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PubMed1998

The strongly conserved lysine 256 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase is essential for phosphoryl transfer.

Lysine 256, a conserved amino acid of Saccharomycescerevisiae phosphoenolpyruvate (PEP) carboxykinase located in the consensus kinase 1a sequence of the enzyme, was changed to alanine, arginine, or glutamine by site-directed mutagenesis. These substitutions did not result in gross changes in the protein structure, as indicated by circular dichroism, tryptophan fluorescence spectroscopy, and gel-exclusion chromatography. The three variant enzymes showed almost unaltered Km for MnADP but about a 20 000-fold decrease in Vmax for the PEP carboxylation reaction, as compared to wild-type PEP carboxykinase. The variant enzymes presented oxaloacetate decarboxylase activity at levels similar to those of the native protein; however, they lacked pyruvate kinase-like activity. The dissociation constant for the enzyme-MnATP complex was 1.3 +/- 0.3 microM for wild-type S. cerevisiae PEP carboxykinase, and the corresponding values for the Lys256Arg, Lys256Gln, and Lys256Ala mutants were 2.0 +/- 0.6 microM, 17 +/- 2 microM, and 20 +/- 6 microM, respectively. These results collectively show that a positively charged residue is required for proper binding of MnATP and that Lys256 plays an essential role in transition state stabilization during phosphoryl transfer for S. cerevisiae PEP carboxykinase.

Adenosine Triphosphate↗
PubMed1985

Pyruvoyl-dependent histidine decarboxylases. Preparation and amino acid sequences of the beta chains of histidine decarboxylase from Clostridium perfringens and Lactobacillus buchneri.

Histidine decarboxylase (HisDCase) from Lactobacillus buchneri was purified to homogeneity. Its subunit structure, (alpha beta)6, and enzymatic properties resemble closely those of the immunologically cross-reactive HisDCase of Lactobacillus 30a (Recsei, P. A., and Snell, E. E. (1984) Annu. Rev. Biochem. 53, 357-387). The complete amino acid sequences of the beta chains of the HisDCase from L. buchneri (81 residues) and Clostridium perfringens (86 residues) were then determined to be a and b, respectively. (a) SEFDKKLNTLGVDRISVSPYKKWSRGYMEPGNIGNGYVSGLKVDAG VVDKTDDMVLDGIGSYDRAETKNAYIGQINMTTAS. (b) TLSEGIHKNIKNIKVRAP KIDKTAISPYDRYCDGYGMPGAYGDGYVSVLKVSVGTVKK TDDILLDGIVSYDRAEINDAYVGQINMLTAS. SEFDKKLNTLGVDRISVSPYKKWSRGYMEPGNIGNGYVSGLKVDAGVV. Although these sequences differ substantially near the NH2-terminal ends, there is striking homology near the COOH termini and also near the NH2 terminus of the two alpha chains (pyruvoyl-Phe-X-Gly-Val-, where X is Ser or Cys). If the four known pyruvoyl-dependent HisDCases arise from inactive proenzymes by the mechanism previously demonstrated for the HisDCase of Lactobacillus 30a (Recsei, P. A., Huynh, Q. K. and Snell, E. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 973-977), then each of these proenzymes has the sequence -Thr-Ala-Ser-Ser-Phe- at the activation site (where -Ser- becomes the COOH terminus of the beta chain and -Ser- becomes the pyruvoyl group blocking the NH2 terminus of the alpha chain), and the sequences around this activation site are highly conserved in all four enzymes. These facts support the assumptions that the four enzymes have evolved from a common ancestral protein, are formed from inactive pyruvate-free proenzymes by similar mechanisms, and have similar catalytic mechanisms.

Amino Acid Sequence↗
PubMed1977

Identification of a pyruvoyl residue in S-adenosylmethionine decarboxylase from Saccharomyces cerevisiae.

S-Adenosylmethionine decarboxylase from Saccharomyces cerevisiae has been purified to homogeneity. Acid hydrolysis of NaB3H4-reduced enzyme released 2.2 mol of tritiated lactate per mol of dimeric enzyme, indicating that a pyruvate moiety is present. Inhibition of enzymatic activity by NaBH4 reduction and by carbonyl-binding reagents indicates that this pyruvoyl residue is required for the activity of the enzyme. This is the first example reported of a eukaryotic enzyme containing a covalently linked pyruvoyl residue.

Adenosylmethionine Decarboxylase↗
PubMed1991

Amino acid residues necessary for putrescine stimulation of human S-adenosylmethionine decarboxylase proenzyme processing and catalytic activity.

Human S-adenosylmethionine (AdoMet) decarboxylase is synthesized as a 38-kDa proenzyme that is autocatalytically cleaved, forming the covalently attached pyruvate cofactor and the two subunits (67 and 267 amino acid residues) of the mature enzyme. Both the cleavage reaction and the catalytic activity of the mature enzyme are stimulated by putrescine. Using site-specific mutagenesis and in vitro transcription followed by translation of the resultant RNA in a cellfree system, we have examined the importance of several amino acid residues on the processing reaction rate and catalytic activity and on stimulation of each of these by putrescine. Changing Cys82 to Ala decreased the stimulatory effect of putrescine on processing and completely eliminated catalytic activity, indicating a probable role in the active site of the enzyme, whereas changing Cys49 or Cys226 to Ala had minimal effects on processing and activity or the putrescine stimulation of either. Since Cys49 is the only cysteine residue in the smaller subunit, this indicates that disulfide-bond formation between the two subunits cannot be necessary for maintenance of the conformation for proenzyme processing or catalytic activity. Changing Glu8 or Glu11 to Gln produced an AdoMet decarboxylase that processed, but was catalytically inactive. Mutation at Glu11 also completely eliminated the putrescine stimulation of proenzyme processing, as did double mutation at Glu8 and Glu11, whereas the single mutation at Glu8 had no effect on the putrescine stimulation of proenzyme processing. Changing Glu15, Glu61, Glu67, Glu247, or Glu247 and Glu249 to Gln had a minimal effect on processing and activity or putrescine stimulation of either. These results demonstrate a role for the smaller subunit in the catalytic activity of the mature AdoMet decarboxylase enzyme and show that Glu8, Glu11, and Cys82 are essential for catalytic activity, with Glu11 also being essential for the putrescine stimulation of AdoMet decarboxylase proenzyme processing.

Adenosylmethionine Decarboxylase↗
PubMedDate not supplied

Activity of biotin-dependent and GABA metabolizing enzymes in chorionic villus samples: potential for 1st trimester prenatal diagnosis.

We have documented the presence of five mitochondrial enzymes in samples of chorionic villus tissue and measured the levels of activity. Three of the enzymes catalyse biotin-dependent reactions. These are propionyl-CoA carboxylase, 3-methylcrotonyl-CoA carboxylase and pyruvate carboxylase. The other enzymes, 4-aminobutyric acid aminotransferase and succinic semialdehyde dehydrogenase, are involved in the degradation of the central inhibitory neurotransmitter GABA. Distinct diseases in which there is deficiency of each of these enzymes have been documented in man. Significant levels of activity were observed for all five enzymes in chorionic villus tissue. This methodology should permit early prenatal diagnosis of deficiencies of these enzymes by chorionic villus biopsy in the first trimester.

4-Aminobutyrate Transaminase↗
PubMed1985

Regulation of S-adenosylmethionine decarboxylase activity in rat liver and prostate.

Two methods were used for the quantitation of S-adenosylmethionine decarboxylase protein. The first involved titrating the active site of the enzyme by reduction of the Schiff base between 3H-decarboxylated S-adenosylmethionine and the pyruvate prosthetic group with sodium cyanoborohydride. The second method was radioimmunoassay with rabbit antiserum which was used to determine the total immunoreactive enzyme protein. It was found that the increased S-adenosylmethionine decarboxylase activity produced in rat prostate by treatment with alpha-difluoromethylornithine and in both prostate and liver by methylglyoxal bis(guanylhydrazone) were due entirely to increases in the amount of enzyme protein. The ratio of enzyme activity to protein (measured by either method) remained constant in rats treated with the drugs. Treatment with 2% alpha-difluoromethylornithine in the drinking water for 3 days increased prostatic S-adenosylmethionine decarboxylase protein by 5-fold. A substantial part, but not all, of this increase could be accounted for by a slowing of the rate of degradation of the enzyme. The half-life for loss of activity and titratable protein after inhibition of protein synthesis by cycloheximide was increased from 35 to 108 min by treatment with alpha-difluoromethylornithine. However, the half-life for loss of immunoreactive protein which was considerably longer was only increased from 139 to 213 min. The molecular weight of the S-adenosylmethionine decarboxylase subunit determined by immunoblotting was 32,000, and no smaller immunoreactive fragments were detected. These results indicate that spermidine depletion produced by alpha-difluoromethylornithine affects the degradation of S-adenosylmethionine decarboxylase at an early step involving the loss of the active site without substantial breakdown of the protein.

Adenosylmethionine Decarboxylase↗
PubMed2006

Mitochondrial function and toxicity: role of the B vitamin family on mitochondrial energy metabolism.

The B vitamins are water-soluble vitamins required as coenzymes for enzymes essential for cell function. This review focuses on their essential role in maintaining mitochondrial function and on how mitochondria are compromised by a deficiency of any B vitamin. Thiamin (B1) is essential for the oxidative decarboxylation of the multienzyme branched-chain ketoacid dehydrogenase complexes of the citric acid cycle. Riboflavin (B2) is required for the flavoenzymes of the respiratory chain, while NADH is synthesized from niacin (B3) and is required to supply protons for oxidative phosphorylation. Pantothenic acid (B5) is required for coenzyme A formation and is also essential for alpha-ketoglutarate and pyruvate dehydrogenase complexes as well as fatty acid oxidation. Biotin (B7) is the coenzyme of decarboxylases required for gluconeogenesis and fatty acid oxidation. Pyridoxal (B6), folate and cobalamin (B12) properties are reviewed elsewhere in this issue. The experimental animal and clinical evidence that vitamin B therapy alleviates B deficiency symptoms and prevents mitochondrial toxicity is also reviewed. The effectiveness of B vitamins as antioxidants preventing oxidative stress toxicity is also reviewed.

Animals↗
PubMed1993

Growth and Energy Generation by Lactococcus lactis subsp. lactis biovar diacetylactis during Citrate Metabolism.

Growth of Lactococcus lactis subsp. lactis biovar diacetylactis was observed on media with citrate as the only energy source. At pH 5.6, steady state was achieved in a chemostat on a citrate-containing medium in the absence of a carbohydrate. Under these conditions, pyruvate, acetate, and some acetoin and butanediol were the main fermentation products. This indicated that energy was conserved in L. lactis subsp. lactis biovar diacetylactis during citrate metabolism and presumably during the conversion of citrate into pyruvate. The presumed energy-conserving step, decarboxylation of oxaloacetate, was studied in detail. Oxaloacetate decarboxylase was purified to homogeneity and characterized. The enzyme has a native molecular mass of approximately 300 kDa and consists of three subunits of 52, 34, and 12 kDa. The enzyme is apparently not sodium dependent and does not contain a biotin moiety, and it seems to be different from the energy-generating oxaloacetate decarboxylase from Klebsiella pneumoniae. Energy-depleted L. lactis subsp. lactis biovar diacetylactis cells generated a membrane potential and a pH gradient immediately upon addition of citrate, whereas ATP formation was slow and limited. In contrast, lactose energization resulted in rapid ATP formation and gradual generation of a proton motive force. These data were confirmed during studies on amino acid uptake. alpha-Aminoisobutyrate uptake was rapid but glutamate uptake was slow in citrate-energized cells, whereas lactose-energized cells showed the reverse tendency. These data suggest that, in L. lactis subsp. lactis bv. diacetylactis, a proton motive force could be generated during citrate metabolism as a result of electrogenic citrate uptake or citrate/product exchange together with proton consumption by the intracellular oxaloacetate decarboxylase.

Journal Article↗
PubMed2003

The biotin enzyme family: conserved structural motifs and domain rearrangements.

The biotin carboxylase family is comprised of a group of enzymes that utilize a covalently bound prosthetic group, biotin, as a cofactor. These enzymes, which include acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase, methylcrotonyl-CoA carboxylase, geranoyl-CoA carboxylase, oxaloacetate decarboxylase, methylmalonyl-CoA decarboxylase, transcarboxylase and urea amidolyase, are found in diverse biosynthetic pathways in both pro-karyotes and eukaryotes. The reactions catalyzed by most members of this group of enzymes share two common features: (1) carboxylation of biotin, apparently via the formation of a carboxyphosphate intermediate, followed by (2) transcarboxylation of CO(2) from biotin to specific acceptor molecules to yield different products. Structural determinations by NMR and X-ray crystallography, complemented by mutagenesis studies, have identified some motifs that are structurally or catalytically important. Analysis of the amino acid sequences of a number of biotin carboxylases not only shows remarkable similarities within certain domains but also that there appears to have been domain rearrangements between groups of carboxylases. Acyl-coenzyme A derivatives, which bind either as substrates or as allosteric regulators of the biotin carboxylases, do not appear to share any of the CoA binding motifs that have been identified in other CoA-SH/acyl-CoA binding proteins. Further comparisons of biotin-dependent carboxylases with other groups of enzymes in the protein data bank reveal that this family of biotin enzymes has strong similarities in specific domains to a number of ATP-utilizing enzymes and to the lipoyl-containing enzymes. These structural homologies are so extensive as to be highly suggestive of evolutionary relationships between biotin carboxylases and these other enzymes.

Acetyl-CoA Carboxylase↗
PubMed2004

Biotin deficiency blocks thymocyte maturation, accelerates thymus involution, and decreases nose-rump length in mice.

Biotin deficiency in experimental animals causes low body weight as well as several phenomena suggestive of an altered immune system. We reported previously that chronic biotin deficiency in mice decreases body weight and alters the number and proportion of lymphocyte subpopulations in the spleen. To further characterize the effects of biotin deficiency, we studied in detail the maturation of thymocytes and the status of biotin in the thymus, as well as the body length of biotin-deficient mice. Male Balb/cAnN mice were fed for up to 20 wk either standard control diet, a biotin-deficient diet, or a biotin-sufficient diet. At different times, nose-rump length, weight of the thymus, spleen and liver, total number of cells in the spleen and thymus, pyruvate carboxylase (PC) and propionyl CoA carboxylase (PCC) activity in thymus cells, and the proportion of distinct thymocyte subsets were determined. These variables did not differ between mice fed the control and biotin-sufficient diets. In contrast, biotin-deficient mice differed from biotin-sufficient mice in all of the analyzed variables. PC and PCC specific activities of thymocytes of mice fed the biotin-depleting diet decreased during the first 4 wk by 84.5%. The maturation of thymocytes in biotin-deficient mice was arrested at the double-negative stage. Our results suggest that biotin deficiency in mice causes an accelerated involution of the thymus and decreases nose-rump length, but these effects do not correlate in magnitude or in temporality with the sharp decrease in the activity of the biotin-dependent carboxylases. As such, the possibility that the aforementioned effects are not related directly to the prosthetic function of biotin should be considered.

Animals↗
PubMed1993

Insulin-mimetic actions of phorbol ester in cultured adult rat hepatocytes. Lack of phorbol-ester-elicited inhibition of the insulin signal.

The actions of the phorbol ester phorbol 12-myristate 13-acetate (PMA) on glucose metabolism, amino acid transport and enzyme inductions were studied in primary cultures of adult-rat hepatocytes and compared with the effects of insulin. PMA and insulin stimulated glycolysis 5- and 7-fold respectively. The half-maximal effective dose of PMA was 60 nM. Stimulation of glycolysis was accompanied by an insulin- or PMA-dependent and okadaic acid-sensitive activation of 6-phosphofructo-2-kinase and pyruvate kinase, as well as by an increase in fructose 2,6-bisphosphate. Glucose production from glycogen was decreased to 50% by PMA and to 15% by insulin, whereas glycogen synthesis was stimulated 2- and 7-fold respectively. PMA also increased aminoisobutyrate uptake, induced ornithine decarboxylase and counteracted the glucagon-dependent induction of phosphoenolpyruvate carboxykinase. PMA strongly antagonized the hormonal activation of glycogen synthesis, but all other insulin actions assayed were not decreased by the phorbol ester. Whereas additive effects of PMA and insulin were not detected, PMA and a simultaneous increase in the glucose concentration had additive effects on glycolysis and glycogen metabolism. Cell exposure to insulin resulted in receptor autophosphorylation and a more than 10-fold activation of the receptor tyrosine kinase. PMA did not alter these effects, and also had no effect on the receptor phosphorylation status in the absence of insulin. Long-term (15 h) pretreatment of the cells with PMA abolished all PMA effects, but not the insulin effects. It is concluded that PMA does not generally antagonize the action of insulin in differentiated adult hepatocytes, and that insulin and PMA may use related signal-transduction pathways.

Aminoisobutyric Acids↗
PubMed1994

Enzymatic, metabolic and secretory patterns in human islets of type 2 (non-insulin-dependent) diabetic patients.

Islets were isolated by automatic digestion from non-diabetic cadaveric organ donors and from Type 2 (non-insulin-dependent) diabetic subjects. The activity of FAD-glycerophosphate dehydrogenase, but not that of either glutamate dehydrogenase, glutamate-oxalacetate transaminase or glutamate-pyruvate transaminase, was lower in Type 2 diabetic patients than control subjects. Hexokinase, glucokinase and glutamate decarboxylase activities were also measured in islets from control subjects. The utilization of D-[5-3H]glucose, oxidation of D-[6-14C]glucose and release of insulin evoked by D-glucose were all lower in Type 2 diabetic patients than control subjects. The secretory response to the combination of L-leucine and L-glutamine appeared less severely affected. Islets from Type 2 diabetic patients may thus display enzymatic, metabolic and secretory anomalies similar to those often observed in animal models of Type 2 diabetes, including a deficiency of beta-cell FAD-linked glycerophosphate dehydrogenase, the key enzyme of the glycerol phosphate shuttle.

Adolescent↗
PubMed1991

Crystallization and preliminary X-ray diffraction studies of dialkylglycine decarboxylase, a decarboxylating transaminase.

The pyridoxal phosphate-dependent enzyme dialkylglycine decarboxylase (E.C. 4.1.1.64) has been crystallized by vapor diffusion from a 15% polyethyleneglycol solution with sodium pyruvate as coprecipitant. The space group of the crystals is either P6(2)22 or the enantiomorph, P6(4)22, with one subunit of 46,500 Da per asymmetric unit. The unit cell has dimensions a = b = 152.7 A, c = 86.6 A, alpha = beta = 90 degrees, gamma = 120 degrees, and a solvent content of approximately 61%. diffraction extends to 2.3 A resolution.

Burkholderia cepacia↗
PubMed1984

Parasite enzymes as potential targets for antiparasitic chemotherapy.

I have thus far listed a total of 10 potential targets for antiparasitic chemotherapeutic consideration. This is by no means a completed list. Many more will be added to it with time and with more future findings. Among these 10 targets (summarized in Table I), however, one may gain some insight and see a few interesting general trends: (1) Nucleic acid metabolism and carbohydrate-energy metabolism in protozoan parasites appear to be targets for fruitful chemotherapeutic attacks. Their being useful targets results generally from the deficient metabolism in the protozoan parasites. Thus, the main vulnerability among the protozoan parasites is closely associated with their parasitic nature. (2) Microtubules and nervous systems appear to be the main chemotherapeutic targets in helminths. They differ from those in the host not because of their parasitic nature but, more likely, because of the evolutionary distance separating the mammalian hosts and the primitive metazoa. Thus, free-living nematodes, such as Caenorhabditis elegans, have their microtubules just as susceptible to the benzimidazole anthelmintics as those from the parasitic worms. The motoneuronal map of C. elegans is identical with that of Ascaris lumbricoides. Both worms are similarly immobilized by levamisole, piperazine, avermectins, etc. The dual insecticidal and antiexoparasite activities found in the avermectins and milbemycins may also suggest that the free-living insects and the ticks and lice may have the same GABA nervous system. This main discrepancy between protozoan parasites and metazoan parasites may be partly attributable to the higher mutation rates and higher frequencies of genetic recombination among the protozoa, evidenced by the higher rates of development of drug resistance among them. The fast adaptation to a new environment may be essential for survival, but it would also lead to metabolic deficiencies after the protozoa lived in a luxurious environment for a while. This revelation may suggest that future chemotherapeutic studies on parasitic helminths can utilize free-living helminths as models to eliminate many unnecessary technical difficulties. Also, there perhaps could be a further classification among the parasites to term the protozoa "true parasites" and the helminth "pseudo-parasites" from the viewpoint of chemotherapy.

Animals↗