1-Carboxyallenyl phosphate, an allenic analogue of phosphoenolpyruvate

1-Carboxyallenyl phosphate, the allenic homologue of phosphoenolpyruvate, has been synthesized in six steps. The key step in the synthesis is the isomerization of methyl 2-hydroxy-3-butynoate to the corresponding allenol and phosphorylation of this material. The allene is an excellent substrate for pyruvate kinase, undergoing reaction at more than half the rate of phosphoenolpyruvate. The allene is also a substrate for phosphoenolpyruvate carboxylase, being hydrolyzed by the enzyme rather than carboxylated. With both enzymes, the organic product is 2-oxo-3-butenoate, which gradually inactivates the enzymes by reaction with one or more sulfhydryl groups not at the active site.     

Structure of the Ring Cleavage Product of 1-Hydroxy-2-Naphthoate, an Intermediate of the Phenanthrene-Degradative Pathway of Nocardioides sp. Strain KP7

1-Hydroxy-2-naphthoate (compound I) is a metabolite of the phenanthrene-degradative pathway in Nocardioides sp. strain KP7. This singly hydroxylated aromatic compound is cleaved by 1-hydroxy-2-naphthoate dioxygenase. In this study, the structure of the ring cleavage product generated by the action of homogeneous 1-hydroxy-2-naphthoate dioxygenase was determined upon separation by high-performance liquid chromatography at pH 2.5 by using nuclear magnetic resonance (NMR) and mass spectroscopic techniques. The ring cleavage product at this pH existed in equilibrium between two forms, 2-oxo-3-(3-oxo-1,3-dihydro-1-isobenzofuranyl)propanoate (compound III) and 2,2-dihydroxy-3-(3-oxo-1,3-dihydro-1-isobenzofuranyl)propanoate (compound IV). After the pH of the solution was raised to 7.5, the structure of the major species became (E)-4-(2-carboxylatophenyl)-2-oxo-3-butenoate (compound II; common name, trans-2′-carboxybenzalpyruvate), which was in equilibrium with compound III. Direct monitoring of the enzymatic formation of the ring cleavage product by ¹H-NMR in a deuterated potassium phosphate buffer (pH 7.5) detected only compound II as a product, and the proton on carbon 3 of compound II was not exchanged with deuterium. Thus, compound II is likely to be the first stable product of dioxygenation of 1-hydroxy-2-naphthoate.     

Synthesis and study of (Z)-3-chlorophosphoenolpyruvate

(Z)-3-Chlorophosphoenolpyruvate has been synthesized by the reaction of 3,3-dichloropyruvic acid with trimethylphosphite, followed by deesterification. This compound is a competitive inhibitor of pyruvate kinase and phosphoenolpyruvate carboxylase. Pyruvate kinase is not inactivated upon prolonged incubation with the compound, but phosphoenolpyruvate carboxylase is slowly inactivated (t1/2 = 5 h). The compound is a substrate for both enzymes, being acted upon by pyruvate kinase approximately 0.1% as rapidly as phosphoenolpyruvate itself. In the case of phosphoenolpyruvate carboxylase, the compound is converted into a 3:1 mixture of chloropyruvate and chlorooxalacetate, at an overall rate that is about 25% the carboxylation rate for phosphoenolpyruvate.     

Suppression of the TRIF-dependent signaling pathway of toll-like receptors by (E)-isopropyl 4-oxo-4-(2-oxopyrrolidin-1-yl)-2-butenoate

Toll-like receptors (TLRs) are pattern recognition receptors that recognize molecular structures derived from microbes and initiate innate immunity. TLRs have two downstream signaling pathways, the MyD88- and TRIF-dependent pathways. Dysregulated activation of TLRs is closely linked to increased risk of many chronic diseases. Previously, we synthesized fumaryl pyrrolidinone, (E)-isopropyl 4-oxo-4-(2-oxopyrrolidin-1- yl)-2-butenoate (IPOP), which contains a fumaric acid isopropyl ester and pyrrolidinone, and demonstrated that it inhibits the activation of nuclear factor kappa B by inhibiting the MyD88-dependent pathway of TLRs. However, the effect of IPOP on the TRIF-dependent pathway remains unknown. Here, we report the effect of IPOP on signal transduction via the TRIF-dependent pathway of TLRs. IPOP inhibited lipopolysaccharide- or polyinosinic-polycytidylic acid-induced interferon regulatory factor 3 activation, as well as interferon- inducible genes such as interferon inducible protein-10. These results suggest that IPOP can modulate the TRIF-dependent signaling pathway of TLRs, leading to decreased inflammatory gene expression.     

Inactivation of phosphoenolypyruvate carboxykinase (GTP) by liver extracts.

1. The inactivation of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) in liver extracts was catalysed by the microsomal fraction, and led to the enzyme becoming bound to the microsomal membranes. 2. Inactivation by microsomal fraction, typsin or heating at 48degreesC was accelerated by L-cystine, D-cystine and oxidized glutathione and decreased by dithiothreitol. 3. MnC1(2) and CoC1(2) protected the enzyme from inactivation by heat or microsomal fraction, but did not affect the inactivation caused by trypsin. 4. Several proteinase inhibitors had no effect on the microsomal inactivation reaction, suggesting that proteolysis was not involved. 5. It is argued that the initial step in the degradation of phosphoenolpyruvate carboxykinase (GTP) is an inactivation reaction, perhaps involving oxidized thiol compounds.     

(E)-3-Cyanophosphoenolpyruvate, a new inhibitor of phosphoenolpyruvate-dependent enzymes

(E)-3-Cyanophosphoenolpyruvate has been synthesized by reacting dimethyl chlorophosphate with the potassium enolate of ethyl cyanopyruvate. The resulting trialkyl ester was deesterified with bromotrimethylsilane followed by potassium hydroxide. Subsequent treatment with Dowex-50-H+ resin and cyclohexylamine afforded the tricyclohexylammonium salt; only the E geometric isomer was obtained. This compound can be photoisomerized to a 70:30 E:Z mixture. (E)-3-Cyanophosphoenolpyruvate is an excellent competitive inhibitor of phosphoenolpyruvate carboxylase [KI(Mn2+) = 16 microM, KI(Mg2+) = 1360 microM], pyruvate kinase [KI(Mn2+) = 0.085 microM, KI(Mg2+) = 0.76 microM], and enolase [KI(Mn2+) = 360 microM, KI(Mg2+) = 280 microM]. The compound is a substrate for pyruvate kinase (Vmax approximately 1% of phosphoenolpyruvate rate), but not for the other two enzymes. No irreversible inactivation is observed with phosphoenolpyruvate carboxylase of pyruvate kinase.     

An Alternate Synthesis of 2-(4-Isobutylphenyl)-propionic Acid

2-(4-Isobutylphenyl)-propionic acid, which is known to have a high anti-inflammatory activity and has widely been used in the treatment of diseases caused by inflammation, such as rheumatism, was synthesized from methyl 3-methyl-3-(4-isobutylphenyl)-glycidate, via methyl 2-hydroxy-3-(4-isobutylphenyl)-3-butenoate (VI) in four steps.     

Affinity labeling of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase with the 2',3'-dialdehyde derivative of ATP

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] is completely inactivated by the 2',3'-dialdehyde derivative of ATP (oATP) in the presence of Mn2+. The dependence of the pseudo-first-order rate constant on reagent concentration indicates the formation of a reversible complex with the enzyme (Kd = 60 +/- 17 microM) prior to covalent modification. The maximum inactivation rate constant at pH 7.5 and 30 degrees C is 0.200 +/- 0.045 min-1. ATP or ADP plus phosphoenolpyruvate effectively protect the enzyme against inactivation. oATP is a competitive inhibitor toward ADP, suggesting that oATP interacts with the enzyme at the substrate binding site. The partially inactivated enzyme shows an unaltered Km but a decreased V as compared with native phosphoenolpyruvate carboxykinase. Analysis of the inactivation rate at different H+ concentrations allowed estimation of a pKa of 8.1 for the reactive amino acid residue in the enzyme. Complete inactivation of the carboxykinase can be correlated with the incorporation of about one mole of [8-14C]oATP per mole of enzyme subunit. The results indicate that oATP can be used as an affinity label for yeast phosphoenolpyruvate carboxykinase.     

Studies on 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe)from Escherichia coli K12. 2. Kinetic properties.

1. Co2+ is not a cofactor for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe). 2. The following analogues of phosphoenolpyruvate were tested as inhibitors of 3-deoxy-D-arabinoheptolosonate-7-phosphate synthetase(phe): pyruvate, lactate, glycerate, 2-phosphoglycerate, 2,3-bisphosphoglycerate, 3-methylphosphoenolpyruvate, 3-ethylphosphoenolpyruvate and 3,3-demethylphosphoenolpyruvate. The rusults obtained indicate that the binding of phosphoenolpyruvate to the enzyme requires a phosphoryl group on the C-2 position of the substrate and one free hydrogen atom at the C-3 position. 3. The dead-end inhibition pattern observed with the substrate analogue 2-phosphoglycerate when either phosphoenolpyruvate or erythrose 4-phosphate was the variable substrate is inconsistent with a ping-pong mechanism and indicates that the reaction mechanism for this enzyme must be sequential. The following kinetic constants were determined:Km for phosphoenolpyruvate, 0.08 +/- 0.04 mM; Km for erythrose 4-phosphate, 0.9 +/- 0.3 mM; K is for competitive inhibition by 2-phosphoglycerate with respect to phosphoenolpyruvate, 1.0 +/- 0.1 mM. 4. The enzyme was observed to have a bell-shaped pH PROFILE WITH A PH OPTIMUM OF 7.0. The effects of pH ON V and V/(Km for phosphoenolpyruvate) indicated that an ionizing group of pKa 8.0-8.1 is involved in the catalytic activity of the enzyme. The pKa of this group is unaffected by the binding of phosphoenolpyruvate.     

Mechanistic studies of phosphoenolpyruvate carboxylase from Zea mays with (Z)- and (E)-3-fluorophosphoenolpyruvate as substrates.

The catalytic mechanism of phosphoenolpyruvate (PEP) carboxylase from Zea mays has been studied using (Z)- and (E)-3-fluorophosphoenolpyruvate (F-PEP) as substrates. Both (Z)- and (E)-F-PEP partition between carboxylation to produce 3-fluorooxalacetate and hydrolysis to produce 3-fluoropyruvate. Carboxylation accounts for 3% of the reaction observed with (Z)-F-PEP, resulting in the formation of (R)-3-fluorooxalacetate, and for 86% of the reaction of (E)-F-PEP forming (S)-3-fluorooxalacetate. Carboxylation of F-PEP occurs on the 2-re face, which corresponds to the 2-si face of PEP. The partitioning of F-PEP between carboxylation and hydrolysis is insensitive to pH but varies with metal ion. Use of 18O-labeled bicarbonate produces phosphate that is multiply labeled with 18O; in addition, 18O is also incorporated into residual (Z)- and (E)-F-PEP. The 13(V/K) isotope effect on the carboxylation of F-PEP catalyzed by PEP carboxylase at pH 8.0, 25 degrees C, is 1.049 +/- 0.003 for (Z)-F-PEP and 1.009 +/- 0.006 for (E)-F-PEP. These results are consistent with a mechanism in which carboxylation of PEP occurs via attack of the enolate of pyruvate on CO2 rather than carboxy phosphate. In this mechanism phosphorylation of bicarbonate to give carboxy phosphate and decarboxylation of the latter are reversible steps. An irreversible step, however, precedes partitioning between carboxylation to give oxalacetate and release of CO2, which results in hydrolysis of PEP.     

In Vitro antifungal activity of 2-(4-substituted phenyl)-3(2H)-isothiazolones

The in vitro antifungal activity of several N2-phenyl-3(2H)-isothiazolones substituted at C4 of the phenyl moiety with heterocyclic nucleus or groups of different physico-chemical properties against four human pathogenic fungi was determined by broth macrodilution method; results were compared with those obtained with itraconazole and ketoconazole. These isothiazolones showed moderate to high activity against some or all tested strains and in comparison with the reference drugs, 5-chloro-2-(4-nitrophenyl)isothiazol-3-one (1g), 5-chloro-2-phenylisothiazol-3-one (1c), 4-[4-(5-chloro-3-oxo-3H-isothiazol-2-yl)phenyl]-1,4-dihydrotriazol-5-one (1s) and 2-(4-nitrophenyl)isothiazol-3-one (2g) against Aspergillus niger, 5-chloro-2-(4-nitrophenyl)isothiazol-3-one (1g) and 4-[4-(5-chloro-3-oxo-3H-isothiazol-2-yl)phenyl]piperazine-1-carboxamide (1q) against Trichophyton mentagrophytes had comparable activity, compounds 1g and 2g showing higher activity against Microsporum canis. Antifungal activity was favored by the presence of chlorine at C5 of the isothiazolone and/or the presence of nitro group or heterocyclic nucleus at C4 of the phenyl ring and proper hydrophilicity of the molecule.     

Cysteinyl peptides of rabbit muscle pyruvate kinase labeled by the affinity label 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate

The affinity label 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate (8-BDB-TA-5'-TP) reacts covalently with rabbit muscle pyruvate kinase, incorporating 2 mol of reagent/mol of enzyme subunit upon complete inactivation. Protection against inactivation is provided by phosphoenolpyruvate, K+, and Mn2+ and only 1 mol of reagent/mol of subunit is incorporated [DeCamp, D.L., Lim, S., & Colman, R.F. (1988) Biochemistry 27, 7651-7658]. We have now identified the resultant modified residues. After reaction with 8-BDB-TA-5'-TP at pH 7.0, modified enzyme was incubated with [3H]NaBH4 to reduce the carbonyl groups of enzyme-bound 8-BDB-TA-5'-TP and to introduce a radioactive tracer into the modified residues. Following carboxymethylation and digestion with trypsin, the radioactive peptides were separated on a phenylboronate agarose column followed by reverse-phase high-performance liquid chromatography in 0.1% trifluoroacetic acid with an acetonitrile gradient. Gas-phase sequencing gave the cysteine-modified peptides Asn162-Ile-Cys-Lys165 and Cys151-Asp-Glu-Asn-Ile-Leu-Trp-Leu-Asp-Tyr-Lys161, with a smaller amount of Asn43-Thr-Gly-Ile-Ile-Cys-Thr-Ile-Gly-Pro-Ala-Ser-Arg55. Reaction in the presence of the protectants phosphoenolpyruvate, K+, and Mn2+ yielded Asn-Ile-Cys-Lys as the only labeled peptide, indicating that inactivation is caused by modification of Cys151 and Cys48.     

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.     

Inhibition of the bovine branched-chain 2-oxo acid dehydrogenase complex and its kinase by arylidenepyruvates

A novel class of inhibitors for the branched-chain 2-oxo acid dehydrogenase (BCOAD) complex has been synthesized and studied. The sodium salts of arylidenepyruvates: e.g., furfurylidenepyruvate (compound I), 4-(3-thienyl)-2-oxo-3-butenoate (compound II), cinnamalpyruvate (compound III) and 4-(2-thienyl)-2-oxo-3-butenoate (compound IV) inhibit the overall and kinase reactions of the BCOAD complex from bovine liver. Inhibitions of the overall reaction occur at the decarboxylase (E1) step as determined by a spectrophotometric assay with 2,6-dichlorophenolindophenol as an electron acceptor. Inhibition of the E1 reaction by compound I (Ki = 0.5 microM) is competitive, whereas inhibitions by compounds II (Ki = 150 microM) and III (Ki = 500 microM) are non-competitive with respect to the substrate 2-oxoisovalerate. The Km value for 2-oxoisovalerate is 6.7 microM as measured by the E1 assay. Inhibition of the E1 step by compounds I, II and III are reversible at low inhibitor concentrations based on the Michaelis-Menten kinetics observed. By comparison, compound I does not significantly inhibit pyruvate and 2-oxoglutarate dehydrogenase complexes. The arylidenepyruvates (compounds I, II and IV) inhibit the BCOAD kinase reaction in a manner similar to the substrate 2-oxo acids. The inhibition of the kinase reaction by compound I is non-competitive with respect to ATP, with an apparent Ki value of 4.5 mM. The results suggest that arylidenepyruvates may be useful probes for elucidating the reaction mechanisms of the BCOAD complex and its kinase.     

Proteolytic enzymes in green wheat-leaves III. Inactivation of acid proteinase II by diazoacetyl-DL-norleucine methyl ester and 1,2-epoxy-3-(p-nitrophenoxy)-propane

Acid proteinase II isolated from green wheat leaves in a purifiedform was rapidly inactivated at pH=5.5 to 6.0 by a 50-fold molarexcess of diazoacetyl-DL-norleucine methyl ester (DAN) in thepresence of cupric ions which were essential for inactivation.The acid proteinase was also inactivated by reaction with 1,2-epoxy-3-(p-nitrophenoxy)-propane(EPNP). The inactivation by EPNP was much slower than by DANand the half-life of the activity was 24 hr. (Received February 6, 1978; )     

Dimerization occurs during the reversible acid inactivation of 2-oxo-4-hydroxyglutarate aldolase from Escherichia coli

Exposure of Escherichia coli 2-oxo-4-hydroxyglutarate aldolase (4-hydroxy-2-oxoglutarate glyoxylate-lyase, EC 4.1.3.16) (molecular weight = 63 000) to phosphoric acid at pH 1.6 for 10 min at 4 degrees C causes 95% or greater inactivation. No significant effect on the rate or extent of inactivation is caused by varied aldolase concentrations or the presence of exogenous proteins. Chloride ion (50-100 mM) or 10 mM 2-oxo-4-hydroxyglutarate markedly decreases both the rate and extent of inactivation; good protection is also afforded by 10 mM pyruvate, glyoxylate, glyoxal, 2-oxoglutarate or 2-oxobutyrate. Whereas native aldolase has two free and three buried sulfhydryl groups, all five are exposed in the acid-inactivated enzyme and the molecular weight of this species at pH 1.6 is 126 000. Ultraviolet absorbance difference spectra, circular dichroism spectra and ultracentrifugation studies establish that the inactivation process is characterized by an alteration of secondary and tertiary structure as well as an aggregation to a dimer of the native molecule. Reactivation of enzyme activity to 60-80% of the original level is seen within 20 min at pH 6 to 8; examination of inactivation/reactivation as a function of pH indicates that these two processes occur via kinetically distinct pathways. Native and reactivated enzymes are identical in molecular weight, sulfhydryl titer, Km and alpha-helix content.     

Cysteine 288: an essential hyperreactive thiol of cytosolic phosphoenolpyruvate carboxykinase (GTP)

Phosphoenolpyruvate carboxykinase from the cytosol of rat liver has 13 cysteines, at least one of which is known to be very reactive and essential for catalytic activity (Carlson, G. M., Colombo, G., and Lardy, H. A. (1978) Biochemistry 17, 5329-5338). In order to identify the essential cysteine, this enzyme was modified with the fluorescent sulfhydryl reagent N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide. Incubation of phosphoenolpyruvate carboxykinase with a 10% molar excess of this maleimide at 0 degrees C results in the rapid and nearly complete loss of catalytic activity. Under these conditions, 1 mol of the maleimide is incorporated per mol inactivated enzyme. The substrate GDP provides almost complete protection against inactivation and modification, while phosphoenolpyruvate protects against the rate, but not the extent, of modification. The pH dependence of the rate of enzyme inactivation suggests that the modified residue has a pK alpha of approximately 7.0. Purification and sequencing of the labeled peptide identifies the hyperreactive essential cysteine as Cys-288. This cysteine lies between two putative phosphoryl-binding domains and within a hydrophobic sequence.     

Purification and characterization of a lactonase from Burkholderia sp. R-711, that hydrolyzes (R)-5-oxo-2-tetrahydrofurancarboxylic acid

A lactonase hydrolyzing (R)-5-oxo-2-tetrahydrofurancarboxylic acid to D-alpha-hydroxyglutaric acid was purified 170-fold with 2% recovery to near homogeneity from crude extracts of Burkholderia sp. R-711, which had been isolated as a bacterium able to assimilate (R)-5-oxo-2-tetrahydrofurancarboxylic acid. The molecular mass was estimated to be 33 kDa by gel filtration. The purified preparation migrated as a single band of molecular mass 38 kDa upon SDS-PAGE. The maximum activity was observed at pH 7.0-8.0 and 35-40 degrees C. The enzyme required no added cofactors or metal ions; the activity was inhibited to 60-100% by SH-blocking reagents, but was not affected by metal-chelating reagents. The enzyme showed lower activity and affinity toward (S)-5-oxo-2-tetrahydrofurancarboxylic acid, but did not act on other natural and synthetic lactones tested.     

Effects of pH on inactivation of maize phosphoenolpyruvate carboxylase

Maize leaf phosphoenolpyruvate carboxylase (PEPC) is inactivated by incubation at pH's above neutrality. Both the amount and the rapidity of inactivation increase as the pH rises. The presence of phosphoenolpyruvate (PEP), malate, glucose 6-phosphate and dithiothreitol in the incubation medium give protection to the enzyme. While the presence of PEP during incubation at pH 8 prevents inactivation, the level of PEP in the assay after incubation has no effect on the relative inactivation. When the enzyme is incubated at pH 7 with 5 mM malate (a treatment known to cause dimerization) subsequent assay at saturating levels of MgPEP completely restores activity while assay at less than Km MgPEP produces greater than 99% inhibition of the same sample, showing that high PEP concentration has reconverted the PEPC to the malate-resistant tetramer. Thus the protective effect of PEP against inactivation at high pH probably is not related to its effect on the aggregation state of the enzyme but rather is due to the presence of PEP at the active site. Protection of PEPC at pH 8 by EDTA and its inactivation by low concentrations of Cu2- indicates that the loss of activity at high pH probably is in a sense an artifact resulting from the binding to a deprotinated cysteine of heavy metal ions contaminating the enzyme preparation or present in reagents. This suggests that caution should be used in the interpretation of experiments involving PEPC activity at alkaline pH's.     

Inactivation of chicken mitochondrial phosphoenolpyruvate carboxykinase by o-phthalaldehyde

Chicken liver mitochondrial phosphoenolpyruvate carboxykinase is inactivated by o-phthalaldehyde. The inactivation followed pseudo first-order kinetics, and the second-order rate constant for the inactivation process was 29 M-1 s-1 at pH 7.5 and 25 degrees C. The modified enzyme showed maximal fluorescence at 427 nm upon excitation at 337 nm, consistent with the formation of isoindole derivatives by the cross-linking of proximal cysteine and lysine residues. Activities in the physiologic reaction and in the oxaloacetate decarboxylase reaction were lost in parallel upon modification with o-phthalaldehyde. Plots of (percent of residual activity) versus (mol of isoindole incorporated/mol of enzyme) were biphasic, with the initial loss of enzymatic activity corresponding to the incorporation of one isoindole derivative/enzyme molecule. Complete inactivation of the enzyme was accompanied by the incorporation of 3 mol of isoindole/mol of enzyme. beta-Sulfopyruvate, an isoelectronic analogue of oxaloacetate, completely protected the enzyme from reacting with o-phthalaldehyde. Other substrates provided protection from inactivation, in decreasing order of protection: oxaloacetate greater than phosphoenolpyruvate greater than MgGDP, MgGTP greater than oxalate. Cysteine 31 and lysine 39 have been identified as the rapidly reacting pair in isoindole formation and enzyme inactivation. Lysine 56 and cysteine 60 are also involved in isoindole formation in the completely inactivated enzyme. These reactive cysteine residues do not correspond to the reactive cysteine residue identified in previous iodoacetate labeling studies with the chicken mitochondrial enzyme (Makinen, A. L., and Nowak, T. (1989) J. Biol. Chem. 264, 12148-12157). Protection experiments suggest that the sites of o-phthalaldehyde modification become inaccessible when the oxaloacetate/phosphoenolpyruvate binding site is saturated, and sequence analyses indicate that cysteine 31 is located in the putative phosphoenolpyruvate binding site.