S-adenosylmethionine decarboxylase degradation by the 26 S proteasome is accelerated by substrate-mediated transamination

The short-lived enzyme S-adenosylmethionine decarboxylase uses a covalently bound pyruvoyl cofactor to catalyze the formation of decarboxylated S-adenosylmethionine, which then donates an aminopropyl group for polyamine biosynthesis. Here we demonstrate that S-adenosylmethionine decarboxylase is ubiquitinated and degraded by the 26 S proteasome in vivo, a process that is accelerated by inactivation of S-adenosylmethionine decarboxylase by substrate-mediated transamination of its pyruvoyl cofactor. Proteasome inhibition in COS-7 cells prevents the degradation of S-adenosylmethionine decarboxylase antigen; however, even brief inhibition of the 26 S proteasome caused substantial losses of S-adenosylmethionine decarboxylase activity despite accumulation of S-adenosylmethionine decarboxylase antigen. Levels of the enzyme's substrate (S-adenosylmethionine) increased rapidly after 26 S proteasome inhibition, and this increase in substrate level is consistent with the observed loss of activity arising from an increased rate of inactivation by substrate-mediated transamination. Evidence is also presented that this substrate-mediated transamination accelerates normal degradation of S-adenosylmethionine decarboxylase, as the rate of degradation of the enzyme was increased in the presence of AbeAdo (5'-([(Z)-4-amino-2-butenyl]methylamino]-5'-deoxyadenosine) (a substrate analogue that transaminates the enzyme); conversely, when the intracellular substrate level was reduced by methionine deprivation, the rate of degradation of the enzyme was decreased. Ubiquitination of S-adenosylmethionine decarboxylase is demonstrated by isolation of His-tagged AdoMetDC (S-adenosylmethionine decarboxylase) from COS-7 cells co-transfected with hemagglutinin-tagged ubiquitin and showing bands that were immunoreactive to both anti-AdoMetDC antibody and anti-hemagglutinin antibody. This is the first study to demonstrate that AdoMetDC is ubiquitinated and degraded by the 26 S proteasome, and substrate-mediated acceleration of degradation is a unique finding.     

Alkylation of an active-site cysteinyl residue during substrate-dependent inactivation of Escherichia coli S-adenosylmethionine decarboxylase

S-Adenosylmethionine decarboxylase from Escherichia coli is a member of a small class of enzymes that uses a pyruvoyl prosthetic group. The pyruvoyl group is proposed to form a Schiff base with the substrate and then act as an electron sink facilitating decarboxylation. We have previously shown that once every 6000-7000 turnovers the enzyme undergoes an inactivation that results in a transaminated pyruvoyl group and the formation of an acrolein-like species from the methionine moiety. The acrolein then covalently alkylates the enzyme [Anton, D. L., & Kutny, R. (1987) Biochemistry 26, 6444]. After reduction of the alkylated enzyme with NaBH4, a tryptic peptide with the sequence Ala-Asp-Ile-Glu-Val-Ser-Thr-[S-(3-hydroxypropyl)Cys]-Gly-Val-Ile-Ser-Pro - Leu-Lys was isolated. This corresponds to acrolein alkylation of a cysteine residue in the second tryptic peptide from the NH2 terminal of the alpha-subunit [Anton, D. L., & Kutny, R. (1987) J. Biol. Chem. 262, 2817-2822]. The modified residue derived is from Cys-140 of the proenzyme [Tabor, C. W., & Tabor, H. (1987) J. Biol. Chem. 262, 16037-16040] and lies in the only sequence conserved between rat liver and E. coli S-adenosylmethionine decarboxylase [Pajunen et al. (1988) J. Biol. Chem. 263, 17040-17049]. We suggest that the alkylated Cys residue could have a role in the catalytic mechanism.     

Escherichia coli S-adenosylmethionine decarboxylase. Subunit structure, reductive amination, and NH2-terminal sequences

S-Adenosylmethionine decarboxylase is one of a small group of enzymes that use a pyruvoyl residue as a cofactor. Histidine decarboxylase from Lactobacillus 30a, the best studied pyruvoyl-containing enzyme, has an (alpha beta)6 subunit structure with the pyruvoyl moiety linked through an amide bond to the NH2-terminal of the larger alpha subunit (Recsei, P. A., Huynh, Q. K., and Snell, E. E. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 973-977). To examine potential structural analogies between the two enzymes, we have isolated and partially characterized S-adenosylmethionine decarboxylase. The purified enzyme comprises equimolar amounts of two subunits of Mr = 14,000 and 19,000 (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and has a native molecular weight of 136,000 (by gel filtration). Approximately 4 mol of [methyl-3H] adenosylmethionine are incorporated per mol of enzyme (Mr = 136,000) when the enzyme is inactivated with this substrate and NaCNBH3. These data suggest an (alpha beta)4 structure with 1 pyruvoyl residue for each alpha beta pair. The two subunits have been separated by reversed-phase high performance liquid chromatography after reduction and carboxymethylation. The smaller subunit (beta) has a free amino terminus. The amino terminus of the larger subunit (alpha) appears to be blocked by a pyruvoyl group; this subunit can be sequenced only after this group is converted to an alanyl residue by reduction with sodium cyanoborohydride in the presence of ammonium acetate. This work suggests that S-adenosylmethionine decarboxylase is structurally much more similar to histidine decarboxylase than previously thought.     

Identification of ecdysone 25-O-beta-D-glucopyranoside as a new metabolite of ecdysone in the nematode Parascaris equorum.

The mechanism of inactivation of rodent ornithine decarboxylase by alpha-difluoromethylornithine (DFMO) was studied using the inhibitor labelled with 14C in both the 1 and the 5 positions. [1-14C]DFMO was a substrate and was decarboxylated by the enzyme yielding 14CO2. A radioactive metabolite derived from [5-14C]DFMO was bound to the enzyme, and the extent of binding paralleled the irreversible inactivation of ornithine decarboxylase. The partition ratio of decarboxylation to binding was approx. 3.3. These results provide support for the postulated mechanism of action of DFMO [Metcalf, Bey, Danzin, Jung, Casera & Vevert (1978) J. Am. Chem. Soc. 100, 2551-2553], in which enzymic decarboxylation of the inhibitor leads to the generation of a conjugated imine, which then alkylates a nucleophilic residue on the enzyme.     

Role of cysteine-82 in the catalytic mechanism of human S-adenosylmethionine decarboxylase

S-Adenosylmethionine decarboxylase is a pyruvate-dependent enzyme. The enzyme forms a Schiff base with substrate, S-adenosylmethionine, through the pyruvoyl moiety. This facilitates the release of CO2 from the substrate, which must then be protonated on the alpha carbon in order to permit hydrolysis of the Schiff base to release the product. The catalytic mechanism of human S-adenosylmethionine decarboxylase was investigated via mutagenic and kinetic approaches. The results of enzyme kinetic studies indicated that Cys-82 is a crucial residue for activity and this residue has a basic pKa. Iodoacetic acid inhibited wild-type enzyme activity in a time- and pH-dependent manner but did not affect the already reduced activity of mutant C82A. Reaction of this mutant with iodoacetic acid led to approximately one less mole of reagent being incorporated per mole of enzyme alphabeta dimer than with wild-type S-adenosylmethionine decarboxylase. Both wild-type and C82A mutant S-adenosylmethionine decarboxylases were inactivated by substrate-mediated transamination, but this reaction occurred much more frequently with C82A than with wild-type enzyme. A major proportion of the recombinant C82A mutant protein was in the transaminated form in which the pyruvoyl cofactor is converted into alanine. This suggests that incorrect protonation of the pyruvate, rather than the substrate, occurs much more readily when Cys-82 is altered. On the basis of these results, it was postulated that residue Cys-82 may be the proton donor of the decarboxylation reaction catalyzed by S-adenosylmethionine decarboxylase.     

Pyruvoyl-dependent histidine decarboxylase from Lactobacillus 30a. Covalent modifications of aspartic acid 191, lysine 155, and the pyruvoyl group

The pyruvoyl-dependent histidine decarboxylase from Lactobacillus 30a is rapidly inactivated by incubation with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and glycine ethyl ester. On 90% of inactivation, 1.3 residues of [14C]glycine ethyl ester are incorporated per alpha subunit; nearly 60% of this is linked to the beta-carboxyl group of Asp-191. Histamine, a competitive inhibitor, protects against this inactivation. The KM value of the modified enzyme for histidine (6.2 mM) is much higher than that of the unmodified enzyme (KM = 0.4 mM); catalytic activity is reduced but not eliminated. Thus, Asp-191 is the most reactive accessible carboxyl group under these conditions and is close to the substrate-binding site, but apparently is not essential for catalysis. At pH 8.0, fluorodinitrobenzene inactivates histidine decarboxylase completely with the incorporation of two dinitrophenyl residues/alpha subunit; the modified residues are Lys-155 and Cys-228. Urocanic acid, a competitive inhibitor, protects against inactivation. Treatment with mercaptoethanol restores the free -SH of Cys-228 but does not restore activity. Conversion of Cys-228 to its cyano derivative slows but does not prevent dinitrophenylation of Lys-155; the resulting derivative is catalytically inactive. Thus, Lys-155 is located within the active site and may play an essential role in catalysis. Finally, histidine methyl ester was shown to inhibit this decarboxylase by forming a Schiff's base with the essential pyruvoyl group.     

Rapid Inactivation of Brain Glutamate Decarboxylase by Aspartate

In the absence of its cofactor, pyridoxal 5'-phosphate (pyridoxal-P), glutamate decarboxylase is rapidly inactivated by aspartate. Inactivation is a first-order process and the apparent rate constant is a simple saturation function of the concentration of aspartate. For the beta-form of the enzyme, the concentration of aspartate giving the half-maximal rate of inactivation is 6.1 +/- 1.3 mM and the maximal apparent rate constant is 1.02 +/- 0.09 min-1, which corresponds to a half-time of inactivation of 41 s. The rate of inactivation by aspartate is about 25 times faster than inactivation by glutamate or gamma-aminobutyric acid (GABA). Inactivation is accompanied by a rapid conversion of holoenzyme to apoenzyme and is opposed by pyridoxal-P, suggesting that inactivation results from an alternative transamination of aspartate catalyzed by the enzyme, as previously observed with glutamate and GABA. Consistent with this mechanism pyridoxamine 5'-phosphate, an expected transamination product, was formed when the enzyme was incubated with aspartate and pyridoxal-P. The rate of transamination relative to the rate of decarboxylation was much greater for aspartate than for glutamate. Apoenzyme formed by transamination of aspartate was reactivated with pyridoxal-P. In view of the high rate of inactivation, aspartate may affect the level of apoenzyme in brain.     

Identification and reactivity of the catalytic site of pig liver thioltransferase

The active site cysteine of pig liver thioltransferase was identified as Cys22. The kinetics of the reaction between Cys22 of the reduced enzyme and iodoacetic acid as a function of pH revealed that the active site sulfhydryl group had a pKa of 2.5. Incubation of reduced enzyme with [1-14C]cysteine prevented the inactivation of the enzyme by iodoacetic acid at pH 6.5, and no stable protein-cysteine disulfide was found when the enzyme was separated from excess [1-14C]cysteine, suggesting an intramolecular disulfide formation. The results suggested a reaction mechanism for thioltransferase. The thiolated Cys22 first initiates a nucleophilic attack on a disulfide substrate, resulting in the formation of an unstable mixed disulfide between Cys22 and the substrate. Subsequently, the sulfhydryl group at Cys25 is deprotonated as a result of micro-environmental changes within the active site domain, releasing the mixed disulfide and forming an intramolecular disulfide bond. Reduced glutathione, the second substrate, reduces the intramolecular disulfide forming a transient mixed disulfide which is then further reduced by glutathione to regenerate the reduced enzyme and form oxidized glutathione. The rate-limiting step for a typical reaction between a disulfide and reduced glutathione is proposed to be the reduction of the intramolecular disulfide form of the enzyme by reduced glutathione.     

Binding of radioactive alpha-difluoromethylornithine to rat liver ornithine decarboxylase

Inactivation of rat liver ornithine decarboxylase by incubation with [5-¹⁴C]-α-difluoromethylornithine resulted in the covalent binding of radio-activity to the enzyme. The extent of binding correlated with the degree of inactivation and with the amount of enzyme present. The labeled protein eluted as a single peak which coincided exactly with the active enzyme when chromatographed on Sephadex G-200 and ran as a single band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate at a position corresponding to a M.W. of about 55,000. The stoichiometric binding of [5-¹⁴C]-α-difluoromethylornithine therefore provides a convenient method for quantitating ornithine decarboxylase protein and for determining the purity of preparations of the enzyme. Assuming that 1 molecule of the drug is needed to inactivate each sub-unit, it was calculated that after stimulation with thioacetamide ornithine decarboxylase represents about 0.00014% of the liver soluble protein.     

Site-directed alteration of Glu197 and Glu66 in a pyruvoyl-dependent histidine decarboxylase

Site-directed mutagenesis has been used to explore the role of two carboxylates in the active site of histidine decarboxylase from Lactobacillus 30a. The most striking observation is that conversion of Glu197 to either Gln or Asp causes a major decrease in catalytic rate while enhancing substrate binding. This is consistent with models based on X-ray diffraction results which suggest that the acid may protonate a reaction intermediate during catalysis. The Asp197 protein undergoes a suicide reaction with substrate, apparently triggered by inappropriate protonation of the intermediate. This leads to decarboxylation-dependent transamination which converts the pyruvoyl cofactor to an alanine, inactivating the enzyme. Conversion of Glu66 to Gln affects parameters of kinetic cooperativity. The mutation fixes the Hill number at approximately 1.5, midway between the pH-dependent values of the wild-type enzyme.     

A sensitive radiometric assay for cysteic acid decarboxylase activity in crude enzyme preparations of rat liver and brain.

1. A method is described for the synthesis of L-[U-14C]cysteic acid from L-[U-14C] cysteine hydrochloride and for its subsequent utilisation as a substrate for cysteic acid decarboxylase activity in liver and brain. 2. The enzyme determination relies on the entrapment of radio-labelled carbon dioxide in Hyamine hydroxide. 3. The assay is sensitive, reliable and convenient and is particularly suitable for measuring the activity of the decarboxylase in crude enzyme preparations.     

Effect of alpha-methylation on inactivation of monoamine oxidase by N-cyclopropylbenzylamine

Monoamine oxidase (MAO) was shown previously [Silverman, R. B., & Hoffman, S. J. (1980) J. Am. Chem. Soc. 102, 7126-7128] to catalyze the oxidation of N-cyclopropylbenzylamine (N-CBA) at two sites on the molecule. Oxidation at the benzyl methylene gave benzaldehyde and cyclopropylamine; oxidation of the cyclopropyl group, which involved cyclopropyl ring cleavage, led to inactivation of the enzyme. In this paper it is shown that methylation of the benzyl methylene dramatically alters this partition ratio in favor of enzyme inactivation. Contrary to a previous report [Alles, G., & Heegaard, E. V. (1943) J. Biol. Chem. 147, 487-503], it is shown here that alpha-methylbenzylamine is a substrate for MAO; consequently, N-cyclopropyl-alpha-methylbenzylamine (N-C alpha MBA) is a good candidate for mechanism-based inactivation. N-Cyclopropyl[7-14C]benzylamine, N-cyclopropyl-alpha-methyl[phenyl-14C]benzylamine, N-[1-3H]-cyclopropylbenzylamine, and N-[1-3H]cyclopropyl-alpha-methylbenzylamine are synthesized, and product formation following MAO inactivation is quantified. The results obtained with these compounds indicate that with N-C alpha MBA, alpha-methylbenzyl oxidation (which produces acetophenone and cyclopropylamine) is only 1% that of cyclopropyl oxidation (which gives enzyme inactivation), whereas with N-CBA the amount of oxidation at the corresponding sites is equal. It also is shown that the Ki values for (R)-(+)- and (S)-(-)-alpha-methylbenzylamine are similar, suggesting that dimethylation of N-CBA should not interfere with binding to MAO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the regulation of leucine catabolism in the liver. Stimulation by pyruvate and dichloroacetate

The rate of oxidation of L-[1-14C]leucine to 14CO2 by isolated rat hepatocytes is increased by pyruvate and dichloroacetate. This effect is specific for L-leucine, not being observed for L-valine, L-isoleucine, or D-leucine. Transamination, the rate-limiting step of L-leucine catabolism in the liver, is the site of stimulation, because uptake of L-leucine by the cells and the oxidation of its transamination product, alpha-ketoisocaproate, are not increased. Measurement of steady state levels of alpha-ketoisocaproate indicate that both pyruvate and dichloroacetate promote the transamination of L-leucine, thereby increasing the availability of substrate for decarboxylation by the alpha-ketoisocaproate dehydrogenase complex (EC 1.2.4.3). Pyruvate stimulation of transamination is secondary to the provision of keto acid acceptors for the amino group of L-leucine. The mechanism of the effect of dichloroacetate remains unknown.     

Polyamine-stimulated alteration of the ornithine decarboxylase molecule in Physarum polycephalum.

The molecular mechanism for polyamine-stimulated feedback modification of ornithine decarboxylase isolated from Physarum polycephalum was investigated by using two-dimensional polyacrylamide-gel electrophoresis. Partially purified A-form enzyme was converted into the B-form enzyme by isolated fractions of the Physarum A-B-converting protein, and the substrates and products were subsequently labelled by covalent addition of alpha-difluoro[14C]methylornithine, an enzyme-activated irreversible inhibitor. The active (A-form) and inactive (B-form) states of this enzyme were found to have the same Mr value, 52 000, yet they differed noticeably in their pI values, 5.45 and 5.65 respectively. In further experiments, the use of high-specific-radioactivity [3H]spermidine to stimulate this enzyme modification was shown not to result in the covalent attachment of this polyamine to ornithine decarboxylase. These results demonstrate that the polyamine-induced modification of ornithine decarboxylase in Physarum is not due to any of the mechanisms previously suggested for ornithine decarboxylase inactivation in this and other eukaryotes, namely phosphorylation, covalent polyamine addition or the non-covalent association of a specific low-Mr protein.     

Cysteinesulfonate and beta-sulfopyruvate metabolism. Partitioning between decarboxylation, transamination, and reduction pathways

L-Cysteinesulfonate (L-cysteate) is present in plasma, urine, and tissues in concentrations comparable to that of L-cysteinesulfinate, the primary oxidative metabolite of L-cysteine. Although cysteinesulfonate is known to be decarboxylated to taurine by cysteinesulfinate decarboxylase, the occurrence and importance of other metabolisms has not been examined. The present studies indicate that cysteinesulfonate partitions in vivo between decarboxylation and transamination; the latter reaction is catalyzed by aspartate aminotransferase and yields beta-sulfopyruvate. Whereas beta-sulfinylpyruvate, the product of cysteinesulfinate transamination, decomposes spontaneously, beta-sulfopyruvate is stable and is reduced by malate dehydrogenase to beta-sulfolactate. When L-[1-14C]cysteinesulfonate is given to mice, 60-75% is decarboxylated to taurine and about 25% is excreted in the urine as beta-sulfolactate. beta-Sulfo[1-14C] pyruvate is found to partition about equally between beta-sulfolactate and cysteinesulfonate formation; greater than 90% of the latter is decarboxylated. Parenterally administered beta-sulfo[1-14C]lactate is mostly excreted in the urine, but 12% is metabolized via beta-sulfopyruvate and cysteinesulfonate to 14CO2 and taurine. beta-Sulfopyruvate is not excreted, and only traces of sulfoacetate, perhaps formed by oxidative decarboxylation, are detected. These studies establish that cysteinesulfonate, beta-sulfopyruvate, and beta-sulfolactate are reversibly interconverted in vivo. Since only cysteinesulfonate is directly metabolized to CO2, the rate of 14CO2 formation from L-[1-14C]cysteinesulfonate is a valid measure of total cysteinesulfinate decarboxylase activity in vivo; use of this assay permits inhibitor effects to be accurately determined in intact mice. Thus, whereas in vitro assays indicate that beta-methyleneaspartate inhibits brain, liver, and kidney cysteinesulfinate decarboxylase by 0, greater than 60, and 90%, respectively, in vivo studies with L-[1-14C]cysteinesulfonate show net metabolic inhibition is about 40%.     

Active site directed irreversible inactivation of brewers' yeast pyruvate decarboxylase by the conjugated substrate analogue (E)-4-(4-chlorophenyl)-2-oxo-3-butenoic acid: development of a suicide substrate

(E)-4-(4-Chlorophenyl)-2-oxo-3-butenoic acid (CPB) was found to irreversibly inactivate brewers' yeast pyruvate decarboxylase (PDC, EC 4.1.1.1) in a biphasic, sigmoidal manner, as is found for the kinetic behavior of substrate. An expression was derived for two-site irreversible inhibition of allosteric enzymes, and the kinetic behavior of CPB fit the expression for two-site binding. The calculated Ki's of 0.7 mM and 0.3 mM for CPB were assigned to the catalytic site and the regulatory site, respectively. The presence of pyruvic acid at high concentrations protected PDC from inactivation, whereas low concentrations of pyruvic acid accelerated inactivation by CPB. Pyruvamide, a known allosteric activator of PDC, was found to enhance inactivation by CPB. The results can be explained if pyruvamide binds only to a regulatory site, but CPB and pyruvic acid compete for both the regulatory and the catalytic centers. [1-14C]CPB was found to lose 14CO2 concurrently with the inactivation of the enzyme. Therefore, CPB was being turned over by PDC, in addition to inactivating it. CPB can be labeled a suicide-type inactivator for PDC.     

Transamination and oxidation of leucine and valine in rat adipose tissue

Leucine was oxidized by rat adipose tissue at a rate which was not limited by the activity of branched chain amino acid transaminase since high concentrations (10 mM) of [1-14C]leucine and its transamination product, alpha-keto[1-14C]isocaproate, were oxidized at similar rates. Despite the apparent abundance of transaminase activity, however, [1-14C]valine was oxidized at only 10 to 25% of the rate of its transamination product, alpha-keto[1-14C]isovalerate. The net rate at which [1-14C] valine was transaminated by intact tissues was estimated as the sum of the rates of 14CO2 production and alpha-ketoiso[1-14C]valerate release into the medium. Transamination did not limit the rate of valine oxidation since valine was transaminated 3 times as fast as it was oxidized. The rate of valine transamination increased 18-fold when its concentration was raised 100-fold, but the fraction of [1-14C]valine oxidized to 14CO2 remained constant over the range of incubation conditions studied. The oxidation/transamination ratio for leucine was also constant and exceeded the oxidation/transamination ratio for valine unless valine oxidation was stimulated, either by the addition of glucose or leucine. Stimulation of valine oxidation did not increase its transamination but reduced the rate at which alpha-ketoisovalerate was released from the tissue. The faster oxidation of alpha-ketoisocaproate than of alpha-ketoisovalerate may be due to the activation of branched chain alpha-keto acid dehydrogenase by alpha-ketoisocaproate, but the alpha-keto acid oxidation rates do not fully account for the faster transamination of leucine than of valine.     

Role of mitochondrial transamination in branched chain amino acid metabolism

Oxidative decarboxylation and transamination of 1-14C-branched chain amino and alpha-keto acids were examined in mitochondria isolated from rat heart. Transamination was inhibited by aminooxyacetate, but not by L-cycloserine. At equimolar concentrations of alpha-ketoiso[1-14C]valerate (KIV) and isoleucine, transamination was increased by disrupting the mitochondria with detergent which suggests transport may be one factor affecting the rate of transamination. Next, the subcellular distribution of the aminotransferase(s) was determined. Branched chain aminotransferase activity was measured using two concentrations of isoleucine as amino donor and [1-14C]KIV as amino acceptor. The data show that branched chain aminotransferase activity is located exclusively in the mitochondria in rat heart. Metabolism of extramitochondrial branched chain alpha-keto acids was examined using 20 microM [1-14C]KIV and alpha-ketoiso[1-14C]caproate (KIC). There was rapid uptake and oxidation of labeled branched chain alpha-keto acid, and, regardless of the experimental condition, greater than 90% of the labeled keto acid substrate was metabolized during the 20-min incubation. When a branched chain amino acid (200 microM) or glutamate (5 mM) was present, 30-40% of the labeled keto acid was transaminated while the remainder was oxidized. Provision of an alternate amino acceptor in the form of alpha-keto-glutarate (0.5 mM) decreased transamination of the labeled KIV or KIC and increased oxidation. Metabolism of intramitochondrially generated branched chain alpha-keto acids was studied using [1-14C]leucine and [1-14C]valine. Essentially all of the labeled branched chain alpha-keto acid produced by transamination of [1-14C]leucine or [1-14C]valine with a low concentration of unlabeled branched chain alpha-keto acid (20 microM) was oxidized. Further addition of alpha-ketoglutarate resulted in a significant increase in the rate of labeled leucine or valine transamination, but again most of the labeled keto acid product was oxidized. Thus, catabolism of branched chain amino acids will be favored by a high concentration of mitochondrial alpha-ketoglutarate and low intramitochondrial glutamate.     

Inactivation of gamma-aminobutyric acid aminotransferase by (Z)-4-amino-2-fluorobut-2-enoic acid

(Z)-4-Amino-2-fluorobut-2-enoic acid (1) is shown to be a mechanism-based inactivator of pig brain gamma-aminobutyric acid aminotransferase. Approximately 750 inactivator molecules are consumed prior to complete enzyme inactivation. Concurrent with enzyme inactivation is the release of 708 +/- 79 fluoride ions; transamination occurs 737 +/- 15 times per inactivation event. Inactivation of [3H]pyridoxal 5'-phosphate ([3H]PLP) reconstituted GABA aminotransferase by 1 followed by denaturation releases [3H]PMP with no radioactivity remaining attached to the protein. A similar experiment carried out with 4-amino-5-fluoropent-2-enoic acid [Silverman, R. B., Invergo, B. J., & Mathew, J. (1986) J. Med. Chem. 29, 1840-1846] as the inactivator produces no [3H]PMP; rather, another radioactive species is released. These results support an inactivation mechanism for 1 that involves normal catalytic isomerization followed by active site nucleophilic attack on the activated Michael acceptor. A general hypothesis for predicting the inactivation mechanism (Michael addition vs enamine addition) of GABA aminotransferase inactivators is proposed.     

Catalytic properties of the archaeal S-adenosylmethionine decarboxylase from Methanococcus jannaschii

S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl cofactor-dependent enzyme that participates in polyamine biosynthesis. AdoMetDC from the Archaea Methanococcus jannaschii is a prototype for a recently discovered class that is not homologous to the eucaryotic enzymes or to a distinct group of microbial enzymes. M. jannaschii AdoMetDC has a Km of 95 microm and the turnover number (kcat) of 0.0075 s(-1) at pH 7.5 and 22 degrees C. The turnover number increased approximately 38-fold at a more physiological temperature of 80 degrees C. AdoMetDC was inactivated by treatment with the imine reductant NaCNBH3 only in the presence of substrate. Mass spectrometry of the inactivated protein showed modification solely of the pyruvoyl-containing subunit, with a mass increase corresponding to reduction of a Schiff base adduct with decarboxylated AdoMet. The presteady state time course of the AdoMetDC reaction revealed a burst of product formation; thus, a step after CO2 formation is rate-limiting in turnover. Comparable D2O kinetic isotope effects of were seen on the first turnover (1.9) and on kcat/Km (1.6); there was not a significant D2O isotope effect on kcat, suggesting that product release is rate-limiting in turnover. The pH dependence of the steady state rate showed participation of acid and basic groups with pK values of 5.3 and 8.2 for kcat and 6.5 and 8.3 for kcat/Km, respectively. The competitive inhibitor methylglyoxal bis(guanylhydrazone) binds at a single site per (alphabeta) heterodimer. UV spectroscopic studies show that methylglyoxal bis(guanylhydrazone) binds as the dication with a 23 microm dissociation constant. Studies with substrate analogs show a high specificity for AdoMet.