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PubMed1994

Expression of mammalian S-adenosylmethionine decarboxylase in Escherichia coli. Determination of sites for putrescine activation of activity and processing.

Mammalian S-adenosylmethionine decarboxylase (AdoMetDC) is known to be regulated by putrescine in two ways: (a) acceleration of the rate of conversion of the proenzyme into the mature enzyme in a reaction that forms the pyruvate prosthetic group and (b) activation of the mature enzyme activity. To determine sites of putrescine interaction with AdoMetDC, putrescine stimulation of both proenzyme processing and catalytic activity was tested with mutant AdoMetDCs in which specific amino acid residues, conserved between mammalian and yeast AdoMetDCs, had been altered by site-directed mutagenesis. Mutations E178Q or E256Q (and the previously reported mutation E11Q (Stanley, B. A., and Pegg, A. E. (1991) J. Biol. Chem. 266, 18502-18506)) abolished stimulation by putrescine without an effect on the processing rate in the absence of putrescine. Mutations E11K, as well as Y112A and L259Stop, completely abolished processing regardless of putrescine concentration, whereas mutation E133Q conferred an absolute putrescine requirement for processing to occur. Mutation E132Q, E135Q, E183Q, or D185N had no effect on proenzyme processing. The effects of mutations on enzyme activity were determined using AdoMetDC protein produced in Escherichia coli and purified by affinity chromatography. Mutation E11Q completely inactivated the enzyme, mutation E133Q reduced the catalytic constant by > 10(4), and mutation E256Q produced a 20-fold decrease. Putrescine did not stimulate the activity of mutants E178Q and E256Q but did activate mutants E133Q and E183Q. It is concluded that residues Glu-11, Glu-178, and Glu-256 are critical residues in the putrescine stimulation of AdoMetDC proenzyme processing and that Glu-178 and Glu-256 are critical for putrescine stimulation of AdoMetDC catalytic activity.

Adenosylmethionine Decarboxylase↗
PubMed1982

Chemistry of the inactivation of cytosolic aspartate aminotransferase by serine O-sulfate.

The reaction of serine O-sulfate with cytosolic aspartate aminotransferase [John, R.A., & Fasella, P. (1969) Biochemistry 8, 4477] has been reinvestigated. As in the corresponding reaction with beta-chloroalanine [Morino, Y., Osman, A.M., & Okamoto, M. (1974) J. Biol. Chem. 249, 6684], the enzyme is inactivated over a 10-min period, and the absorption maximum at pH 5.4 shifts from 430 to 336 nm. Upon prolonged standing the peak shifts again over a period of 20 h to 455 nm, a behavior entirely similar to that reported by Morino et al. for beta-chloroalanine in the presence of 3 M formate. When the pH of either the 10-min product (1a) or the 20-h product (1b) is raised to 11 or above, a yellow, diffusible compound (2) is released from the protein. This compound as well as its dephosphorylation and reduction products has been isolated and studied by NMR spectroscopy. Compound 2 is identical with a compound formed from serine sulfate and glutamate decarboxylase by a similar reaction sequence [Likos, J.J., Ueno, H., Feldhaus, R.W., & Metzler, D.E. (1982) Biochemistry (preceding paper in this issue)] and is the product of an aldol condensation of pyruvate with pyridoxal phosphate. When the 20-h product 1b is reduced with sodium borohydride and then heated in a boiling water bath, a material identical with the reduction product of 2 is released. We propose that the 20-h product 1b consists of 2 bound to the enzyme. Pathways for the formation of the various compounds are proposed. These findings require a reevaluation of the mechanisms of action of many enzyme-activated inhibitors of pyridoxal phosphate dependent enzymes.

Aspartate Aminotransferases↗
PubMedDate not supplied

[The compensatory function of a GABA shunt in brain energy metabolism in measured craniocerebral trauma in rats].

A severe compression craniocerebral trauma was induced in rats under short-term halothane anesthesia. The activity of pyruvate and 2-oxoglutarate dehydrogenase complexes reduced significantly in the tissue of the damaged hemisphere, ALT activity increased sharply, AST activity grew slowly, the production of GABA in the glutamate decarboxylase reaction was slightly inhibited and its utilization in the GABA transaminase reaction was clearly accelerated. The GABA level in the nerve tissue showed a tendency to reduce, while the glutamate level had a tendency to increase. The observed changes are evidence that the inclusion of the GABA skeleton in the reaction of further oxidation intensifies, which may be of significance in compensation of the transport of the energetically oxidizing succinate and, possibly, in the formation of endogenous GABA possessing a stress-relieving effect.

Acute Disease↗
PubMed1993

Prediction of the three-dimensional structures of the biotinylated domain from yeast pyruvate carboxylase and of the lipoylated H-protein from the pea leaf glycine cleavage system: a new automated method for the prediction of protein tertiary structure.

A new, automated, knowledge-based method for the construction of three-dimensional models of proteins is described. Geometric restraints on target structures are calculated from a consideration of homologous template structures and the wider knowledge base of unrelated protein structures. Three-dimensional structures are calculated from initial partly folded states by high-temperature molecular dynamics simulations followed slow cooling of the system (simulated annealing) using nonphysical potentials. Three-dimensional models for the biotinylated domain from the pyruvate carboxylase of yeast and the lipoylated H-protein from the glycine cleavage system of pea leaf were constructed, based on the known structures of two lipoylated domains of 2-oxo acid dehydrogenase multienzyme complexes. Despite their weak sequence similarity, the three proteins are predicted to have similar three-dimensional structures, representative of a new protein module. Implications for the mechanisms of posttranslational modification of these proteins and their catalytic function are discussed.

Amino Acid Oxidoreductases↗
PubMed1985

Glucose metabolism in insulin-producing tumoral cells.

Homogenates of insulin-producing tumoral cells catalyzed the phosphorylation of glucose, mannose, and fructose. The kinetics of phosphorylation at increasing glucose concentrations, the inhibitory effect of glucose 6-phosphate, and the comparison of results obtained with distinct hexoses indicated the presence of both low-Km hexokinase-like and high-Km enzymatic activities, the results being grossly comparable to those collected in normal pancreatic islets. Relative to protein content, the glucose-phosphorylating enzymatic activity was higher in tumoral than normal islet cells. The activity of other enzymes was either lower (glutamate dehydrogenase), moderately higher (phosphoglucomutase, lactate dehydrogenase) or considerably greater (ornithine decarboxylase) in tumoral than in normal islet cells. In intact tumoral cells, incubated under increasing glucose concentrations, the oxidation of D-[U-14C]glucose and the output of lactic and pyruvic acids reached a close-to-maximal value at 2.8 mM glucose. The ratios for glucose oxidation/utilization and lactate/pyruvate output were much lower in tumoral than in normal islet cells. Although glucose caused a modest increase in insulin output from the tumoral cells, this effect was saturated at a low glucose concentration (2.8 mM) and less marked than that of other secretagogues (e.g., L-leucine, L-ornithine, or forskolin). Thus, despite a close-to-normal enzymatic equipment for glucose phosphorylation, the tumoral cells displayed severe abnormalities in the metabolism and secretory response to this hexose. These findings point to regulatory mechanisms distal to glucose phosphorylation in the control of glucose metabolism in insulin-producing cells.

Animals↗
PubMed1978

Regulation of pyruvate oxidation and the conservation of glucose.

In animals the pyruvate dehydrogenase reaction is mainly responsible for the irreversible loss of glucose carbon by oxidation. Regulation of this reaction is shown to be a major determinant of glucose conservation in starvation and diabetes. Estimates of conservation in man in starvation and diabetes are reviewed. The pyruvate dehydrogenase complex is inhibited by products of its reactions; it is also regulated by a phosphorylation-dephosphorylation cycle catalysed by a kinase intrinsic to the complex and by a more loosely associated phosphatase. Inactivation is largely accomplished by phosphorylation of the tetrameric decarboxylase component (alpha2beta2) to alpha2Pbeta2. Complete phosphorylation produces the (alpha2P3)beta2 form. Both forms are completely reactivated by phosphatase action but the initial rate of reactivation of a complex containing alpha2Pbeta2 is approximately three times that of (alpha2P3)beta2. The proportion of active (dephosphorylated) complex is decreased in rat tissues by starvation and diabetes and in perfused rat heart by oxidation of fatty acids and ketone bodies. In adipose tissue in vitro, insulin increases the proportion of active complex and lipolytic hormones may decrease this proportion. It is suggested that rates of oxidation of lipid fuels may be a major determinant of the activity of pyruvate dehydrogenase in tissues in relation to the actions of insulin and lipolytic hormones and the effects of diabetes and starvation. Phosphorylation and inactivation of the complex are enhanced by high mitochondrial ratios of [acetyl-CoA]/[CoA], [ATP]/[ADP], [NADH]/[NAD+] and low concentrations of pyruvate, Mg2+ and Ca2+, and vice versa.

Animals↗
PubMed2006

A thiamin-bound, pre-decarboxylation reaction intermediate analogue in the pyruvate dehydrogenase E1 subunit induces large scale disorder-to-order transformations in the enzyme and reveals novel structural features in the covalently bound adduct.

The crystal structure of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined with phosphonolactylthiamin diphosphate (PLThDP) in its active site. PLThDP serves as a structural and electrostatic analogue of the natural intermediate alpha-lactylthiamin diphosphate (LThDP), in which the carboxylate from the natural substrate pyruvate is replaced by a phosphonate group. This represents the first example of an experimentally determined, three-dimensional structure of a thiamin diphosphate (ThDP)-dependent enzyme containing a covalently bound, pre-decarboxylation reaction intermediate analogue and should serve as a model for the corresponding intermediates in other ThDP-dependent decarboxylases. Regarding the PDHc-specific reaction, the presence of PLThDP induces large scale conformational changes in the enzyme. In conjunction with the E1-PLThDP and E1-ThDP structures, analysis of a H407A E1-PLThDP variant structure shows that an interaction between His-407 and PLThDP is essential for stabilization of two loop regions in the active site that are otherwise disordered in the absence of intermediate analogue. This ordering completes formation of the active site and creates a new ordered surface likely involved in interactions with the lipoyl domains of E2s within the PDHc complex. The tetrahedral intermediate analogue is tightly held in the active site through direct hydrogen bonds to residues His-407, Tyr-599, and His-640 and reveals a new, enzyme-induced, strain-related feature that appears to aid in the decarboxylation process. This feature is almost certainly present in all ThDP-dependent decarboxylases; thus its inclusion in our understanding of general thiamin catalysis is important.

Amino Acid Substitution↗
PubMed2001

Autoantibodies of sera from patients with primary biliary cirrhosis recognize the alpha subunit of the decarboxylase component of human branched-chain 2-oxo acid dehydrogenase complex.

BACKGROUND/AIMS: The major antigens for anti-mitochondrial autoantibodies in primary biliary cirrhosis (PBC) are the lipoyl-containing components of 2-oxo acid dehydrogenase complexes. Autoantibodies against the E1alpha subunit of the pyruvate dehydrogenase complex (PDH) also have been found, but those against the E1alpha subunit of branched-chain 2-oxo acid dehydrogenase complex (BCKADH) have not been detected. We investigated the occurrence of BCKADH-E1alpha-specific autoantibodies by employing the purified human antigen. METHODS: The reactivities of PBC sera against purified antigens were assessed by ELISA and by immunoblotting analysis. The specificity of immunoreactivity was confirmed by absorption tests and affinity-purified antibodies. RESULTS: Fourteen out of 27 PBC sera reacted with BCKADH-E1alpha, and these same sera also reacted with BCKADH-E2. No PBC sera reacted with BCKADH-E1beta. The reactivity of PBC sera with BCKADH-E1alpha was removed only when the sera were pre-absorbed with this antigen. However, reactivities to BCKADH-E2 and PDH-E1alpha were retained. Affinity-purified antibodies to BCKADH-E1alpha reacted with BCKADH-E1alpha, but not PDH-E1alpha. Thus, it was confirmed that anti-BCKADH-Elalpha did not cross-react with either BCKADH-E2 or PDH-E1alpha. CONCLUSIONS: BCKADH-E1alpha-specific autoantibodies were found in the sera of PBC patients. The antibodies seem to occur subsequent to the anti-BCKADH-E2 antibody production, supporting the concept of intermolecular determinant spreading.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1993

Refined structure of the pyruvoyl-dependent histidine decarboxylase from Lactobacillus 30a.

The crystal structure of the pyruvoyl-dependent histidine decarboxylase from Lactobacillus 30a has been refined to an R-value of 0.15 (for the 5.0 to 2.5 A resolution shell) and 0.17 (for the 10.0 to 2.5 A resolution shell). A description of the overall structure is presented, focusing on secondary structure and subunit association. The enzyme is a hexamer of alpha beta subunits. Separate alpha and beta-chains arise from an autocatalytic cleavage reaction between two serine residues, which results in the pyruvoyl cofactor. The central core of the alpha beta subunit is a beta-sandwich which consists of two face-to-face three-stranded antiparallel beta-sheets, flanked by alpha-helices on each side. The beta-sandwich creates a stable fold that allows conformational strain to be introduced across an internal cleavage region between the alpha and beta chains and places the pyruvoyl cofactor in a position for efficient electron withdrawal from the substrate. Three alpha beta subunits are related by a molecular three-fold symmetry axis to form a trimer whose interfaces have complementary surfaces and extensive molecular interactions. Each of the interfaces contains an active site and a solvent channel that leads from the active site to the exterior of the molecule. The trimers are related by a crystallographic two-fold symmetry axis to form the hexamer with an overall dumbbell shape. The interface between trimers has few molecular interactions.

Amino Acid Sequence↗
PubMed1994

CGP 48664, a new S-adenosylmethionine decarboxylase inhibitor with broad spectrum antiproliferative and antitumor activity.

Inhibitors of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (SAMDC), derived from methylglyoxal-bis(guanylhydrazone) (MGBG), have been shown to have significant antitumor activity in several human solid tumor systems (U. Regenass et al., Cancer Res., 52:4712-4718, 1992). From an ongoing effort to synthesize derivatives with increased enzyme specificity and potency and improved antitumor efficacy, we have now identified CGP 48664, a 4-amidinoindan-1-one 2'-amidinohydrazone (J. Stanek et al., J. Med. Chem., 36:2168-2171, 1993). The compound displays potent inhibition of SAMDC (50% inhibitory concentration, 5 nM), modest inhibition of diamine oxidase (50% inhibitory concentration, 4 microM), and no detectable inhibition of ornithine decarboxylase. CGP 48664 inhibits the growth of a panel of human and mouse tumor cell lines, including one which expresses the multidrug resistance phenotype, with 50% inhibitory concentrations ranging between 0.3 and 3 microM. CGP 48664 does not seem to utilize the polyamine transport carrier system since it competes poorly with spermidine for uptake into L1210 cells (Ki 161 microM) and inhibits the growth of polyamine transport-deficient Chinese hamster ovary cells. Relative to MGBG or previously described MGBG analogues, CGP 48664 accumulates to much lower intracellular concentrations. Treatment of the L1210 cell for 48 h with 3 microM CGP 48664 decreases SAMDC activity to < 10% of control and initiates a compensatory 3-fold rise in ornithine decarboxylase. Consistent with SAMDC inhibition, putrescine pools increase 10-fold, whereas spermidine and spermine pools fall to < 10% of control. In contrast to MGBG, CGP 48664 displays attenuated antimitochondrial activity as indicated by a lack of effect on pyruvate oxidation and mitochondrial DNA levels under treatment conditions which inhibit cell proliferation. Specificity of drug action was indicated further by prevention of L1210 cell growth inhibition by exogenous spermidine or spermine. More convincingly, Chinese hamster ovary cells made approximately 1000-fold resistant by chronic exposure to the analogue were found to selectively overexpress SAMDC mRNA due to gene amplification. The new SAMDC inhibitor showed potent antitumor activity against syngeneic tumors (B16 melanoma and Lewis lung carcinoma) and nude mouse human tumor xenografts (T-24 bladder carcinoma, SK MEL-24 melanoma, and MALME-3M melanoma). On the basis of its novel structure, its apparent specificity of action, and its potent antitumor activity, CGP 48664 is the candidate drug for further preclinical development.

Adenosylmethionine Decarboxylase↗
PubMed1996

Imbalance of leucine flux in Lactococcus lactis and its use for the isolation of diacetyl-overproducing strains.

Diacetyl is a by-product of pyruvate metabolism in Lactococcus lactis, where pyruvate is first converted to alpha-acetolactate, which is slowly decarboxylated to diacetyl in the presence of oxygen. L. lactis usually converts alpha-acetolactate to acetoin enzymatically, by alpha-acetolactate decarboxylase encoded by the aldB gene. We took advantage of the fact that this enzyme also has a central role in the regulation of branched-chain amino acids, to select spontaneous aldB mutants in an unbalanced concentration of leucine versus those of valine and isoleucine in the medium. Industrial dairy strains of L. lactis subsp. lactis biovar diacetylactis containing point mutations and deletions of aldB were isolated and characterized. Their growth in milk was not affected, but they rapidly accumulated a large amount of alpha-acetolactate instead of acetoin from citrate in milk. Under aerated condition, strains devoid of AldB produced about 10 times more diacetyl than did the parental strains.

Amino Acid Sequence↗
PubMed1978

Purification and regulatory properties of the oxaloacetate decarboxylase of Acetobacter xylinum.

The oxaloacetate (OAA) decarboxylase (EC 4.1.1.3) activity of Acetobacter xylinum cells grown on glucose or glycerol is the same as that of cells grown on intermediates of the citrate cycle. The enzyme was purified 92-fold from extracts, and its molecular weight was determined to be 100,000 by gel filtration. Initial velocity studies revealed marked positive cooperativity for OAA (Hill coefficient [n(H)] = 1.8; S(0.5) = 21 mM). The affinity of the enzyme for OAA was markedly increased upon addition of nicotinamide adenine dinucleotide (NAD), NAD phosphate (NADP), and some other pyridine nucleotides. S(0.5(OAA)) decreased to 1 mM but n(H) and V(max) were unchanged. Saturation kinetics for the pyridine nucleotides were hyperbolic, and a half-maximal effect was obtained with 8 muM NAD and 30 muM NADP. The enzyme also catalyzed the exchange of (14)CO(2) into OAA but not the net carboxylation of pyruvate. Exchange activity, too, exhibited sigmoidal kinetics for OAA and was strongly stimulated by NAD at low substrate concentrations. The enzyme was inhibited by acetate competitively with respect to OAA. The K(I) for acetate (12 mM) was well within the physiological range of this compound inside the cell. The regulatory properties of the decarboxylase with respect to OAA cooperativity, NAD activation, and acetate inhibition were retained in situ within permeabilized cells. These properties seem to provide for a control mechanism which could insure the maintenance of OAA and the citrate cycle during growth of cells on glucose and, conversely, the required supply of pyruvate during growth on intermediates of the citrate cycle.

Carbon Dioxide↗
PubMed1992

Purification of human S-adenosylmethionine decarboxylase expressed in Escherichia coli and use of this protein to investigate the mechanism of inhibition by the irreversible inhibitors, 5'-deoxy-5'-[(3-hydrazinopropyl)methylamino]adenosine and 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine.

Human S-adenosylmethionine decarboxylase (AdoMetDC) was expressed in high yield in Escherichia coli using the pIN-III(lppP-5) expression vector and purified to apparent homogeneity using affinity chromatography on methylglyoxal bis(guanylhydrazone)-Sepharose. The inactivation of the purified enzyme by 5'-deoxy-5'-[(3-hydrazinopropyl)methylamino]adenosine (MHZPA) was accompanied by an increase in absorbance at 260 nm of the large subunit. This increase was equivalent to the addition of 1 molecule of MHZPA. After digestion with the protease Lys-C, a peptide that contained the bound MHZPA was isolated and found to have the amino acid composition consistent with that expected from the amino terminus of the large subunit. These results indicate that MHZPA inactivates AdoMetDC by forming a hydrazone derivative at the pyruvate prosthetic group. Inactivation of AdoMetDC by 5'-([(Z)-4-amino-2-butenyl]methylamino]-5'-deoxyadenosine (AbeAdo) led to the appearance of a new peptide peak in the Lys-C protease digest. This peptide had the sequence ASMFVSK. This agrees with the expected sequence from the amino terminus, which is pyruvoyl-SMFVSK, with the exception that the pyruvate has been converted to alanine. Direct gas-phase sequencing of the large subunit of the enzyme also indicated the presence of alanine at the amino terminus after inactivation with AbeAdo. These results indicate that this inhibitor leads to transamination of the pyruvate prosthetic group. Since the pyruvate is covalently linked to the protein, its replacement by alanine leads to an irreversible inactivation of AdoMetDC.

Adenosylmethionine Decarboxylase↗