Structural and functional organization of the animal fatty acid synthase

The entire pathway of palmitate synthesis from malonyl-CoA in mammals is catalyzed by a single, homodimeric, multifunctional protein, the fatty acid synthase. Each subunit contains three N-terminal domains, the beta-ketoacyl synthase, malonyl/acetyl transferase and dehydrase separated by a structural core from four C-terminal domains, the enoyl reductase, beta-ketoacyl reductase, acyl carrier protein and thiosterase. The kinetics and specificities of the substrate loading reaction catalyzed by the malonyl/acetyl transferase, the condensation reaction catalyzed by beta-ketoacyl synthase and chain-terminating reaction catalyzed by the thioesterase ensure that intermediates do not leak off the enzyme, saturated chains exclusively are elongated and palmitate is released as the major product. Only in the fatty acid synthase dimer do the subunits adopt conformations that facilitate productive coupling of the individual reactions for fatty acid synthesis at the two acyl carrier protein centers. Introduction of a double tagging and dual affinity chromatographic procedure has permitted the engineering and isolation of heterodimeric fatty acid synthases carrying different mutations on each subunit. Characterization of these heterodimers, by activity assays and chemical cross-linking, has been exploited to map the functional topology of the protein. The results reveal that the two acyl carrier protein domains engage in substrate loading and condensation reactions catalyzed by the malonyl/acetyl transferase and beta-ketoacyl synthase domains of either subunit. In contrast, the reactions involved in processing of the beta-carbon atom, following each chain elongation step, together with the release of palmitate, are catalyzed by the cooperation of the acyl carrier protein with catalytic domains of the same subunit. These findings suggest a revised model for the fatty acid synthase in which the two polypeptides are oriented such that head-to-tail contacts are formed both between and within subunits.     

Isolation of a functional transferase component from the rat fatty acid synthase by limited trypsinization of the subunit monomer. Formation of a stable functional complex between transferase and acyl carrier protein domains

Limited trypsinization of rat fatty acid synthase monomers results in cleavage at sites protected in the native dimer. A 47,000-Da polypeptide containing the transferase component was isolated from the digest and its location in the multifunctional polypeptide established. Both acetyl and malonyl moieties are transferred stoichiometrically from CoA ester to this polypeptide and each can replace the other, confirming that a single common site is utilized in the loading of these substrates onto the fatty acid synthase. Transferase activity of the 47,000-Da polypeptide decreases with increasing acyl donor chain length (malonyl = acetyl greater than butyryl greater than hexanoyl greater than octanoyl). Activity is inhibited by certain thiol-directed reagents, and protection is afforded by substrate suggesting the presence of a sensitive cysteine residue near the substrate binding site. The transferase was also able to utilize as acyl acceptor the Escherichia coli acyl carrier protein and the acyl carrier protein domain of the multifunctional fatty acid synthase. When the fatty acid synthase monomer was trypsinized under milder conditions, the 47,000-Da transferase domain could be isolated in association with the 8,000-Da acyl carrier protein domain. The transferase was capable of translocating substrate moieties from CoA ester donors to the associated acyl carrier protein. The results provide the first direct evidence that, in the head-to-tail oriented fatty acid synthase homodimer, functional communication between the transferase domain located near the end of one polypeptide and the acyl carrier protein domain located at the opposite end of the other polypeptide is facilitated by a stable physical interaction between these domains.     

Mapping the functional topology of the animal fatty acid synthase by mutant complementation in vitro

An in vitro mutant complementation approach has been used to map the functional topology of the animal fatty acid synthase. A series of knockout mutants was engineered, each mutant compromised in one of the seven functional domains, and heterodimers generated by hybridizing all possible combinations of the mutated subunits were isolated and characterized. Heterodimers comprised of a subunit containing either a beta-ketoacyl synthase or malonyl/acetyltransferase mutant, paired with a subunit containing mutations in any one of the other five domains, are active in fatty acid synthesis. Heterodimers in which both subunits carry a knockout mutation in either the dehydrase, enoyl reductase, keto reductase, or acyl carrier protein are inactive. Heterodimers comprised of a subunit containing a thioesterase mutation paired with a subunit containing a mutation in either the dehydrase, enoyl reductase, beta-ketoacyl reductase, or acyl carrier protein domains exhibit very low fatty acid synthetic ability. The results are consistent with a model for the fatty acid synthase in which the substrate loading and condensation reactions are catalyzed by cooperation of an acyl carrier protein domain of one subunit with the malonyl/acetyltransferase or beta-ketoacyl synthase domains, respectively, of either subunit. The beta-carbon-processing reactions, responsible for the complete reduction of the beta-ketoacyl moiety following each condensation step, are catalyzed by cooperation of an acyl carrier protein domain with the beta-ketoacyl reductase, dehydrase, and enoyl reductase domains associated exclusively with the same subunit. The chain-terminating reaction is carried out most efficiently by cooperation of an acyl carrier protein domain with the thioesterase domain of the same subunit. These results are discussed in the context of a revised model for the fatty acid synthase.     

Rat mammary gland fatty acid synthase: localization of the constituent domains and two functional polyadenylation/termination signals in the cDNA.

The rat fatty acid synthase (FAS) is active only as a dimer, although the eight component functions are contained in a single polypeptide chain. Using mRNA from lactating rat mammary glands a cDNA expression library was established. With the overlapping immunologically positive clones we have an 8.9kb cDNA sequence for rat FAS. In the 3'-nontranslated region of the rat FAS cDNA we find a prototype polyadenylation/termination signal and 779 nucleotides upstream, a mutated one. Both of these polyadenylation/termination signals are used and give rise to two equally abundant mRNA species which are coordinately regulated. In the derived amino acid sequence we could locate six of the eight component functions; their order is NH2- beta-ketoacyl synthase - acetyl/malonyl transferases -enoyl reductase - acyl carrier protein - thioesterase -COOH. Comparison of FAS from different sources shows that the primary sequence is conserved only for the active residues and the amino acids in their immediate vicinity.     

The isolation of the two subunits of yeast fatty acid synthetase.

The two subunits that comprise the yeast fatty acid synthetase (designated α and β) have been isolated. The separation was performed using DEAE Biogel A chromatography after first treating yeast fatty acid synthetase with 3,4,5,6 tetrahydrophthalic anhydride. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the fractions eluted from the ion exchange column indicated that the separation of the subunits was essentially complete. It was possible to remove the 3,4,5,6 tetrahydrophthalate derivative from the subunits and regenerate certain of the partial activities. The α subunit was found to have the β-keto reductase activity as well as the acyl carrier protein component associated with it. The β subunit had the acetyl and malonyl transacylases and the palmitoyl transferase activity associated with it. The different extent to which the malonyl and acetyl transacylase activities were regained indicated that these two catalytic sites have separate domains in the β subunit.     

Subunit structure of the mammalian fatty acid synthetase: further evidence for a homodimer.

Immunochemical procedures and limited proteolysis have been used to investigate the subunit structure of fatty acid synthetase from rat mammary gland. Specific antibodies were raised against the two thioesterase I domains obtained from the fatty acid synthetase by treatment with trypsin. The antibodies precipitated both subunits of the dissociated fatty acid synthetase, indicating that both subunits contained a single thioesterase I domain. An analysis of the time course of limited trypsinization of the fatty acid synthetase, labeled in its two thioesterase I domains with [1,3-¹⁴C] diisopropylphosphofluoridate, indicated that each subunit was susceptible to tryptic attack at identical locations and that the thioesterase I domains occupied a terminal locus at one end of each polyfunctional polypeptide chain. The most plausible explanation for these results is that the mammalian fatty acid synthetase is a homodimer.     

Mammalian fatty acid synthetase is a structurally and functionally symmetrical dimer

We have explored a comprehensive experimental approach to determine whether the two condensing-enzyme active centers of the mammalian fatty acid synthetase are simultaneously functional. Our strategy involved utilization of trypsinized fatty acid synthetase, which is a nicked homodimer composed of two pairs of 125 + 95-kDa polypeptides. These core polypeptides lack the chain-terminating thioesterase domains but retain all other functional domains of the native enzyme and can assemble long-chain acyl moieties at a rate equal to that of the native enzyme. The 4'-phosphopantetheine content of these enzyme preparations, estimated from the amount of beta-alanine present, from the amount of taurine formed by performic acid oxidation and from the amount of carboxymethylcysteamine formed by alkylation with iodo[2-14C]acetate, was typically 0.86 mol/mol 95-kDa polypeptide. The stoichiometry of long-chain acyl-enzyme synthesis, measured with radiolabeled precursors, indicated that 0.84 mol acyl-chains were assembled/mol 95-kDa polypeptide. When the small amount of apoenzyme present is taken into account, this stoichiometry translates to 1.94 acyl chains per holoenzyme dimer. The 125-kDa polypeptide of one subunit could be cross-linked to the 95-kDa polypeptide of the other subunit by 1,3-dibromo-2-propanone yielding a single molecular species of 220 kDa. Cross-linking was accompanied by a loss of condensing-enzyme activity. This result is consistent with a structurally symmetrical model for the animal fatty acid synthetase [J.K. Stoops and S.J. Wakil (1981) J. Biol. Chem. 256, 5128-5133] in which the juxtaposed 4'-phosphopantetheine and cysteine thiols of opposing subunits that form the two potential catalytic centers for condensing activity are readily susceptible to cross-linking. Both half-maximal cross-linking and 50% inhibition of activity were observed with 1 mol 1,3-dibromo-2-propanone bound/mol enzyme. After assembly of long-chain acyl moieties on the 4'-phosphopantetheine residues, no vacant condensing-enzyme active sites were demonstrable either by cross-linking with 1,3-dibromo-2-propanone or by formation of carboxymethylcysteamine on treatment with iodoacetate. These results are consistent with a structurally and functionally symmetrical model for the mammalian fatty acid synthetase in which the two condensation sites are simultaneously active.     

Characterization of recombinant thioesterase and acyl carrier protein domains of chicken fatty acid synthase expressed in Escherichia coli

Fatty acid synthase of animal tissue is a multifunctional enzyme comprised of two identical subunits, each containing seven partial activities and a site for the prosthetic group, 4'-phosphopantetheine (acyl carrier protein). We have recently isolated cDNA clones of chicken fatty acid synthase coding for the dehydratase, enoyl reductase, beta-ketoacyl reductase, acyl carrier protein, and thioesterase domains (Chirala, S.S., Kasturi, R., Pazirandeh, M., Stolow, D.T., Huang, W.Y., and Wakil, S.J. (1989) J. Biol. Chem. 264, 3750-3757). To gain insight into the structure and function of the various domains, the portion of the cDNA coding for the acyl carrier protein and thioesterase domains was expressed in Escherichia coli by using an expression vector that utilizes the phage lambda PL promoter. The recombinant protein was efficiently expressed and purified to near homogeneity using anion-exchange and hydroxyapatite chromatography. As expected from the coding capacity of the cDNA expressed, the protein has a molecular weight of 43,000 and reacts with antithioesterase antibodies. The recombinant thioesterase was found to be enzymatically active and has the same substrate specificity and kinetic properties as the native enzyme of the multifunctional synthase. Treatment of the recombinant protein with alpha-chymotrypsin results in the cleavage of the acyl carrier protein and thioesterase domain junction sequence at exactly the same site as with native fatty acid synthase. The amino acid composition of the purified recombinant protein revealed the presence of 0.6 mol of beta-alanine/mol of protein, indicating partial pantothenylation of the recombinant acyl carrier protein domain. These results indicate that the expressed protein has a conformation similar to the native enzyme and that its folding into functionally active domains is independent of the remaining domains of the multifunctional synthase subunit. These conclusions are consistent with the proposal that the multifunctional synthase gene has evolved from fusion of component genes.     

Characterization of a genomic and cDNA clone coding for the thioesterase domain and 3' noncoding region of the chicken liver fatty acid synthase gene

The fatty acid synthase (FAS) of animal tissue is a dimer of two identical subunits, each with a Mr of 260,000. The subunit is a single multifunctional protein having seven catalytic activities and a site for binding of the prosthetic group 4'-phosphopantetheine. The mRNA coding for the subunit has an estimated size of 10-16 kb, which is about twice the number of nucleotides needed to code for the estimated 2300 amino acids. We have isolated a positive clone, lambda CFAS, containing FAS gene sequences by screening a chicken genomic library with a segment of a 3' untranslated region of goose fatty acid synthase cDNA clone, pGFAS3, as a hybridization probe. The DNA insert in lambda CFAS hybridizes with synthetic oligonucleotide probes prepared according to the known amino acid sequence of the thioesterase component of the chicken liver fatty acid synthase [Yang, C.-Y., Huang, W.-Y., Chirala, S., & Wakil, S.J. (1988) Biochemistry (preceding paper in this issue)]. Further characterization of the DNA insert shows that the lambda CFAS clone contains about a 4.7-kbp segment from the 3' end of the chicken FAS gene that codes for a portion of the thioesterase domain. Complete sequence analyses of this segment including S1 nuclease mapping, showed that the lambda CFAS clone contains the entire 3' untranslated region of the chicken FAS gene and three exons that code for 162 amino acids of the thioesterase domain from the COOH-terminal end of the fatty acid synthase. Using the exon region of the genomic clone, we were able to isolate a cDNA clone that codes for the entire thioesterase domain of chicken liver fatty acid synthase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium-dependent conformational changes in the 36-kDa subunit of intestinal protein I related to the cellular 36-kDa target of Rous sarcoma virus tyrosine kinase

Protein I from intestinal epithelium is biochemically and immunologically related to the fibroblast 36-kDa substrate of the Rous sarcoma virus-encoded tyrosine protein kinase (Gerke and Weber (1984) EMBO J. 3, 227-233). Protein I is a Ca2+-binding protein containing two copies each of a 36- and 10-kDa subunit. Denaturation/renaturation experiments show that the 36-kDa subunit is a monomer, whereas the 10-kDa subunit forms a dimer. Mixing of the subunits leads to reconstituted protein I. Physicochemical properties of protein I and its isolated subunits reveal a Ca2+-dependent conformational change in the 36-kDa subunit which involves the exposure of 1 or more tyrosine residues to a more aqueous environment. This change points to a Ca2+ binding constant of about 10(4) M-1 in the presence of 2 mM Mg2+ and induces the ability of protein I and the 36-kDa subunit to bind in vitro to F-actin and nonerythroid spectrin. The same high Ca2+ requirement has been reported for the in vitro tyrosine phosphorylation of a 35-kDa protein from A-431 carcinoma cells by the epidermal growth factor receptor kinase (Fava and Cohen (1984) J. Biol. Chem. 259, 2636-2645). Here we show that this 35-kDa substrate is biochemically and immunologically related to the 36-kDa subunit of protein I, which in turn corresponds to the substrate of the Rous sarcoma virus kinase. The protein of A-431 cells exists not only as a monomer but also as a dimer. The latter fraction contains a 10-kDa polypeptide immunologically related to the corresponding subunit of protein I. Given past results on the A-431 system, we speculate that the monomer rather than the dimer is the preferred in vitro substrate for the epidermal growth factor receptor kinase. Thus, the 10-kDa subunit, which induces dimerization of the phosphorylatable large subunit, may act as an inhibitor.     

The architecture of the animal fatty acid synthetase complex. IV. Mapping of active centers and model for the mechanism of action

The fatty acid synthetase of animal tissue consists of two subunits, each containing seven catalytic centers and an acyl carrier site. Proteolytic cleavage patterns indicate that the subunit is arranged into three major domains, I, II, and III. Domain I contains the NH2-terminal end of the polypeptide and the catalytic sites of beta-ketoacyl synthetase (condensing enzyme) and the acetyl-and malonyl-transacylases. This domain, therefore, functions as a site for acetyl and malonyl substrate entry into the process of fatty acid synthesis and acts in part as the site of carbon-carbon condensation, resulting in chain elongation. Domain II is the medial domain and contains the beta-ketoacyl and enoyl reductases, probably the dehydratase, and the 4'-phosphopantetheine prosthetic group of the acyl carrier protein site. Domain II, therefore, is designated as the reduction domain where the keto carbon is reduced to methylene carbon by sequential processes of reduction, dehydration, and reduction again. Throughout these processes, the acyl group is attached to the pantetheine-SH of the acyl carrier protein. The latter site is distal to the cysteine-SH of the beta-ketoacyl synthetase, constitutes the 15000-dalton polypeptide at the COOH-terminal end of Domain II, and connects to Domain III. When the growing chain reaches C16 carbon length, the fatty acyl group is released by the thioesterase activity, which is contained in Domain III. A functional model is proposed based on the aforementioned results and the recent evidence that the synthetase subunits are arranged in a head-to-tail fashion, such that the pantetheine-SH of the acyl carrier protein of one subunit and the cysteine-SH of the beta-ketoacyl synthetase of the second subunit are juxtaposed. In this model, a palmitate synthesizing site contains Domain I of one subunit and Domains II and III of the second subunit. Therefore, even though each subunit contains all of the partial activities of the reaction sequence, the actual palmitate synthesizing unit consists of one-half of a subunit interacting with the complementary half of the other subunit.     

New experiments of biotin enzymes.

The objects of structural studies on biotin-enzymes were acetyl CoA-carboxylase and pyruvate carboxylase of Saccharomyces cerevisiae and beta-methylcrotonyl CoA-carboxylase and acetyl CoA-carboxylase of Achromobacter IV S. It was found that these enzymes can be arranged in three groups. In the first group, as represented by acetyl CoA-carboxylase of Achromobacter, the active enzyme could be resolved in three types of functional components: (1) the biotin-carboxyl carrier protein, (2) the biotin carboxylase, and (3) the carboxyl transferase. In the second group, as represented by beta-methylcrotonyl CoA-carboxylase from Achromobacter only two types of polypeptides are present. The one carries the biotin carboxylase activity together with the biotin-carboxyl-carrier protein, the other one carries the carboxyl transferase activity. In this third group, as represented by the two enzymes of yeast, all three catalytic functions are incorporated in one multifunctional polypeptide chain. The evolution of the different enzymes is discussed. The animal tissues acetyl CoA-carboxylase is under metabolic control, as known from previous studies. It thus has to be expected that the levels of malonyl CoA in livers of rats in all states of depressed fatty acid synthesis are much lower than under normal conditions because the carboxylation of acetyl CoA is strongly reduced and cannot keep pace with the consumption of malonyl CoA by fatty acid synthetase. A new highly sensitive assay method for malonyl CoA was developed which uses tritiated NADPH and measures the incorporation of radioactivity into the fatty acids formed from malonyl CoA in the presence of purified fatty acid synthetase. The application of this method to liver extracts showed that the level of malonyl CoA which amounts to about 7 nmoles per gram of wet liver drops to less than 10% within a starvation period of 24 hr and even further if the starvation period is extended to 48 hr. A low malonyl CoA concentration is also found in the alloxan diabetic animals and in animals being fed a fatty diet after starvation. On the other hand, feeding a carbohydrate rich diet leads to malonyl CoA levels surpassing the levels found after feeding a balanced diet. These observations reconfirm the concept that fatty acid synthesis is principally regulated by the carboxylation of acetyl CoA.     

Topography of prostaglandin H synthase. Antiinflammatory agents and the protease-sensitive arginine 253 region

Prostaglandin H synthase catalyzes two reactions: the bis-dioxygenation of arachidonic acid to form prostaglandin G2 (cyclooxygenase activity), and the reduction of hydroperoxides to the corresponding alcohols (peroxidase activity). The cyclooxygenase activity can be selectively inhibited by many nonsteroidal antiinflammatory agents including indomethacin. In the native synthase, there is a single prominent protease-sensitive region, located near Arg253; binding of the heme prosthetic group makes the synthase resistant to proteases. To investigate the spatial relationship between the area of the synthase which interacts with indomethacin and the protease-sensitive region, the effects of indomethacin and similar agents on the protease sensitivity of the two enzymatic activities and of the synthase polypeptide were examined. Incubation of the synthase apoenzyme with trypsin (3.6% w/w) resulted in the time-dependent coordinate loss (75% at 1 h) of both enzymatic activities and the cleavage (85% at 1 h) of the 70-kDa subunit into 38- and 33-kDa fragments, indicating that proteolytic cleavage of the polypeptide at Arg253, destroyed both activities of the synthase simultaneously. Indomethacin, (S)-flurbiprofen, or meclofenamate (each at 20 microM) rendered both activities and the synthase polypeptide (at 5 microM subunit) resistant to attack by trypsin or proteinase K; these agents also inhibited the cyclooxygenase activity of the intact synthase. Two reversible cyclooxygenase inhibitors, ibuprofen and flufenamate, also made both of the activities and the synthase polypeptide more resistant to trypsin. Titration of the apoenzyme with indomethacin (0-3 mol/mol of synthase dimer) resulted in proportional increases in the inhibition of the cyclooxygenase and in the resistance to attack by trypsin. (R)-Flurbiprofen did not increase the resistance to protease or appreciably inhibit the cyclooxygenase. These results suggest that the same stereospecific interaction of these agents with the synthase that produced inhibition of the cyclooxygenase led to a decreased accessibility of the Arg253 region to proteases. Aspirin treatment made the synthase less resistant to trypsin; aspirin-treated synthase became more resistant to trypsin when it was incubated with indomethacin before addition of the protease. The presence of 50 microM arachidonate during digestion of apoenzyme or aspirin-treated apoenzyme with trypsin did not decrease the cleavage of the synthase subunit.(ABSTRACT TRUNCATED AT 400 WORDS)     

Double mutation at the subunit interface of glutathione transferase rGSTM1-1 results in a stable, folded monomer

Canonical glutathione (GSH) transferases are dimeric proteins with subunits composed of an N-terminal GSH binding region (domain 1) and a C-terminal helical region (domain 2). The stabilities of several GSH transferase dimers are dependent upon two groups of interactions between domains 1 and 2 of opposing subunits: a hydrophobic ball-and-socket motif and a buried charge cluster motif. In rGSTM1-1, these motifs involve residues F56 and R81, respectively. The structural basis for the effects of mutating F56 to different residues on dimer stability and function has been reported (Codreanu et al. (2005) Biochemistry 44, 10605-10612). Here, we show that the simultaneous disruption of both motifs in the F56S/R81A mutant causes complete dissociation of the dimer to a monomeric protein on the basis of gel filtration chromatography and multiple-angle laser light scattering. The fluorescence and far-UV CD properties of the double mutant as well as the kinetics of amide H/D exchange along the polypeptide backbone suggest that the monomer has a globular structure that is similar to a single subunit in the native protein. However, the mutant monomer has severely impaired catalytic activity, suggesting that the dimer interface is vital for efficient catalysis. Backbone amide H/D exchange kinetics in the F56S and F56S/R81A mutants indicate that a reorganization of the loop structure between helix alpha2 and strand beta3 near the active site is responsible for the decreased catalytic activity of the monomer. In addition, the junction between the alpha4 and alpha5 helices in F56S/R81R shows decreased H/D exchange, indicating another structural change that may affect catalysis. Although the native subunit interface is important for dimer stability, urea-induced unfolding of the F56S/R81A mutant suggests that the interface is not essential for the thermodynamic stability of individual subunits. The H/D exchange data reveal a possible molecular basis for the folding cooperativity observed between domains 1 and 2.     

Conversion of a beta-ketoacyl synthase to a malonyl decarboxylase by replacement of the active-site cysteine with glutamine.

beta-Ketoacyl synthases involved in the biosynthesis of fatty acids and polyketides exhibit extensive sequence similarity and share a common reaction mechanism, in which the carbanion participating in the condensation reaction is generated by decarboxylation of a malonyl or methylmalonyl moiety; normally, the decarboxylation step does not take place readily unless an acyl moiety is positioned on the active-site cysteine residue in readiness for the ensuing condensation reaction. Replacement of the cysteine nucleophile (Cys-161) with glutamine, in the beta-ketoacyl synthase domain of the multifunctional animal fatty acid synthase, completely inhibits the condensation reaction but increases the uncoupled rate of malonyl decarboxylation by more than 2 orders of magnitude. On the other hand, replacement with Ser, Ala, Asn, Gly, and Thr compromises the condensation reaction without having any marked effect on the decarboxylation reaction. The affinity of the beta-ketoacyl synthase for malonyl moieties, in the absence of acetyl moieties, is significantly increased in the Cys161Gln mutant compared to that in the wild type and is similar to that exhibited by the wild-type beta-ketoacyl synthase in the presence of an acetyl primer. These results, together with modeling studies of the Cys --> Gln mutant from the crystal structure of the Escherichia coli beta-ketoacyl synthase II enzyme, suggest that the side chain carbonyl group of the Gln-161 can mimic the carbonyl of the acyl moiety in the acyl-enzyme intermediate so that the mutant adopts a conformation analogous to that of the acyl-enzyme intermediate. Catalysis of the decarboxylation of malonyl-CoA requires the dimeric form of the Cys161Gln fatty acid synthase and involves prior transfer of the malonyl moiety from the CoA ester to the acyl carrier protein domain and subsequent release of the acetyl product by transfer back to a CoA acceptor. These results suggest that the role of the Cys --> Gln beta-ketoacyl synthases found in the loading domains of some modular polyketide synthases likely is to act as malonyl, or methylmalonyl, decarboxylases that provide a source of primer for the chain extension reactions catalyzed by associated modules containing fully competent beta-ketoacyl synthases.     

Proteolytic studies on the structure of bovine von Willebrand factor

Bovine von Willebrand factor (vWF) was digested with protease I (P-I), a metalloprotease isolated from rattlesnake venom. Digestion of vWF for 24 h with P-I yielded a terminal digest consisting of an equimolar mixture of two major fragments (apparent Mr 250K and 200K). The 250-kilodalton (kDa) fragment consists of a 125-kDa chain from one subunit and a 45- and 78-kDa polypeptide chain from an adjacent subunit. The 200-kDa fragment consists of a 97-kDa chain from one subunit and a 35- and 61-kDa polypeptide chain from an adjacent subunit. The 200-kDa fragment binds to heparin, and the heparin binding domain is located on the 97-kDa polypeptide chain. This fragment also competes with labeled, native vWF for binding to formalin-fixed human platelets, with an IC50 of 12.5 micrograms/mL (65 nM). However, native vWF has an IC50 of 2.5 micrograms/mL, indicating that the affinity of the 200-kDa fragment for platelets is approximately one-fifth that of vWF. The 200-kDa fragment agglutinates platelets, but its agglutinating ability is only 5% that of the native molecule. Only the 200-kDa fragment is recognized by monoclonal antibodies 2 and H-9, which are directed against vWF and inhibit vWF binding to platelet glycoprotein Ib (GPIb). Immunological studies, using nine monoclonal antibodies directed against vWF, and the demonstration that the heparin and GPIb binding domains are located on only one fragment suggest that the two fragments are composed of different regions of the vWF subunit. Analysis of the P-I cleavage pattern suggests that all vWF subunits are not cleaved in the same fashion. The first cleavage on half of the subunits generates the 45-kDa terminal and 175-kDa intermediate digest products. The 175-kDa chain is again cleaved, producing the 97- and 78-kDa terminal polypeptide chains. However, the first cleavage of the other subunits generates the 35-kDa terminal and the 186-kDa intermediate digest product, which upon cleavage produces the 125- and 61-kDa terminal polypeptide chains. Immunological data support the asymmetric cleavage pattern. An epitope for a monoclonal antibody is present on both the 186- and 175-kDa intermediate digest products but is only found on one terminal digest fragment, the 78-kDa polypeptide chain, suggesting that the 186- and 175-kDa polypeptides are cleaved at different sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cross-linking and labeling of the Escherichia coli F1F0-ATP synthase reveal a compact hydrophilic portion of F0 close to an F1 catalytic subunit

The subunit arrangement of the F0 sector of the Escherichia coli ATP synthase is examined using hydrophilic and hydrophobic (cleavable) cross-linking reagents and the water-soluble labeling reagent [35S] diazoniumbenzenesulfonate ( [35S]DABS). Cross-linking is performed on purified ATP synthase and inverted minicell membranes. ATP synthase incorporated into liposomes is labeled with [35S]DABS. Three cross-linked products involving the F0 subunits (a, b, and c) are observed with the purified ATP synthase in solution: a-b, b2, and c2 dimers. A cross-link between the F0 and F1 is detected and occurs between the a and beta subunits. A cross-linker independent association between the b and beta subunits is also evident, suggesting that the two subunits are close enough to form a disulfide bridge. A cross-linking reagent stable to reducing agents produces a b-beta dimer, as detected by immunoblotting with anti-beta serum. The c subunit does not cross-link with any F1 polypeptide. Minicell membranes containing ATP synthase polypeptides radioactively labeled in vivo similarly show b2 and c2 dimers after cross-linking. [35S]DABS labels the a and b, but not c, subunits, showing that the a and b, but not c, subunits possess hydrophilic domains. Thus, certain domains of subunits a and b extend from the membrane and are in close proximity to one another and the F1 catalytic subunit beta.     

Purification and characterization of five forms of glutathione transferase from human uterus

Five glutathione transferase (GST) forms were purified from human uterus by glutathione-affinity chromatography followed by chromatofocusing, and their structural, kinetic and immunological properties were investigated. Upon SDS/polyacrylamide slab gel electrophoresis all forms resulted composed of two subunits of identical molecular size. GST V (pI 4.5) is a dimer of 23-kDa subunits. GST I (pI 6.8) and GST IV (pI 4.9) are dimers of 24-kDa subunits whereas GST II (pI 6.1) and GST III (pI 5.5) are dimers of 26.5-kDa subunits. GST V accounts for about 85-90% of the activity whereas the other isoenzymes are present in trace quantities. On the basis of the molecular mass of the subunits, amino acid composition, substrate specificities, sensitivities to inhibitors, CD spectra and immunological studies, GST V appeared very similar to transferase pi. Structural and immunological studies provide evidence that GST IV is closely related to the less 'basic' transferase (GST pI 8.5) of human skin. Extensive similarities have been found between GST II and GST III. The comparison includes amino acid compositions, subunits molecular size and immunological properties. The two enzymes, however, are kinetically distinguishable. The data presented also indicate that GST II and GST III are related to transferase mu and to transferase psi of human liver. Even though GST I has a subunit molecular mass identical to GST IV, several lines of evidence, including catalytic and immunological properties, indicate that they are different from each other. GST I seems not to be related to any of known human transferases, suggesting that it may be specific for the uterus.     

Structure of the 116-kDa polypeptide of the clathrin-coated vesicle/synaptic vesicle proton pump.

A 116-kDa polypeptide has recently been found to be a common component of vacuolar proton pumps isolated from a variety of sources. The 116-kDa subunit of the proton pump was purified from clathrin-coated vesicles of bovine brain, and internal sequences were obtained from proteolytic peptides. Oligonucleotide probes designed from these peptide sequences were utilized in polymerase chain reactions to isolate partial bovine cDNA clones for the protein. Sequences from these were then utilized to isolate rat brain cDNA clones containing the full-length coding region. RNA blots indicate the presence of an abundant 3.9-kilobase message for the 116-kDa subunit in brain, and primer extension analysis demonstrates that the cloned sequence is full-length. The rat cDNA sequences predict synthesis of a protein of 96,267 Da. Analysis of the deduced amino acid sequence of the 116-kDa subunit suggests that it consists of two fundamental domains: a hydrophilic amino-terminal half that is composed of greater than 30% charged residues, and a hydrophobic carboxyl-terminal half that contains at least six transmembrane regions. The structural properties of the 116-kDa proton pump polypeptide agree well with its proposed function in coupling ATP hydrolysis by the cytoplasmic subunits to proton translocation by the intramembranous components of the pump.