Oxygenase side reactions of acetolactate synthase and other carbanion-forming enzymes

Abell, Lynn M.; Schloss, John V.

doi:10.1021/bi00246a002

Cited by 93 publications

(118 citation statements)

References 16 publications

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“…Because of the spin-forbidden interaction of triplet oxygen with singlet-state molecules, oxygen is generally unreactive in the absence of an activating cofactor such as a transition metal or flavin, so its presence in protein active sites is usually unnoticed and inconsequential. One exception is the reduction of oxygen to superoxide by carbanions (28)(29)(30). Although alternative explanations for the mechanism of firefly luciferase have been offered (18), we propose that formation of the luciferin-AMP ester within the enzyme allows access to a resonance-stabilized carbanion that can reduce molecular dioxygen to superoxide by single-electron transfer and then react by subsequent recombination of the radical pair after spin inversion to form a peroxide (Fig.…”

Section: Discussionmentioning

confidence: 92%

Latent luciferase activity in the fruit fly revealed by a synthetic luciferin

Mofford

Reddy

Miller

2014

Proc. Natl. Acad. Sci. U.S.A.

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Beetle luciferases are thought to have evolved from fatty acyl-CoA synthetases present in all insects. Both classes of enzymes activate fatty acids with ATP to form acyl-adenylate intermediates, but only luciferases can activate and oxidize D-luciferin to emit light. Here we show that the Drosophila fatty acyl-CoA synthetase CG6178, which cannot use D-luciferin as a substrate, is able to catalyze light emission from the synthetic luciferin analog CycLuc2. Bioluminescence can be detected from the purified protein, live Drosophila Schneider 2 cells, and from mammalian cells transfected with CG6178. Thus, the nonluminescent fruit fly possesses an inherent capacity for bioluminescence that is only revealed upon treatment with a xenobiotic molecule. This result expands the scope of bioluminescence and demonstrates that the introduction of a new substrate can unmask latent enzymatic activity that differs significantly from an enzyme's normal function without requiring mutation.evolution | enzymatic promiscuity | imaging | firefly luciferase | chemical biology

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Section: Discussionmentioning

confidence: 92%

Latent luciferase activity in the fruit fly revealed by a synthetic luciferin

Mofford

Reddy

Miller

2014

Proc. Natl. Acad. Sci. U.S.A.

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“…In addition, rate constants derived from the distribution of intermediates are net rate constants, which reflect the true forward rate constant multiplied by the fraction of the resulting enzyme form that partitions onto products as opposed to undergoing reversal (15). The decarboxylation step (kЈ 3 ) is presumably irreversible, but we know that steps 4 and 5 are reversible, because chemically synthesized AL can be converted to, e.g., peracetate (22) or phenylacetyl carbinol (ref. 23 and D.M.C., unpublished results) by AHAS II.…”

Section: Discussionmentioning

confidence: 99%

“…23 and D.M.C., unpublished results) by AHAS II. On the basis of Schloss and coworkers' observations on the peracetate-forming oxygenase reaction (22,24), one also can assume that there is reversible binding of the acceptor ketoacid to the enzyme-HEThDP complex before its reaction (as opposed to Theorell-Chance kinetics for step 4): Both pyruvate and 2-keto- butyrate can compete with oxygen for enzyme-bound HEThDP, but the competition saturates at Ͻ100% inhibition of the oxygenase reaction. Despite the difference in microscopic rate constants for ligation of ketobutyrate and pyruvate to bound HEThDP Ϫ , the slow reduction of FAD observed during substrate turnover by AHAS II is also only slightly dependent on the second substrate (14).…”

Section: Discussionmentioning

confidence: 99%

The carboligation reaction of acetohydroxyacid synthase II: Steady-state intermediate distributions in wild type and mutants by NMR

Tittmann

Vyazmensky

Hübner

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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The thiamin diphosphate (ThDP)-dependent enzyme acetohydroxyacid synthase (AHAS) catalyzes the first common step in branched-chain amino acid biosynthesis. By specific ligation of pyruvate with the alternative acceptor substrates 2-ketobutyrate and pyruvate, AHAS controls the flux through this branch point and determines the relative rates of synthesis of isoleucine, valine, and leucine, respectively. We used detailed NMR analysis to determine microscopic rate constants for elementary steps in the reactions of AHAS II and mutants altered at conserved residues Arg-276, Trp-464, and Met-250. In Arg276Lys, both the condensation of the enzyme-bound hydroxyethyl-ThDP carbanion͞enamine (HEThDP) with the acceptor substrates and acetohydroxyacid release are slowed several orders of magnitude relative to the wild-type enzyme. We propose that the interaction of the guanidinium moiety of Arg-264 with the carboxylate of the acceptor ketoacid provides an optimal alignment of substrate and HEThDP orbitals in the reaction trajectory for acceptor ligation, whereas its interaction with the carboxylate of the covalent HEThDP-acceptor adduct plays a similar role in product release. Both Trp-464 and Met-250 affect the acceptor specificity. The high preference for ketobutyrate in the wild-type enzyme is lost in Trp464Leu as a consequence of similar forward rate constants of carboligation and product release for the alternative acceptors. In Met250Ala, the turnover rate is determined by the condensation of HEThDP with pyruvate and release of the acetolactate product, whereas the parallel steps with 2-ketobutyrate are considerably faster. We speculate that the specificity of carboligation and product liberation may be cumulative if the former is not completely committed.mechanism ͉ thiamin diphosphate ͉ amino acid biosynthesis A cetohydroxyacid (AHA) synthase (AHAS), which catalyzes the first common step in the biosynthesis of branched-chain amino acids, belongs to a homologous family of thiamin diphosphate (ThDP)-dependent enzymes whose initial step is decarboxylation of pyruvate or another 2-ketoacid (1-3). In the process catalyzed by AHAS, the decarboxylation of pyruvate to form the hydroxyethyl-ThDP Ϫ anion͞enamine (HEThDP Ϫ ) is followed by the specific condensation of the intermediate with a second aliphatic ketoacid to form an AHA (Fig. 1). Whereas the role of the enzyme in the first steps in AHAS catalysis, i.e., activation of ThDP, decarboxylation of pyruvate, and formation of HEThDP (3), is comparable with the function of other members of its homologous family (4), the function of the protein in controlling the fate of HEThDP is poorly understood.The competition between the alternative ''second substrates'' 2-ketobutyrate and pyruvate, leading to partition of the flux through AHAS between acetohydroxybutyrate (AHB) and acetolactate (AL), determines the relative rates of formation of isoleucine and of valine, leucine, and pantothenate, respectively ( Fig. 1) (5). Most AHASs show a strong preference for 2-ketobutyrate as the s...

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“…the release of CO # . It is of interest that Abell and Schloss [18] have demonstrated that several enzymes mediating carbanion chemistry are capable of reacting with molecular oxygen, thus catalysing oxygen-consuming side reactions. Among these enzymes, a PLP-dependent enzyme, GAD, has been shown to exhibit oxygenase activity.…”

Section: Discussionmentioning

confidence: 99%

Reaction of dopa decarboxylase with L-aromatic amino acids under aerobic and anaerobic conditions

Bertoldi

Voltattorni

2000

Biochemical Journal

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Analysis of the reaction of dopa decarboxylase (DDC) with L-dopa reveals that loss of decarboxylase activity with time is observed at enzyme concentrations approximately equal to the binding constant, Kd, of the enzyme for pyridoxal 5ƀ-phosphate (PLP). Instead, at enzyme concentrations higher than Kd the course of product formation proceeds linearly until complete consumption of the substrate. Evidence is provided that under both experimental conditions no pyridoxamine 5ƀ-phosphate (PMP) is formed during the reaction and that dissociation of coenzyme occurs at low enzyme concentration, leading to the formation of a PLP-L-dopa Pictet–Spengler cyclic adduct. Taken together, these results indicate that decarboxylation-dependent transamination does not accompany the decarboxylation of L-dopa proposed previously [O'Leary and Baughn (1977) J. Biol. Chem. 252, 7168–7173]. Nevertheless, when the reaction of DDC with L-dopa is studied under anaerobic conditions at an enzyme concentration higher than Kd, we observe that (1) the enzyme is gradually inactivated and inactivation is associated with PMP formation and (2) the initial velocity of decarboxylation is approximately half of that in the presence of O2. Similar behaviour is observed by comparing the reaction with L-5-hydroxytryptophan occurring in aerobiosis or in anaerobiosis. Therefore the reaction of DDC with L-aromatic amino acids seems to be under O2 control. In contrast, the reactivity of the enzyme with D-aromatic amino acids does not change in the presence or absence of O2. These and other results, together with previous results on the effect exerted by O2 on reaction specificity of DDC towards aromatic amines [Bertoldi, Frigeri, Paci and Borri Voltattorni (1999) J. Biol. Chem. 274, 5514–5521], suggest a productive effect of O2 on an intermediate complex of the reaction of the enzyme with L-aromatic amino acids or aromatic amines.

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Oxygenase side reactions of acetolactate synthase and other carbanion-forming enzymes

Cited by 93 publications

References 16 publications

Latent luciferase activity in the fruit fly revealed by a synthetic luciferin

Latent luciferase activity in the fruit fly revealed by a synthetic luciferin

The carboligation reaction of acetohydroxyacid synthase II: Steady-state intermediate distributions in wild type and mutants by NMR

Reaction of dopa decarboxylase with L-aromatic amino acids under aerobic and anaerobic conditions

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