The carboxyl group in the active center of transketolase

Transketolase from baker's yeast is rapidly inactivated in the presence of 1-ethyl-3 (3'-dimethylaminopropyl)-carbodiimide or Woodward's reagent K. In both cases the kinetics of inactivation is biphasic, which agrees with the presence of two active centers in the enzyme molecule differing in their sensitivity to the inhibitors. There is some evidence that inactivation of transketolase is due to modification of carboxyl groups of enzyme. Complete inactivation is achieved by modification of one carboxyl per active site of the enzyme. The experimental results suggest that the carboxyl group is essential for the enzymatic activity of transketolase.     

Specific modification of the carboxyl groups of hemoglobin S

The reactivity of the carboxyl groups of hemoglobin S to form amide bonds with glycine ethyl ester by carbodiimide-activated coupling, and the influence of this derivatization on the functional properties of the protein have been investigated. Incubation of carbonmonoxy or oxyhemoglobin S with 20 mM 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide in the presence of 100 mM [14C]glycine ethyl ester, at pH 6.0 and 23 degrees C for 1 h resulted in the modification of, on an average, three carboxyl groups of the protein. The Hill coefficient of the modified hemoglobin S was 2.7, indicating normal subunit interactions. The derivatization increased the oxygen affinity of the molecule (the P50 was lowered from 8.0 to 5.0). The derivatization also resulted in an increase in the minimum gelling concentration of hemoglobin S from 16 to 24 g/100 ml. The reaction conditions used for the derivatization of the carboxyl groups of hemoglobin S are very selective for the protein carboxyl groups; very little of the label is associated with the heme carboxyls. Tryptic peptide mapping of the modified hemoglobin S indicated that the peptide beta T5, i.e. the segment representing amino acid residues 41 to 59 of beta-chain, accounted for nearly 75% of the label associated with the globin, demonstrating the high selectivity of the derivatization. Sequence analysis of the derivatized beta T5 demonstrated that at least 65% of the label incorporated into hemoglobin S is targeted toward the carboxyl group of Glu-43(beta), identifying it as the most reactive carboxyl group in hemoglobins. The results suggest that modification of the carboxyl group of hemoglobins S, presumably the gamma-carboxyl of Glu-43(beta), reduces the propensity of deoxyhemoglobin S to polymerize.     

The carboxy-terminal tail of pyruvate dehydrogenase kinase 2 is required for the kinase activity

Pyruvate dehydrogenase kinase 2 (PDK2) is a prototypical mitochondrial protein kinase that regulates the activity of the pyruvate dehydrogenase complex. Recent structural studies have established that PDK2 consists of a catalytic core built of the B and K domains and the relatively long amino and carboxyl tails of unknown function. Here, we show that the carboxy-terminal truncation variants of PDK2 display a greatly diminished capacity for phosphorylation of holo-PDC. This effect is due largely to the inability of the transacetylase component of PDC to promote the phosphorylation reaction catalyzed by the truncated PDK2 variants. Furthermore, the truncated forms of PDK2 bind poorly to the lipoyl-bearing domain(s) provided by the transacetylase component. Taken together, these data strongly suggest that the carboxyl tails of PDK isozymes contribute to the lipoyl-bearing domain-binding site of the kinase molecule. We also show that the carboxyl tails derived from isozymes PDK1, PDK3, and PDK4 are capable of supporting the kinase activity of the kinase core derived from PDK2 as well as binding of the respective PDK2 chimeras to the lipoyl-bearing domain. Furthermore, the chimera carrying the carboxyl tail of PDK3 displays a stronger response to the addition of the transacetylase component along with a better binding to the lipoyl-bearing domain, suggesting that, at least in part, the differences in the amino acid sequences of the carboxyl tails account for the differences between PDK isozymes.     

Insensitivity of perturbed carboxyl pK(a) values in the ovomucoid third domain to charge replacement at a neighboring residue

A number of carboxyl groups in turkey ovomucoid third domain (OMTKY3) have low pK(a) values. A previous study suggested that neighboring amino groups were primarily responsible for the low carboxyl pK(a) values. However, the expected elevation in pK(a) values for these amino groups was not observed. In the present study, site-directed mutagenesis is used to investigate the origins of perturbed carboxyl pK(a) values in OMTKY3. Electrostatic calculations suggest that Lys 34 has large effects, 0.4-0.6 unit, on Asp 7, Glu 10, and Glu 19 which are 5-11 A away from Lys 34. Two-dimensional (1)H NMR techniques were used to determine pK(a) values of the acidic residues in OMTKY3 mutants in which Lys 34 has been replaced with threonine and glutamine. Surprisingly, the pK(a) values in the mutants are very close to those of the wild-type protein. The insensitivity of the acidic residues to replacement of Lys 34 suggests that long-range electrostatic interactions play less of a role in perturbing carboxyl pK(a) values than originally thought. We hypothesize that hydrogen bonds play a key role in perturbing some of the carboxyl ionization equilibria in OMTKY3.     

Pitfalls in the measurement of protein carboxyl methylation during chemotaxis of Dictyostelium discoideum

In Dictyostelium discoideum, a chemoacttractant-stimulated incorporation of radioactivity from [methyl-³H]methionine into protein in the presence of cycloheximide has previously been assumed to represent carboxyl methylation. In this paper, however, evidence is presented which demonstrates it to be a non-covalent binding of methionine to structural components of the cell. Certain pitfalls in the assay of carboxyl methylation by measurement of methanol production are described, and the assay has been optimized to avoid measurement of other volatile compounds. When carboxyl methylation is measured as the amount of methanol formed from methylated protein, methanol production is near the limit of detection in all current methods of assay. No increase of methanol production could be observed upon a single or repeated stimulation of aggregative cells with their chemoattractant, cyclic AMP. We conclude that carboxyl methylation is either absent in Dictyostelium, or that it involves only a small amount of specific methyl acceptors.     

Carboxyl-terminal truncations of the melibiose carrier of Escherichia coli

The melibiose carrier of Escherichia coli is predicted to possess a short NH2 terminus, 11 transmembrane segments joined by short hydrophilic regions, and a 40-residue hydrophilic carboxyl terminus of unknown function. This paper describes truncations of the carboxyl terminus at eight locations using site-specific mutagenesis to introduce stop codons. Measurement of sugar transport and cation-coupling characteristics indicate that the carboxyl tail plays no direct role in substrate recognition or energy transduction. Thirty-six amino acids could be removed from the hydrophilic carboxyl domain without the loss of sugar specificity, facilitated diffusion, uphill transport, H+-coupling or Na+-coupling characteristics. These results are consistent with the hypothesis that the sugar/cation binding site is formed by the interaction of the transmembrane helices 3, 4, 6, 9, and 10 and does not involve the carboxyl-terminal portion of the protein. When truncations were made within the hydrophobic domain of transmembrane helix 11 (truncations of 41 or more residues), the carrier was no longer found in the membrane. This suggests that the carboxyl terminus may be involved in the membrane insertion process, stabilization of the carrier within the membrane following insertion, or protection of the inserted carrier from proteolytic scavenging. A new plasmid that expresses the temperature-resistant isoform of the melibiose carrier under inducible control of a tac promoter, designated pKKMB, is also described.     

Assembly and processing of procollagen type III in chick embryo blood vessels

The processing of [3H]proline-labeled procollagen III in excised chick embryo blood vessels was found to differ significantly from that of procollagen I in the same tissue. While first the amino propeptides and then the carboxyl propeptides were fairly rapidly cleaved from procollagen I, only the carboxyl propeptides were split off procollagen III, leaving pN-collagen III. This intermediate, which is only slowly converted to collagen III by loss of amino propeptides, was characterized by its sedimentation properties, isolation of the amino propeptide, and reaction with purified antibodies that are specific against bovine amino propeptide III. It is interchain disulfide-linked, both through the amino propeptide and the carboxyl ends of the collagen chains. The conversion of procollagen III to pN-collagen III either in blood vessels, or after isolation by a carboxyl procollagen peptidase obtained from chick tendon fibroblast cultures, is inhibited by 50 mM arginine. Underhydroxylated procollagen III was isolated from blood vessels treated with alpha, alpha'-dipyridyl. Its amino propeptides reacted with the above antibodies but were not linked to each other. In contrast, its carboxyl propeptides were interchain disulfide-bridged, supporting previous suggestions that the carboxyl propeptides play a role in the assembly of procollagen trimer.     

Non-equivalence of the carboxyl groups of glucagon in the carbodiimide-promoted reaction with nucleophiles and the role of carboxyl groups in the ability of glucagon to stimulate the adenyl cyclase of rat liver

The polypeptide hormone glucagon can react with the nucleophiles; glycinamide, taurine or ethylenediamine in the presence of 1-ethyl-3-(3-dimethylaminopropylcarbodiimide). The number of carboxyl groups which are modified depend on the concentration of guanidine hydrochloride in the reaction media. These results demonstrate an additional property which glucagon possesses in common with larger globular proteins and suggests that the hormone has a specific, folded structure in dilute aqueous solution. In the absence of guanidine hydrochloride only one taurine residue is incorporated into the terminal carboxyl group of the peptide. In 7 M guanidine hydrochloride all four of the carboxyl groups react with glycinamide or taurine while only two and a half residues of ethylenediamine are incorporated. All of these derivatives and glucagon have identical circular dichroism spectra in dilute aqueous solution. The taurine modified derivative has greatly enhanced solubility compared with glucagon but still associates to structures of higher helical content. Both of the taurine derivatives of glucagon have the ability to stimulate the adenyl cyclase of rat liver membranes but at concentrations several fold higher than is needed for the native hormone. It is suggested that each carboxyl group contributes to the binding of the hormone to the specific membrane receptor sites.     

Carboxyl ester lipase cofractionates with scavenger receptor BI in hepatocyte lipid rafts and enhances selective uptake and hydrolysis of cholesteryl esters from HDL3

Cholesteryl esters are selectively removed from high density lipoproteins by hepatocytes and steroidogenic cells through a process mediated by scavenger receptor BI. In the liver this cholesterol is secreted into bile, primarily as free cholesterol. Previous work showed that carboxyl ester lipase enhanced selective uptake of cholesteryl ether from high density lipoprotein by an unknown mechanism. Experiments were performed to determine whether carboxyl ester lipase plays a role in scavenger receptor BI-mediated selective uptake. When added to cultures of HepG2 cells, carboxyl ester lipase cofractionated with scavenger receptor BI and [(3)H]cholesteryl ether-labeled high density lipoprotein in lipid raft fractions of cell homogenates. Confocal microscopy of immunostained carboxyl ester lipase and scavenger receptor BI showed a close association of these proteins in HepG2 cells. The enzyme and receptor also cofractionated from homogenates of mouse liver using two different fractionation methods. Antibodies that block scavenger receptor BI function prevented carboxyl ester lipase stimulation of selective uptake in primary hepatocytes from carboxyl ester lipase knockout mice. Heparin blockage of cell-surface proteoglycans also prevented carboxyl ester lipase stimulation of cholesteryl ester uptake by HepG2 cells. Inhibition of carboxyl ester lipase activity in HepG2 cells reduced hydrolysis of high density lipoprotein-cholesteryl esters approximately 40%. In vivo, hydrolysis was similarly reduced in lipid rafts from the livers of carboxyl ester lipase-null mice compared with control animals. Primary hepatocytes from these mice yielded similar results. The data suggest that carboxyl ester lipase plays a physiological role in hepatic selective uptake and metabolism of high density lipoprotein cholesteryl esters by direct and indirect interactions with the scavenger receptor BI pathway.     

Evolution of Cinnamate/p-coumarate carboxyl methyltransferases and their role in the biosynthesis of methylcinnamate

Methylcinnamate, which is widely distributed throughout the plant kingdom, is a significant component of many floral scents and an important signaling molecule between plants and insects. Comparison of an EST database obtained from the glandular trichomes of a basil (Ocimum basilicum) variety that produces high levels of methylcinnamate (line MC) with other varieties producing little or no methylcinnamate identified several very closely related genes belonging to the SABATH family of carboxyl methyltransferases that are highly and almost exclusively expressed in line MC. Biochemical characterization of the corresponding recombinant proteins showed that cinnamate and p-coumarate are their best substrates for methylation, thus designating these enzymes as cinnamate/p-coumarate carboxyl methyltransferases (CCMTs). Gene expression, enzyme activity, protein profiling, and metabolite content analyses demonstrated that CCMTs are responsible for the formation of methylcinnamate in sweet basil. A phylogenetic analysis of the entire SABATH family placed these CCMTs into a clade that includes indole-3-acetic acid carboxyl methyltransferases and a large number of uncharacterized carboxyl methyltransferase-like proteins from monocots and lower plants. Structural modeling and ligand docking suggested active site residues that appear to contribute to the substrate preference of CCMTs relative to other members of the SABATH family. Site-directed mutagenesis of specific residues confirmed these findings.     

Oligomerization,F-actin interaction,and membrane association of the ubiquitous mammalian coronin 3 are mediated by its carboxyl terminus

Coronin 3 is a ubiquitously expressed member of the coronin protein family in mammals. In fibroblasts and HEK 293 cells, it is localized both in the cytosol and in the submembranous cytoskeleton, especially at lamellipodia and membrane ruffles. The carboxyl terminus of all coronins contains a coiled coil suggested to mediate dimerization. We show here that in contrast to other coronin homologues, the recombinant human coronin 3 carboxyl terminus forms oligomers rather than dimers, and that this part is sufficient to bind to and cross-link F-actin in vitro. The carboxyl terminus alone also conferred membrane association in vivo, and removal of the coiled coil abolished membrane localization but not in vitro F-actin binding. Coronin 3 is exclusively extracted as an oligomer from both the cytosol and the membrane fraction. Because oligomerization was not reported for other coronins, it might be a key feature governing coronin 3-specific functions. Cytosolic coronin 3 showed a high degree of phosphorylation, which is likely to regulate the subcellular localization of the protein.     

The essential activated carboxyl group of inorganic pyrophosphatase.

1. A carboxyl group of high reactivity has been found in inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) from yeast. This group interacts with agents which react neither with carboxyl groups of low molecular weight compounds nor with other carboxyl groups of the protein. 2. The reaction of this activated carboxyl group with inorganic phosphate, hydroxylamine, N-methyl- and O-methylhydroxylamines, and glycine methyl ester has been studied. 3. Homoserine and homoserine lactone were found in the hydrolyzate of phosphorylated and NaBH4-reduced pyrophosphatase, indicating that an aspartyl residue is phosphorylated. 4. Hydroxylamine and other nucleophilic agents cause inactivation of pyrophosphatase as a result of interaction with a carboxyl group. Both diaminobutyric and diaminopropionic acids were seen in the acid hydrolyzate of the protein treated with hydroxylamine and subjected to rearrangement in the presence of carbodiimide. 5. The ways in which the activation of a carboxyl group in the enzyme is achieved and the presumed mechanism of action of inorganic pyrophosphatase are discussed.     

Chemical modification as a probe of the topography and reactivity of horse-spleen apoferritin.

In apoferritin, but not in ferritin, 1.0 +/- 0.1 cysteine residue per subunit can be modified. In ferritin 3.3 +/- 0.3 lysine residues and 7.1 +/- 0.7 carboxyl groups per subunit can be modified, whilst the corresponding values for apoferritin are 4.4 +/- 0.4 lysine residues and 11.0 +/- 0.4 carboxyl groups per subunit. Modification of lysine residues which maleic anhydride and carboxyl groups with glycineamide in apoferritin which has been dissociated and denatured in guanidine hydrochloride leads to the introduction of 9.1 +/- 0.5 maleyl groups per subunit and 22.0 +/- 0.9 glycineamide residues per subunit. Whereas unmodified apoferritin subunit can be reassociated from guanidine hydrochloride to apoferritin monomer, the ability of maleylated apoferritin to reassociate is impaired. Apoferritin in which all the carboxyl groups have been blocked with glycineamide cannot be reassociated to apoferritin and exists in solution as stable subunits. The modification of one cysteine residue per subunit, of 3 or 4 lysine residues per subunit or of 7 carboxyl groups per subunit has no effect on the catalytic activity of apoferritin. In contrast the modification of 11 carboxyl groups per subunit completely abolishes the catalytic properties of the protein. We conclude that one or more carboxyl groups are essential for the catalytic activity of horse spleen apoferritin.     

The function of free carboxyl groups in the action of peroxidase and indole-3-acetic acid oxidase

The extracellular peroxidase from cultures of Inonotus radiatus and of peanut (Arachis hypogeaL.) cells as well as the mycelial peroxidase from Trametes versicolor were used for studies of immobilizing this protein either by its free amino or its carboxyl groups. The immobilization process was carried out either on keratin proteins derived from feathers or on polyamide coated over silica gel. Coupling was established either through the free amino or carboxyl groups. In general the indolyl-3-acetic acid oxidase activity of fungal peroxidases exceeds that of peanut peroxidase. When the peroxidase of the three sources was immobilized on the matrices by the free amino groups, little if any effect on the IAA oxidase activity could be measured. However, immobilization through the carboxyl groups resulted in a drastic reduction of indole-3-acetic acid oxidase activity. Since identical amounts of peroxidase were linked in all cases, the loss of indolyl-3-acetic acid oxidase activity implies that the carboxyl group is essential for the active site.     

A novel UbcH10-binding protein facilitates the ubiquitinylation of cyclin B in vitro

UbcH10 is known to act as a ubiquitin-conjugating enzyme (E2) for anaphase-promoting complex/cyclosome. Since some E2s support different ubiquitin ligases (E3), it is possible that UbcH10 interacts with other proteins. We cloned a novel protein named H10BH by using a yeast two-hybrid screening method with UbcH10 as bait. The carboxyl terminus of H10BH showed a weak homology to the HECT (homologous to E6-AP carboxyl terminus) domain, which is conserved in one of the families of E3. H10BH bound UbcH10, and the amino acid sequence between 235 and 257 was necessary for this binding. H10BH showed a self-ubiquitinylation activity in a HECT-like sequence-dependent manner. The carboxyl terminal half (amino acids 188-389) showed stronger activity than the full-length H10BH. Furthermore, the carboxyl terminal half of H10BH was able to bind cyclin B and ubiquitinylate cyclin B in vitro. These results suggest that H10BH functions as an E3 using UbcH10 for its E2.     

The carboxyl terminus of v-Abl protein can augment SH2 domain function

Abelson murine leukemia virus (Ab-MLV) transforms NIH 3T3 and pre-B cells via expression of the v-Abl tyrosine kinase. Although the enzymatic activity of this molecule is absolutely required for transformation, other regions of the protein are also important for this response. Among these are the SH2 domain, involved in phosphotyrosine-dependent protein-protein interactions, and the long carboxyl terminus, which plays an important role in transformation of hematopoietic cells. Important signals are sent from each of these regions, and transformation is most likely orchestrated by the concerted action of these different parts of the protein. To explore this idea, we compared the ability of the v-Src SH2 domain to substitute for that of v-Abl in the full-length P120 v-Abl protein and in P70 v-Abl, a protein that lacks the carboxyl terminus characteristic of Abl family members. Ab-MLV strains expressing P70/S2 failed to transform NIH 3T3 cells and demonstrated a greatly reduced capacity to mediate signaling events associated with the Ras-dependent mitogen-activated protein (MAP) kinase pathway. In contrast, Ab-MLV strains expressing P120/S2 were indistinguishable from P120 with respect to these features. Analyses of additional mutants demonstrated that the last 162 amino acids of the carboxyl terminus were sufficient to restore transformation. These data demonstrate that an SH2 domain with v-Abl substrate specificity is required for NIH 3T3 transformation in the absence of the carboxyl terminus and suggest that cooperativity between the extreme carboxyl terminus and the SH2 domain facilitates the transmission of transforming signals via the MAP kinase pathway.     

Nucleotide Sequence of Canine Smad3

The whole genome sequence of a Boxer dog suggested that the amino acid sequence of the carboxyl terminus of a putative Smad3 is SSVF-_COOH, not SSVS-_COOH as in all Smad3 sequences identified in many species. Because phosphorylation of the last two serines at the carboxyl terminus is generally indispensable for Smad3-mediated signaling, the role of Smad3 may be unique in dogs. The present study determines the nucleotide sequence of the coding region of canine Smad3 and deduces the carboxyl terminal amino acids of Smad3 in several breeds. Except for the Boxer, the deduced amino acid sequence was SSVS-_COOH in all dogs examined. In addition, the nucleotide at position 1204 in the Boxer was different from that of the other dogs. Furthermore, there was a SNP at nt 240. The present study indicates that the carboxyl terminal amino acid of canine Smad3 is not unique, although it is unknown in the Boxer breed.     

pH dependence of the reverse reaction catalyzed by phosphofructokinase I from Escherichia coli: implications for the role of Asp 127.

The kinetics of the reverse reaction catalyzed by Escherichia coli phosphofructokinase, i.e., the synthesis of ATP and fructose-6-phosphate from ADP and fructose-1,6-bisphosphate, have been studied at different pH values, from pH 6 to pH 9.2. Hyperbolic saturations of the enzyme are observed for both substrates. The affinity for fructose-1,6-bisphosphate decreases with pH following the ionization of a group with a pK of 6.6, whereas the catalytic rate constant and perhaps the affinity for ADP are controlled by the ionization of a group with a pK of 6. Several arguments show that the pK of 6.6 is probably that of the carboxyl group of Asp 127, whereas the pK of 6 is tentatively attributed to the carboxyl group of Asp 103. The pK of 6.6 is assigned to the carboxyl group of Asp 127 in the free enzyme, and a simple model suggests that the same group would have an abnormally high pK, above 9.6, in the complex between phosphofructokinase and fructose-1,6-bisphosphate. It is proposed that the large pK shift of more than 3 pH units upon binding of fructose-1,6-bisphosphate is due to an electrostatic repulsion that could exist between the 1-phosphate group and the carboxyl group of Asp 127, which are close to each other in the crystal structure of phosphofructokinase (Shirakihara, Y. & Evans, P.R., 1988, J. Mol. Biol. 204, 973-994). The same interpretation would also explain the much higher affinity of the enzyme for fructose-1,6-bisphosphate when Asp 127 is protonated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of the human gamma-fibrinogen gene. Alternate mRNA splicing near the 3' end of the gene produces gamma A and gamma B forms of gamma-fibrinogen

The gamma chain of human fibrinogen exists in 2 nonallelic forms, gamma A and gamma B, which differ only in their carboxyl termini. We have found that only one genomic locus exists for gamma-fibrinogen and that the gamma A and gamma B chains arise by alternate mRNA splicing near the 3' end of this gene. In contrast to the rat gamma B mRNA which is produced by alternate splicing with identical polyadenylation sites, human gamma B is produced when the eighth intervening sequence remains unspliced and a polyadenylation signal within this intron is used. The new carboxyl terminus is 16 amino acids longer than the gamma A protein, and although there is only minimal homology between the rat and human carboxyl termini they share a very high proportion of acidic amino acids.     

Intracellular translocation of the decapeptide carboxyl terminal of Gi3 alpha induces the dual phosphorylation of p42/p44 MAP kinases

The carboxyl terminal of heterotrimeric G protein alpha subunits binds both G protein-coupled receptors and mastoparan (MP), a tetradecapeptide allostere. Moreover, peptides corresponding to the carboxyl domains of G(i)3alpha and G(t) display intrinsic biological activities in cell-free systems. Thus, the purpose of this study was to develop a cell penetrant delivery system to further investigate the biological properties of a peptide mimetic of the G(i)3alpha carboxyl terminal (G(i)3alpha(346-355); H-KNNLKECGLY-NH2). Kinetic studies, using a CFDA-conjugated analogue of G(i)3alpha(346-355), confirmed the rapid and efficient intracellular translocation of TP10-G(i)3alpha(346-355) (t(0.5) = 3 min). Translocated G(i)3alpha(346-355), but not other bioactive cargoes derived from PKC and the CB1 cannabinoid receptor, promoted the dual phosphorylation of p42/p44 MAPK without adverse changes in cellular viability. The relative specificity of this novel biological activity was further confirmed by the observation that translocated G(i)3alpha(346-355) did not influence the exocytosis of beta-hexoseaminidase from RBL-2H3, a secretory event stimulated by other cell penetrant peptide cargoes and MP. We conclude that TP10-G(i)3alpha(346-355) is a valuable, non-toxic research tool with which to study and modulate signal transduction pathways mediated by heterotrimeric G proteins and MAPK.