomer. In the presence of its cofactor Mg(2+), the denaturated enzyme could be successfully reconstituted into the active enzyme with a yield of approximately 70-90%. Refolding kinetic data indicate that rapid refolding and reassociation of the monomers into a nativelike tetramer and reactivation of the tetramer are sequential events, the latter involving slow and small conformational rearrangements in the refolded enzyme.     

Calcium triggers folding of lipoprotein lipase into active dimers

The active form of lipoprotein lipase (LPL) is a noncovalent homodimer of 55-kDa subunits. The dimer is unstable and tends to undergo irreversible dissociation into inactive monomers. We noted that a preparation of such monomers slowly regained traces of activity under assay conditions with substrate, heparin, and serum or in cell culture medium containing serum. We therefore studied the refolding pathway of LPL after full denaturation in 6 M guanidinium chloride or after dissociation into monomers in 1 M guanidinium chloride. In crude systems, we identified serum as the factor promoting reactivation. Further investigations demonstrated that Ca2+ was the crucial component in serum for reactivation of LPL and that refolding involved at least two steps. Studies of far-UV circular dichroism, fluorescence, and proteolytic cleavage patterns showed that LPL started to refold from the C-terminal domain, independent of calcium. The first step was rapid and resulted in formation of an inactive monomer with a completely folded C-terminal domain, whereas the N-terminal domain was in the molten globule state. The second step was promoted by Ca2+ and converted LPL monomers from the molten globule state to dimerization-competent and more tightly folded monomers that rapidly formed active LPL dimers. The second step was slow, and it appears that proline isomerization (rather than dimerization as such) is rate-limiting. Inactive monomers isolated from human tissue recovered activity under the influence of Ca2+. We speculate that Ca2+-dependent control of LPL dimerization might be involved in the normal post-translational regulation of LPL activity.     

Interaction between the Component Enzymes of the Glycine Decarboxylase Multienzyme Complex

The glycine decarboxylase multienzyme complex comprises about one-third of the soluble protein of the matrix of pea (Pisum sativum) leaf mitochondria where it exists at a concentration of approximately 130 milligrams protein/milliliter. Under these conditions the complex is stable with an approximate subunit ratio of 2 P-protein dimers:27 H-protein monomers:9 T-protein monomers:1 L-protein dimer. When the complex is diluted it tends to dissociate into its component enzymes. This prevents the purification of the intact complex by gel filtration or ultracentrifugation. In the dissociated state the H-protein acts as a mobile cosubstrate that commutes between the other three enzymes and shows typical substrate kinetics. When the complex is reformed, the H-protein no longer acts as a substrate but as an integrated part of the enzyme complex.     

Subunit structure and hybrid formation of bovine pyruvate kinases

After denaturing either type M or L pyruvate kinase by guanidine hydrochloride, urea, or low pH, enzymatic activity and quaternary structure can be recovered by diluting the enzyme into buffer containing beta-mercaptoethanol. After denaturation of type M pyruvate kinase by guanidine hydrochloride, the yield and polarization of the intrinsic protein fluorescence, as well as most of the circular dichroism characteristic of the native enzyme, were regained very rapidly, while enzymatic activity was recovered much more slowly. Under the conditions used, about 50% of the original M and 30-50% of the original type L activity were typically recovered. Average half-times for recovery of enzymatic activity were 37 min for type M and 104 min for type L but depended somewhat on the renaturation buffer and on protein concentrations in the renaturation medium. If types M and L pyruvate kinases are renatured together, an approximately random recombination of the two subunits types results in a five-membered hybrid set. We have used this hybridizability to determine the kinetics of reformation of the native tetramer by denaturing each isozyme and beginning its renaturation separately at various times mixing the two isozymes and continuing their renaturation together. These studies indicate that reformation of stable tetramers occurs relatively slowly, qualitatively paralleling the regain of enzymatic activity, and that tetramer formation may be necessary for enzymatic activity. Using a similar technique to test for spontaneous dissociation of the native isozymes in buffer, we find that type L, but not type M, reversibly dissociates into dimers and monomers in buffer solutions. This dissociation is decreased by the presence of the substrate, phosphoenolpyruvate, by Mg2+ ions, or by the allosteric effector, fructose bisphosphate.     

Dissociation and in vitro reconstitution of bovine liver uridine diphosphoglucose dehydrogenase. The paired subunit nature of the enzyme

Uridine diphosphoglucose dehydrogenase (EC 1.1.1.22: UDPglucose dehydrogenase) at pH 5.5-7.8 is a stable homohexamer of 305 +/- 7 kDa that does not undergo concentration-dependent dissociation at enzyme concentrations greater than 5 micrograms/mL. Chemical cross-linking of the native enzyme at varying glutaraldehyde concentrations yields dimers, tetramers, and hexamers; at greater than 2% (w/v) glutaraldehyde, plateau values of 21% monomers, 16% dimers, 5% tetramers, and 58% hexamers are obtained. Dissociation at acid pH (pH 2.3) or in 4-6 M guanidine hydrochloride leads to inactive monomers (Mr 52,000). Denaturation at increasing guanidine hydrochloride concentration reveals separable unfolding steps suggesting the typical domain structure of dehydrogenases holds for the present enzyme. At greater than 4 M guanidine hydrochloride complete randomization of the polypeptide chains is observed after 10-min denaturation. Reconstitution of the native hexamer after dissociation/denaturation has been monitored by reactivation and glutaraldehyde fixation. The kinetics may be described in terms of a sequential uni-bimolecular model, governed by rate-determining folding and association steps at the monomer level. Trimeric intermediates do not appear in significant amounts. Reactivation is found to parallel hexamer formation. Structural changes during reconstitution (monitored by circular dichroism) are characterized by complex kinetics, indicating the rapid formation of "structured monomers" (with most of the native secondary structure) followed by slow "reshuffling" prior to subunit association. The final product of reconstitution is indistinguishable from the initial native enzyme.     

Alternate mRNA splicing in multiple human tryptase genes is predicted to regulate tetramer formation

Tryptases are serine proteases that are thought to be uniquely and proteolytically active as tetramers. Crystallographic studies reveal that the active tetramer is a flat ring structure composed of four monomers, with their active sites arranged around a narrow central pore. This model explains why many of the preferred substrates of tryptase are short peptides; however, it does not explain how tryptase cleaves large protein substrates such as fibronectin, although a number of studies have reported in vitro mechanisms for generating active monomers that could digest larger substrates. Here we suggest that alternate mRNA splicing of human tryptase genes generates active tryptase monomers (or dimers). We have identified a conserved pattern of alternate splicing in four tryptase alleles (alphaII, betaI, betaIII, and deltaI), representing three distinct tryptase gene loci. When compared with their full-length counterparts, the splice variants use an alternate acceptor site within exon 4. This results in the deletion of 27 nucleotides within the central coding sequence and 9 amino acids from the translated protein product. Although modeling suggests that the deletion can be easily accommodated by the enzymes structurally, it is predicted to alter the specificity by enlarging the S1' or S2' binding pocket and results in the complete loss of the "47 loop," reported to be critical for the formation of tetramers. Although active monomers can be generated in vitro using a range of artificial conditions, we suggest that alternate splicing is the in vivo mechanism used to generate active tryptase that can cleave large protein substrates.     

Resolution of brewer's yeast pyruvate decarboxylase into multiple isoforms with similar subunit structure and activity using high-performance liquid chromatography.

A method for the purification of brewer's yeast pyruvate decarboxylase (EC 4.1.1.1) that resolves the enzyme into multiple active isoforms was developed. Seven activity fractions are resolved by DEAE HPLC chromatography. Among these fractions, three distinct subunit composition isoforms are apparent by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: alpha 4, a homotetrameric holoenzyme consisting of the lower mass subunit; alpha 2 beta 2, a heterotetrameric holoenzyme consisting of lower and higher mass subunits; and beta 4, a homotetrameric holoenzyme consisting of the higher mass subunit. Beta 4 is a heretofore unreported form which may represent the unproteolyzed form of the enzyme. The Km and Vmax for the alpha 4 and beta 4 isoforms are identical within the limits of experimental error, as is their behavior vis-à-vis the allosteric regulator pyruvamide. All active isoforms exist as tetramers according to gel filtration analysis under native conditions. The purification has been successfully applied to pyruvate decarboxylase isolated from two different species of yeast and therefore is likely to be of general utility for purification of this enzyme from other yeast sources. Conditions under which all three isoforms demonstrate exceptional stability, making them amenable to prolonged physicochemical studies at 4 degrees C and even at room temperature are reported.     

Pyridoxal phosphate inhibits dynamic subunit interchange among serine hydroxymethyltransferase tetramers

Cytoplasmic serine hydroxymethyltransferase (cSHMT) is a tetrameric, pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the reversible interconversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. The enzyme has four active sites and is best described as a dimer of obligate dimers. Each monomeric subunit within the obligate dimer contributes catalytically important amino acid residues to both active sites. To investigate the interchange of subunits among cSHMT tetramers, a dominant-negative human cSHMT enzyme (DNcSHMT) was engineered by making three amino acid substitutions: K257Q, Y82A, and Y83F. Purified recombinant DNcSHMT protein was catalytically inactive and did not bind 5-formyltetrahydrofolate. Coexpression of the cSHMT and DNcSHMT proteins in bacteria resulted in the formation of heterotetramers with a cSHMT/DNcSHMT subunit ratio of 1. Characterization of the cSHMT/DNcSHMT heterotetramers indicates that DNcSHMT and cSHMT monomers randomly associate to form tetramers and that cSHMT/DNcSHMT obligate dimers are catalytically inactive. Incubation of recombinant cSHMT protein with recombinant DNcSHMT protein did not result in the formation of hetero-oligomers, indicating that cSHMT subunits do not exchange once the tetramer is assembled. However, removal of the active site PLP cofactor does permit exchange of obligate dimers among preformed cSHMT and DNcSHMT tetramers, and the formation of heterotetramers from cSHMT and DNcSHMT homodimers does not affect the activity of the cSHMT homodimers. The results of these studies demonstrate that PLP inhibits dimer exchange among cSHMT tetramers and suggests that cellular PLP concentrations may influence the stability of cSHMT protein in vivo.     

Binding of diphosphoglycerate and ATP to oxyhemoglobin dimers

The relative affinity of diphosphoglycerate and ATP for hemoglobin dimers and tetramers can be measured under conditions where the protein is in large molar excess over the polyphosphate. Binding of both compounds to dimers was about 25 times stronger than to tetramers in the case of the three low-spin hemoglobins, oxyhemoglobin, carboxyhemoglobin and cyanomethemoglobin. The mutation in hemoglobin Kansas leads to an increased dissociation into alpha beta dimers. The increase in diphosphoglycerate binding by this hemoglobin was in good agreement with that expected from the dimer-tetramer dissociation constant over a wide range of hemoglobin concentrations. In contrast to the liganded hemoglobins, both deoxyhemoglobin and aquomethemoglobin bind the two polyanions as tetramers.     

The origin of the molecular diversity and functional anchoring of cholinesterases

Vertebrates possess two cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) which both hydrolyze acetylcholine, but differ in their specificity towards other substrates, and in their sensitivity to inhibitors. In mammals, the AChE gene produces three types of coding regions through the choice of 3' splice acceptor sites, generating proteins which possess the same catalytic domain, associated with distinct C-terminal peptides. AChE subunits of type R ('readthrough') produce soluble monomers; they are expressed during development and induced by stress in the mouse brain. AChE subunits of type H ('hydrophobic') produce GPI-anchored dimers, but also secreted molecules; they are mostly expressed in blood cells. Subunits of type T ('tailed') exist for both AChE and BChE. They represent the enzyme forms expressed in brain and muscle. These subunits generate a variety of quaternary structures, including homomeric oligomers (monomers, dimers, tetramers), as well as hetero-oligomeric assemblies with anchoring proteins, ColQ and PRiMA. Mutations in the four-helix bundle (FHB) zone of the catalytic domain indicate that subunits of type H and T use the same interaction for dimerization. On the other hand, the C-terminal T peptide is necessary for tetramerization. Four T peptides, organized as amphiphilic alpha helices, can assemble around proline-rich motifs of ColQ or PRiMA. The association of AChE(T) or BChE subunits with ColQ produces collagen-tailed molecules, which are inserted in the extracellular matrix, e.g. in the basal lamina of neuromuscular junctions. Their association with PRiMA produces membrane-bound tetramers which constitute the predominant form of cholinesterases in the mammalian brain; in muscles, the level of PRiMA-anchored tetramers is regulated by exercise, but their functional significance remains unknown. In brain and muscles, the hydrolysis of acetylcholine by cholinesterases, in different contexts, and their possible noncatalytic functions clearly depend on their localization by ColQ or PRiMA.