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PubMed2000

Identification of a Drosophila vitamin K-dependent gamma-glutamyl carboxylase.

Using reduced vitamin K, oxygen, and carbon dioxide, gamma-glutamyl carboxylase post-translationally modifies certain glutamates by adding carbon dioxide to the gamma position of those amino acids. In vertebrates, the modification of glutamate residues of target proteins is facilitated by an interaction between a propeptide present on target proteins and the gamma-glutamyl carboxylase. Previously, the gastropod Conus was the only known invertebrate with a demonstrated vitamin K-dependent carboxylase. We report here the discovery of a gamma-glutamyl carboxylase in Drosophila. This Drosophila enzyme is remarkably similar in amino acid sequence to the known mammalian carboxylases; it has 33% sequence identity and 45% sequence similarity to human gamma-glutamyl carboxylase. The Drosophila carboxylase is vitamin K-dependent, and it has a K(m) toward a model pentapeptide substrate, FLEEL, of about 4 mm. However, unlike the human gamma-glutamyl carboxylase, it is not stimulated by human blood coagulation factor IX propeptides. We found the mRNA for Drosophila gamma-glutamyl carboxylase in virtually every embryonic and adult stage that we investigated, with the highest concentration evident in the adult head.

Adult↗

PubMed2005

The vitamin K-dependent carboxylase has been acquired by Leptospira pathogens and shows altered activity that suggests a role other than protein carboxylation.

Leptospirosis is an emerging infectious disease whose pathology includes a hemorrhagic response, and sequencing of the Leptospira interrogans genome revealed an ortholog of the vitamin K-dependent (VKD) carboxylase as one of several hemostatic proteins present in the bacterium. Until now, the VKD carboxylase was known to be present only in the animal kingdom (i.e. metazoans that include mammals, fish, snails, and insects), and this restricted distribution and high sequence similarity between metazoan and Leptospira orthologs strongly suggests that Leptospira acquired the VKD carboxylase by horizontal gene transfer. In metazoans, the VKD carboxylase is bifunctional, acting as an epoxidase that oxygenates vitamin K to a strong base and a carboxylase that uses the base to carboxylate Glu residues in VKD proteins, rendering them active in hemostasis and other physiologies. In contrast, the Leptospira ortholog showed epoxidase but not detectable carboxylase activity and divergence in a region of identity in all known metazoan VKD carboxylases that is important to Glu interaction. Furthermore, although the mammalian carboxylase is regulated so that vitamin K epoxidation does not occur unless Glu substrate is present, the Leptospira VKD epoxidase showed unfettered epoxidation in the absence of Glu substrate. Finally, human VKD protein orthologs were not detected in the L. interrogans genome. The combined data, then, suggest that Leptospira exapted the metazoan VKD carboxylase for some use other than VKD protein carboxylation, such as using the strong vitamin K base to drive a new reaction or to promote oxidative damage or depleting vitamin K to indirectly inhibit host VKD protein carboxylation.

Amino Acid Sequence↗

PubMed1975

Loss of Ribulose 1,5-Diphosphate Carboxylase and Increase in Proteolytic Activity during Senescence of Detached Primary Barley Leaves.

Symptoms typical of senescence occurred in green detached primary barley (Hordeum vulgare L.) leaves placed in darkness and in light. Chlorophyll, total soluble protein, ribulose 1,5-diphosphate carboxylase protein and activity each progressively decreased in darkness and to a lesser extent in light. In all treatments most of the total soluble protein lost was accounted for by a decrease in ribulose 1,5-diphosphate carboxylase protein, suggesting that the chloroplast was a major site of degradation early in senescence.Loss of ribulose 1,5-diphosphate carboxylase protein was negatively correlated with an increase in proteolytic activity measured against azocasein. Both rates were exponential, with about a 30% difference in apparent rate constants. Cycloheximide essentially prevented the loss of chlorophyll, ribulose 1,5-diphosphate carboxylase protein, and activity and completely inhibited the increase in proteolytic activity against azocasein. Since chloramphenicol had little effect on the loss of ribulose 1,5-diphosphate carboxylase protein or chlorophyll, or on proteolytic activity against azocasein, it is suggested that the proteolytic activity was developed on cytoplasmic 80 S ribosomes.Kinetin greatly retarded the onset of such symptoms of senescence by inhibiting the losses of chlorophyll and ribulose 1,5-diphosphate carboxylase protein and protected against inactivation of enzymic activity. It also prevented the increase in proteolytic activity measured against azocasein. Incorporation of labeled amino acids into ribulose 1,5-diphosphate carboxylase during its rapid degradation showed that the enzyme was under turnover. The changes in ribulose 1,5-diphosphate carboxylase protein and activity, chlorophyll, soluble protein other than ribulose 1,5-diphosphate carboxylase, proteolytic and esterolytic activity during senescence indicate that senescence is a selective, sequential process.

Journal Article↗

PubMed1981

Ribulose 1,5-bisphosphate and activation of the carboxylase in the chloroplast.

Ribulose 1,5-bisphosphate in the chloroplast has been suggested to regulate the activity of the ribulose bisphosphate carboxylase/oxygenase. To generate high levels of ribulose bisphosphate, isolated and intact spinach chloroplasts were illuminated in the absence of CO(2). Under these conditions, chloroplasts generate internally up to 300 nanomoles ribulose 1,5-bisphosphate per milligram chlorophyll if O(2) is also absent. This is equivalent to 12 millimolar ribulose bisphosphate, while the enzyme, ribulose bisphosphate carboxylase, offers up to 3.0 millimolar binding sites for the bisphosphate in the chloroplast stroma. During illumination, the ribulose bisphosphate carboxylase is deactivated, due mostly to the absence of CO(2) required for activation. The rate of deactivation of the ribulose bisphosphate carboxylase was not affected by the chloroplast ribulose bisphosphate levels. Upon addition of CO(2), the carboxylase in the chloroplast was completely reactivated. Of interest, addition of 3-phosphoglycerate stopped deactivation of the carboxylase in the chloroplast while ribulose bisphosphate accumulated. With intact chloroplasts in light, no correlation between deactivation of the carboxylase and ribulose bisphosphate levels could be shown.In contrast, incubation of purified ribulose bisphosphate carboxylase with ribulose bisphosphate irreversibly inhibited activation, especially in the absence of CO(2). Addition of the same amount of ribulose bisphosphate to lysed chloroplasts did cause some deactivation of the carboxylase in the extract, but full activation returned when the ribulose bisphosphate was consumed. The ribulose bisphosphate carboxylase in the chloroplast is not irreversibly inhibited by high levels of ribulose bisphosphate.

Journal Article↗

PubMed1983

Photosynthesis and Ribulose 1,5-Bisphosphate Carboxylase in Rice Leaves: Changes in Photosynthesis and Enzymes Involved in Carbon Assimilation from Leaf Development through Senescence.

Changes in photosynthesis and the ribulose 1,5-bisphosphate (RuBP) carboxylase level were examined in the 12th leaf blades of rice (Oryza sativa L.) grown under different N levels. Photosynthesis was determined using an open infrared gas analysis system. The level of RuBP carboxylase was measured by rocket immunoelectrophoresis. These changes were followed with respect to changes in the activities of RuBP carboxylase, ribulose 5-phosphate kinase, NADP-glyceraldehyde 3-phosphate dehydrogenase, and 3-phosphoglyceric acid kinase.RuBP carboxylase activity was highly correlated with the net rate of photosynthesis (r = 0.968). Although high correlations between the activities of other enzymes and photosynthesis were also found, the activity per leaf of RuBP carboxylase was much lower than those of other enzymes throughout the leaf life. The specific activity of RuBP carboxylase on a milligram of the enzyme protein basis remained fairly constant (1.16 +/- 0.07 micromoles of CO(2) per minute per milligram at 25 degrees C) throughout the experimental period.Kinetic parameters related to CO(2) fixation were examined using the purified carboxylase. The K(m)(CO(2)) and V(max) values were 12 micromolar and 1.45 micromoles of CO(2) per minute per milligram, respectively (pH 8.2 and 25 degrees C). The in vitro specific activity calculated at the atomospheric CO(2) level from the parameters was comparable to the in situ true photosynthetic rate per milligram of the carboxylase throughout the leaf life.The results indicated that the level of RuBP carboxylase protein can be a limiting factor in photosynthesis throughout the life span of the leaf.

Journal Article↗

PubMed1989

Interference by phosphatases in the spectrophotometric assay for phosphoenolpyruvate carboxylase.

The aim of this work was to discover the extent of interference by phosphoenolpyruvate (PEP) phosphatase in spectrophotometric assays of PEP carboxylase (EC 4.1.1.31) in crude extracts of plant organs. The presence of PEP phosphatase and lactate dehydrogenase (EC 1.1.1.27) in extracts leads to PEP-dependent NADH oxidation that is independent of PEP carboxylase activity, and hence to overestimation of PEP carboxylase activity. In extracts of three organs of pea (Pisum sativum L.: leaves, developing embryos, and Rhizobium nodules), two organs of wheat (Triticum aestivum L.: developing grain and endosperm), and leaves of Moricandia arvensis (L.) D.C., lactate dehydrogenase activity was at most only 16% of that of PEP carboxylase at the pH optimum for PEP carboxylase activity. Endogenous PEP phosphatase and lactate dehydrogenase are thus unlikely to interfere seriously with the assay for PEP carboxylase at its optimum pH. Addition of lactate dehydrogenase to PEP carboxylase assays- a proposed means of correcting for nonenzymic decarboxylation of oxaloacetate to pyruvate-resulted in increases in PEP-dependent NADH oxidation from zero (Rhizobium nodules) to 131% (wheat grains). There was no obvious relationship between the magnitude of this increase and conditions in the assay that might promote oxaloacetate decarboxylation. However, the magnitude of the increase was highly positively correlated with the activity of PEP phosphatase in the extract. Addition of lactate dehydrogenase to PEP carboxylase assays can thus result in very large overestimations of PEP carboxylase activity, and should only be used as a means of correction for oxaloacetate decarboxylation for extracts with negligible PEP phosphatase activity.

Journal Article↗

PubMed2003

Characterization of a bifunctional archaeal acyl coenzyme A carboxylase.

Acyl coenzyme A carboxylase (acyl-CoA carboxylase) was purified from Acidianus brierleyi. The purified enzyme showed a unique subunit structure (three subunits with apparent molecular masses of 62, 59, and 20 kDa) and a molecular mass of approximately 540 kDa, indicating an alpha(4)beta(4)gamma(4) subunit structure. The optimum temperature for the enzyme was 60 to 70 degrees C, and the optimum pH was around 6.4 to 6.9. Interestingly, the purified enzyme also had propionyl-CoA carboxylase activity. The apparent K(m) for acetyl-CoA was 0.17 +/- 0.03 mM, with a V(max) of 43.3 +/- 2.8 U mg(-1), and the K(m) for propionyl-CoA was 0.10 +/- 0.008 mM, with a V(max) of 40.8 +/- 1.0 U mg(-1). This result showed that A. brierleyi acyl-CoA carboxylase is a bifunctional enzyme in the modified 3-hydroxypropionate cycle. Both enzymatic activities were inhibited by malonyl-CoA, methymalonyl-CoA, succinyl-CoA, or CoA but not by palmitoyl-CoA. The gene encoding acyl-CoA carboxylase was cloned and characterized. Homology searches of the deduced amino acid sequences of the 62-, 59-, and 20-kDa subunits indicated the presence of functional domains for carboxyltransferase, biotin carboxylase, and biotin carboxyl carrier protein, respectively. Amino acid sequence alignment of acetyl-CoA carboxylases revealed that archaeal acyl-CoA carboxylases are closer to those of Bacteria than to those of Eucarya. The substrate-binding motifs of the enzymes are highly conserved among the three domains. The ATP-binding residues were found in the biotin carboxylase subunit, whereas the conserved biotin-binding site was located on the biotin carboxyl carrier protein. The acyl-CoA-binding site and the carboxybiotin-binding site were found in the carboxyltransferase subunit.

Amino Acid Sequence↗

PubMed1983

[3H]biotin-labeled proteins in cultured human skin fibroblasts from patients with pyruvate carboxylase deficiency.

Biotin containing carboxylases in cultured human skin fibroblasts were radioactively labeled by addition of [8,9-3H]biotin to biotin-depleted cell cultures. Three major bands were visualized by fluorography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fibroblast proteins. These bands corresponded to pyruvate carboxylase (Mr = 125,000), the biotin-containing subunit of methyl crotonyl-CoA carboxylase (Mr = 75,000) and the biotin-containing subunit of propionyl-CoA carboxylase (Mr = 73,000) as judged by molecular weight markers, purified carboxylase protein standards, and interaction with monospecific antisera. Four out of 5 cell lines from patients with classical pyruvate carboxylase deficiency (less than 5% of normal activity) labeled with this technique displayed a normal band in the position of pyruvate carboxylase while one cell line showed complete absence of any labeled protein in this area. These results demonstrate heterogeneity in the etiology of pyruvate carboxylase deficiency.

Biotin↗

PubMed1981

Biochemical characterization of biotin-responsive multiple carboxylase deficiency: heterogeneity within the bio genetic complementation group.

Three biotin-dependent enzymes, pyruvate carboxylase (PC), propionyl CoA carboxylase (PCC), and beta-methylcrotonyl CoA carboxylase (beta MCC), were biochemically characterized in fibroblasts from two patients with neonatal multiple carboxylase deficiency. Genetic complementation analyses indicated that both cell lines, designated lines 1 and 2, were deficient in the various carboxylase activities and belonged to the bio complementation group. The activities of the three carboxylases became normal when line 2 cells were incubated in medium supplemented with biotin (1 mg/l) for 24 hrs, whereas 4-6 days were required to achieve maximum activities of PC, PCC, and beta MCC (57%, 46%, and 29% of mean normal enzyme activity, respectively) in line 1 cells incubated in medium containing up to 10 mg/1 biotin. Furthermore, PC activity in line 2 continued to increase under apparent gluconeogenic conditions in culture, but not in line 1. Thermostability studies suggested that biotin stabilizes PC and beta MCC in both cell lines. PC in line 1 cells incubated with or without biotin was less stable than that in normal or line 2 cells, and the less than normal increase of enzyme activities in line 1, especially that of PC, may represent incomplete biotination. These results indicate that there is biochemical heterogeneity within the bio complementation group. Immunotitration with antibodies prepared against purified pig heart PCC demonstrated normal quantities of cross-reacting material in both lines and no differences in the amount of this material after incubation with supplemental biotin, despite the seven- to 20-fold increase in PCC activity. Thus, the increase in carboxylase activity in both bio lines appears to represent activation of rpe-existing apocarboxylase rather than de novo enzyme synthesis. The primary defect in this form of multiple carboxylase deficiency may be in a common holocarboxylase synthetase or in biotin transport. If the defect is in the synthetase, the differences noted between the two bio lines could be explained by a difference in the enzyme's Km for biotin.

Biotin↗

PubMed1980

Induction of pyruvate carboxylase apoenzyme and holoenzyme in 3T3-L1 cells during differentiation.

The specific activity of pyruvate carboxylase [pyruvate:carbon-dioxide ligase (ADP-forming); EC 6.4.1.1] in 3T3-L1 cells increases approximately 20-fold when these cells differentiate to an adipocyte-like form [Mackall, J. C. & Lane, M. D. (1977) Biochem. Biophys. Res. Commun. 79, 720-725]. A specific antibody to the purified rat liver enzyme quantitatively precipitated pyruvate carboxylase from 3T3-L1 crude homogenates. Use of this immunological technique permitted us to demonstrate that the increase in pyruvate carboxylase activity is due to an increase in the intracellular concentration of the enzyme. The content of pyruvate carboxylase in differentiated 3T3-L1 cells is sufficiently high (1-2% of total protein) that the increase in this large protein (subunit M(r) = 130,000) can be visualized when 3T3-L1 crude extracts are subjected to electrophoresis on sodium dodecyl sulfate/polyacrylamide gels. When 3T3-L1 cells differentiated in the presence of avidin, they contained less than 5% of the pyruvate carboxylase activity of cells that differentiated in the absence of avidin. However, the immunoprecipitable pyruvate carboxylase content of the avidin-treated cells was essentially the same as that of cells that differentiated without avidin. Full activity of the enzyme was rapidly restored in the avidin-treated cells upon the addition of excess biotin. The recovery of activity was closely correlated with the incorporation of [(14)C]biotin into immunoprecipitable pyruvate carboxylase. The rapidity with which the activity was restored and the insensitivity of the process to inhibitors of protein synthesis strongly suggest that the apoenzyme of pyruvate carboxylase accumulates during differentiation in the presence of avidin.

Adipose Tissue↗

PubMed1980

Cell-free translation and regulation of Candida lipolytica acetyl-coenzyme-A carboxylase messenger RNA.

Messenger RNA from Candida lipolytica directs the synthesis of complete acetyl-coenzyme-A carboxylase in the mRNA-dependent reticulocyte lysate cell-free translation system. The identity of the translation product is evidenced by the following results: first, it is immunoprecipitated with antibody to acetyl-CoA carboxylase and competes with authentic acetyl-CoA carboxylase for binding to the antibody; secondly, it co-migrates with authentic acetyl-CoA carboxylase (Mr 230000) upon dodecylsulfate/polyacrylamide gel electrophoresis; finally, the peptide fragments formed by its partial proteolysis with papain or alpha-chymotrypsin are identical with those formed from authentic acetyl-CoA carboxylase. With the use of this assay system, it has been demonstrated that the level of acetyl-CoA carboxylase mRNA activity in C. lipolytica cells decreases with increasing concentrations of oleic acid in culture medium and that the changes in the mRNA activity parallel those in the cellular level of acetyl-CoA carboxylase. This finding, in conjunction with our previous study, indicates that the diminished synthesis of acetyl-CoA carboxylase in cells grown in the presence of fatty acid is due to a reduced level of the mRNA coding for the enzyme.

Acetyl-CoA Carboxylase↗

PubMed1994

The modulation of enzyme reaction rates within multi-enzyme complexes. 2. Information transfer within a chloroplast multi-enzyme complex containing ribulose bisphosphate carboxylase-oxygenase.

Octameric ribulose bisphosphate carboxylase-oxygenase binds in an independent manner its substrate (ribulose bisphosphate) and a substrate analog (6-phosphogluconate). The eight active sites of the free enzyme are thus independent. The kinetic behaviour of the active site becomes different if ribulose bisphosphate carboxylase-oxygenase is inserted in the five-enzyme complex previously isolated from chloroplasts. Ribulose bisphosphate carboxylase-oxygenase then becomes more active than the corresponding free enzyme form. By comparing the behaviour of the same enzyme in the free state and in the associated state it then becomes possible to study how the thermodynamics of protein-protein interactions alters the kinetic behaviour of ribulose bisphosphate carboxylase-oxygenase. This alteration may be expressed in terms of stabilization-destabilization energies exerted upon the various intermediate states of the enzyme reaction, within the multi-protein complex. Heterologous interactions within this complex exert a constant stabilization energy on the enzyme ground states along the reaction co-ordinate of -1.68 kJ/mol and a constant stabilization energy of -3.79 kJ/mol on the enzyme transition states. These stabilization energies express how information propagates within the multi-enzyme complex as to increase the apparent affinity of the substrate for the active sites of ribulose bisphosphate carboxylase-oxygenase, as well as to increase the catalytic rate constant. The binding of the substrate analog 6-phosphogluconate to free ribulose bisphosphate carboxylase-oxygenase is non-co-operative. It becomes positively co-operative if this enzyme is inserted in the multi-protein complex. Under these conditions, only one type of enzyme-inhibitor complex is detected experimentally. Here again heterologous interactions stabilize this enzyme-inhibitor complex relative to that expected if ribulose bisphosphate carboxylase oxygenase is free. The extent of stabilization is -1.03 kJ/mol. Neither free nor associated ribulose bisphosphate carboxylase-oxygenase display any co-operativity relative to substrate binding. However, in the presence of the substrate analog 6-phosphogluconate, this enzyme displays positive co-operativity relative to substrate, although not if it is naked. These results can be explained theoretically and show that the maximum value of the Hill coefficient is < or = 2. As 6-phosphogluconate and other substrate analogs are present in chloroplasts under normal conditions, this co-operativity might be of functional importance in vivo.

Binding Sites↗

PubMed1994

Cloning of human acetyl-CoA carboxylase cDNA.

Acetyl-CoA carboxylase is the rate-limiting enzyme in the biogenesis of long-chain fatty acids. In order to understand the mechanisms that regulate human acetyl-CoA carboxylase at the gene level, and the relationship between its structure and function, cDNA clones for human acetyl-CoA carboxylase have been isolated and sequenced. Human acetyl-CoA-carboxylase cDNA contains 7020 nucleotides encoding a protein of 2340 amino acids with a calculated relative molecular mass of 264575. The human enzyme shows approximately 85% identity in nucleotide sequence with previously cloned rat acetyl-CoA carboxylase, and shows 90% identity in the amino acid sequence. Two human acetyl-CoA-carboxylase mRNA species, which differ in the 5' untranslated region with the same coding sequence, have been identified. The sequence analysis reveals that type I and type II acetyl-CoA-carboxylase mRNA contain 313- and 173-base-long 5' untranslated regions, respectively. The first 240 nucleotides in the 5' untranslated region of type I acetyl-CoA-carboxylase mRNA replace the first 100 nucleotides of the (G + C)-rich region of the 5' untranslated region of the type II mRNA. These two species of mRNAs are the only species of human ACC mRNA which have been detected compared to at least five species in rat tissues, and they are expressed in a tissue-specific manner.

Acetyl-CoA Carboxylase↗

PubMed1987

Phosphorylation state of acetyl-coenzyme A carboxylase. II. Variation with nutritional condition.

Acetyl-CoA carboxylase from liver exhibits a linear inverse relationship between the ratio of enzymic activities at 0 and 2 mM citrate and the extent of phosphorylation by its kinase, and this citrate activity ratio method was used to examine the effect of nutritional conditions on the phosphorylation state of the enzyme. This method showed that the calculated phosphorylation state, being the extent of phosphorylation at sites accessible to carboxylase kinase, was highest in the livers of starved rats, lower in those fed normally, and lower still in starved rats which had been refed for 48 h on a fat-free diet. The actual values were 0.44, 0.26, and 0 mol of P/subunit, respectively, provided that liver samples were frozen rapidly to liquid nitrogen temperatures and extracted with stopping buffers at temperatures well below freezing. Normal homogenization with stopping buffers (containing inhibitors for protein kinases and phosphatases) resulted in much higher calculated phosphorylation states. The effect of nutritional conditions on the phosphorylation state as estimated reported above was confirmed by purifying the carboxylase from livers of rats, measuring the amount of phosphate which could be incorporated by carboxylase kinase, and comparing this with the phosphorylation state calculated from the citrate activity ratio method or the specific activity. Furthermore, treatment with protein phosphatase of carboxylase from starved rats resulted in the largest increase in specific activity, that from the starved/refed rats in the least. Finally, the effects of hyperglycemia on carboxylase and phosphorylase characteristics in the livers of intact rats were ascertained by taking liver samples and preparing crude extracts by the rapid freezing method described above. Hyperglycemia caused a rapid increase in the activity of the carboxylase and a rapid decrease in its putative phosphorylation state as measured by the citrate activity ratio method. Phosphorylase was also dephosphorylated, as indicated by a decrease in phosphorylase a activity. We conclude that the citrate activity ratio method is a valid test for the phosphorylation state of acetyl-CoA carboxylase in crude extracts of tissue.

Acetyl-CoA Carboxylase↗

PubMed1980

Interaction of the fluorescent analogue stearoyl-(1,N6)-etheno coenzyme A with chicken liver acetyl coenzyme A carboxylase.

The interaction of stearoyl-(1,N6)-etheno coenzyme A (stearoyl-epsilon-CoA) with acetyl coenzyme A carboxylase was investigated by using fluorescence spectroscopy. The fluorescence emission of stearoyl-epsilon-CoA was partially quenched by acetyl coenzyme A carboxylase. Analysis of the data for dissociation constant (KD) and the stoichiometry of the interaction (n) gave values of 5.06 nM and 1.2, respectively, at pH 7.6 in 50 mM Tris-HCl and 25 degrees C. The KD value is comparable to the inhibition constant (Ki) obtained previously by others for the inhibition of rat liver acetyl coenzyme A carboxylase by long chain fatty acyl-CoAs. Citrate (which is known to polymerize and thus activate carboxylase) caused a partial quenching of the protein fluorescence of carboxylase, presumably due to polymerization of the enzyme. The quenching of the stearoyl-epsilon-CoA fluorescence caused by carboxylase as well as the inhibition of carboxylase activity by stearoyl-epsilon-CoA was reversed by citrate, but only in the presence of 6-O-methylglucose polysaccharide which forms a stable complex with fatty acyl-CoA. This shows that the stearoyl-epsilon-CoA bound to the enzyme is displaced by citrate only in the presence of an acceptor of fatty acyl-CoA. These results support the reciprocal relationship of citrate and fatty acyl-CoA in the regulation of acetyl coenzyme A carboxylase.

Acetyl-CoA Carboxylase↗

PubMed1994

Purification and characterization of 3-methylcrotonyl-coenzyme-A carboxylase from leaves of Zea mays.

3-Methylcrotonyl-CoA carboxylase has been purified to near homogeneity from maize leaves. The resulting preparations of 3-methylcrotonyl-CoA carboxylase have a specific activity of between 200 and 600 nmol.min-1.mg-1 protein, representing an approximately 5000-fold purification of the enzyme. The purified 3-methylcrotonyl-CoA carboxylase has a molecular weight of 853,000 +/- 34,000 and is composed of two types of subunits, a biotin-containing subunit of 80 +/- 2 kDa and a non-biotin-containing subunit of 58.5 +/- 1.5 kDa. These data suggest that the enzyme has an alpha 6 beta 6 configuration. The optimum pH for activity is 8.0. The kinetic constants for the substrates 3-methylcrotonyl-CoA, ATP, and HCO3- are 11 microM, 20 microM, and 0.8 mM, respectively. Kinetic studies of the 3-methylcrotonyl-CoA carboxylase reaction with variable concentrations of two substrates confirmed that ATP and HCO3- bind sequentially to the enzyme and that ATP and 3-methylcrotonyl-CoA bind in ping-pong fashion. However, similar analyses indicate that the binding of HCO3- at the first site is affected by 3-methylcrotonyl-CoA. Kinetic studies of the role of Mg2+ in the 3-methylcrotonyl-CoA carboxylase reaction establish that Mg.ATP is the substrate for the enzyme, that free ATP is an inhibitor, and that free Mg2+ is an activator. Both Mn2+ and Co2+ can substitute somewhat for Mg2+, but Zn2+ is unable to do so. In addition to carboxylating 3-methylcrotonyl-CoA, the maize carboxylase can carboxylate crotonyl-CoA, but not acetoacetyl-CoA. In fact, acetoacetyl-CoA is a potent, noncompetitive inhibitor, which indicates that the enzyme contains an acetoacetyl-CoA binding site that is independent of the active sites. The monovalent cations K+, Cs+, Rb+, and NH4+ activated 3-methylcrotonyl-CoA carboxylase activity, with Rb+ being the most potent activator. The inhibition of 3-methylcrotonyl-CoA carboxylase by sulfhydryl and arginyl modifying reagents could be partly alleviated by the substrates ATP and 3-methylcrotonyl-CoA, which suggests that sulfhydryl and arginyl residues may be involved in catalysis.

Acyl Coenzyme A↗

PubMed1999

Geranoyl-CoA carboxylase: a novel biotin-containing enzyme in plants.

Geranoyl-CoA carboxylase (EC 6.4.1.4) is a biotin-containing enzyme previously described in two genera of bacteria. Here we report the presence of geranoyl-CoA carboxylase in kingdom Plantae. Geranoyl-CoA carboxylase was purified 180-fold from maize leaves. The enzyme has a biotin-containing subunit of 122 kDa. The pH optimum for activity is 8.3. The apparent Km values for the substrates geranoyl-CoA, bicarbonate, and ATP are 64 +/- 5 microM, 0. 58 +/- 0.04 mM, and 8.4 +/- 0.4 microM, respectively. Subcellular fractionations indicate that geranoyl-CoA carboxylase is located in plastids. Geranoyl-CoA carboxylase activity is ubiquitous in organs of monocots and dicots and varies with development. We postulate that geranoyl-CoA carboxylase plays an important role in isoprenoid catabolism in plants, in a pathway analogous to that shown in Psuedomonas sp. In plants, this catabolic pathway would require the interaction of at least three subcellular compartments (plastids, microbodies, and mitochondria) and two biotin-containing enzymes, geranoyl-CoA carboxylase and 3-methylcrotonyl-CoA carboxylase.

Biotin↗

PubMed1995

Localization of the factor IX propeptide binding site on recombinant vitamin K dependent carboxylase using benzoylphenylalanine photoaffinity peptide inactivators.

The propeptide binding/activation site on the vitamin K dependent carboxylase has been localized to a region of carboxylase between residues Arg +50 and Glu +225 by photoinactivation studies using [125I]tyrosyl-labeled benzoylphenylalanine (Bpa)-containing analogs of proFIX19, a peptide containing residues -18 to +1 of factor IX. Four proFIX19 analogs with Bpa substituents at -16, -13, -7, and -6 were synthesized. These peptides were specific photoinactivators of carboxylase and were used to label a His6-carboxylase construct produced in baculovirus-infected insect cells. Fragments of the labeled carboxylase produced by V8 protease digestion were analyzed by peptide-specific antibodies and by autoradiography. The propeptide recognition site was localized to the N-terminal one-third of the 94 kDa carboxylase. This is consistent with previous studies using a carboxylase substrate affinity label, N-(bromoacetyl)-FLEELY [Kuliopulos, A., Nelson, N.P., Yamada, M., Walsh, C.T., Furie, B., Furie, B.C., & Roth, D.A. (1994) J. Biol. Chem. 269, 21364-21370], indicating that the propeptide binding site and the FLEEL binding site are both located within the N-terminal one-third of the vitamin K dependent carboxylase.

Affinity Labels↗