The carboxyl group in the active center of transketolase

Transketolase from baker's yeast is rapidly inactivated in the presence of 1-ethyl-3 (3'-dimethylaminopropyl)-carbodiimide or Woodward's reagent K. In both cases the kinetics of inactivation is biphasic, which agrees with the presence of two active centers in the enzyme molecule differing in their sensitivity to the inhibitors. There is some evidence that inactivation of transketolase is due to modification of carboxyl groups of enzyme. Complete inactivation is achieved by modification of one carboxyl per active site of the enzyme. The experimental results suggest that the carboxyl group is essential for the enzymatic activity of transketolase.     

Functional arginine residues and carboxyl groups in the adenosine triphosphatase of the thermophilic bacterium PS-3

Treatment of purified ATPase of the thermophilic bacterium PS-3 with the arginine reagent phenylglyoxal or with Woodward's reagent K, gave complete inactivation of the enzyme. The inactivation rates followed apparent first-order kinetics. The apparent order of reaction with respect to inhibitor concentrations gave values near to 1 with both reagents, suggesting that inactivation was a consequence of modifying one arginine or carboxyl group per active site. ADP and ATP strongly protected the thermophilic ATPase against both reagents. GDP and IDP protected less, whilst CTP did not protect. Experiments in which the incorporation of [14C]phenylglyoxal into the enzyme was measured show that extrapolation of incorporation to 100% inactivation of the enzyme gives 8-9 mol [14C]phenylglyoxal per mol ATPase, whilst ADP or ATP prevent modification of about one arginine per mol.     

The benzoylarginine peptidase from Treponema denticola (strain ASLM), a human oral spirochaete: evidence for active-site carboxyl groups

The benzoylarginine peptidase of Treponema denticola (strain ASLM; a human oral spirochaete) was progressively and irreversibly inactivated by 1-(ethoxycarbonyl)-2-ethoxy-1, 2-dihydroquinoline, a carboxyl-group reagent. At acidic pH values, reaction of one mole of the modifier per active site of the enzyme resulted in total inactivation of the enzyme. Assuming that this modifier is a specific carboxyl reagent, the data suggest that the inactivation of the T. denticola benzoylarginine peptidase was caused by the modification of one carboxyl group located close to the active site of the enzyme. Results obtained with Woodward's reagent K (N-ethyl-5-phenylisoxazolium 3'-sulphonate) supported these findings. Carbethoxylation with diethylpyrocarbonate effectively inactivated the enzyme, and addition of hydroxylamine at pH 7.0 restored the activity almost totally, suggesting that the pyrocarbonate had reacted with tyrosyl or histidyl residues.     

Modification of maize phosphoenolpyruvate carboxylase by Woodward's reagentK

Maize leaf phosphoenolpyruvate carboxylase was completely and irreversibly inactivated by treatment with micromolar concentrations of Woodward's reagentK (WRK) for about 1 min. The inactivation followed pseudo-first-order reaction kinetics. The order of reaction with respect to WRK showed that the reagent causes formation of reversible enzyme inhibitor complex before resulting in irreversible inactivation. The loss of activity was correlated to the modification of a single carboxyl group per subunit, even though the reagent reacted with 2 carboxyl groups per protomer. Substrate PEP and PEP + Mg²⁺ offered substantial protection against inactivation by WRK. The modified enzyme showed a characteristic absorbance at 346 nm due to carboxyl group modification. The modified enzyme exhibited altered surface charge as seen from the elution profile on FPLC Mono Q anion exchange column. The modified enzyme was desensitized to positive and negative effectors like glucose-6-phosphate and malate. Pretreatment of PEP carboxylase with diethylpyrocarbonate prevented WRK incorporation into the enzyme, suggesting that both histidine and carboxyl groups may be closely physically related. The carboxyl groups might be involved in metal binding during catalysis by the enzyme.     

Modification of carboxyl groups at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase

Ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach was inactivated by a carboxyl-directed reagent, Woodward's reagent K ( WRK ). The inactivation followed pseudo-first-order kinetics. The reaction order with respect to inactivation by WRK was 1.1, suggesting that inactivation was the consequence of modifying a single residue per active site. The substrate ribulose 1,5-bisphosphate (RBP), two competitive inhibitors, fructose 1,6-bisphosphate (FBP) and sedoheptulose 1,7-bisphosphate (SBP), and a number of sugars-phosphate protected against inactivation by WRK . SBP was a strong protector, displaying a dissociation constant (Kd) of 3 microM with native RBP carboxylase. Pretreatment of RBP carboxylase with diethyl pyrocarbonate prevented WRK incorporation into the enzyme. The enol ester derivative produced by reaction of WRK with RBP carboxylase has a maximal absorbance at 346 nm, and the extinction coefficient was found to be 12300 +/- 700 M-1 cm-1. Spectrophotometric titration of the number of carboxyl groups modified by WRK in RBP carboxylase/oxygenase in the presence and in the absence of SBP suggests that inactivation was associated with the modification of one carboxyl group per active site.     

Modification of a carboxyl group that appears to cross the permeability barrier in the red blood cell anion transporter

A recently developed method for converting protein carboxyl groups to alcohols has been used to examine the functional role of carboxyl groups in the red blood cell inorganic anion-transport protein (band 3). A major goal of the work was to investigate the carboxyl group that is protonated during the proton-sulfate cotransport that takes place during net chloride-sulfate exchange. Three kinds of evidence indicate that the chemical modification (Woodward's reagent K followed by borohydride) converts this carboxyl to an alcohol. First, monovalent anion exchange is inhibited irreversibly. Second, the modification stimulates sulfate influx into chloride-loaded cells and nearly eliminates the extracellular pH dependence of the sulfate influx. (The stimulated sulfate influx in the modified cells is inhibitable by stilbenedisulfonate.) Third, the proton influx normally associated with chloride-sulfate exchange is inhibited by the modification. These results would all be expected if the titratable carboxyl group were converted into the untitratable, neutral alcohol. In addition to altering the extracellular pH dependence of sulfate influx, the chemical modification removes the intracellular pH dependence of sulfate efflux. The modification is performed under conditions in which the reagent does not cross the permeability barrier. The large effect on the intracellular pH dependence of sulfate transport suggests that a single carboxyl group can at different times be in contact with the aqueous medium on each side of the permeability barrier.     

The nature of functional groups in the active center of antitumor glutamin-(asparagin-)ase

The effect of two reagents on glutamin (asparagin) ase from Pseudomonas aurantiaca-548 has been studied. 2,3-butanedione which modified arginine residues was ineffective for the inactivation of the enzyme. The enzyme was completely inactivated in the presence of N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K). The effects of pH, reagent concentration, competitive inhibitors and their analogues on the rate or degree of enzyme inactivation were studied. The experimental results suggest that the carboxyl groups localized at the active site of glutamin (asparagin) ase are probably essential for the substrate binding.     

Effect of protein-modifying reagents on ecto-apyrase from rat brain

We have tested several chemical modifiers to investigate which amino acid residues, present in the primary structure of the ecto-apyrase, could be involved in catalysis. Synaptosomes from cerebral cortex of rats were prepared and the ATP diphosphohydrolase activity was assayed in absence or the presence of the modifiers. Percentages of residual activity for ATPase and ADPase obtained when the following reagents were tested, are respectively: phenylglyoxal (an arginine group modifier) 17 and 30%; Woodward's reagent (a carboxylic group modifier) 33 and 23%; Koshland's reagent (a tryptophan group modifier) 10 and 12%; maleic anhidride (an amino group modifier) 11 and 25% and carbodiimide reagent (a carboxylic group modifier) 56 and 72%. Otherwise, PMSF, a seryl protein modifier and DTNB, a SH-group modifier did not affect either ATPase or ADPase activity. Inhibitions observed after treatment with phenylglyoxal and Woodward's reagent were significantly prevented when the synaptosomal fraction was preincubated with ATP and ADP, indicating that the arginine and the side chain of glutamate or aspartate (carboxyl groups) participate in the structure of the active site. This interpretation was confirmed by using GTP and GDP, two other apyrase substrates. Phenylglyoxal and Woodward's reagent also inhibited the GTPase and GDPase activities and this inhibition was prevented by preincubation with these substrates.     

Yeast enolase carboxyl modification using Woodward's reagent K

Yeast enolase is inactivated by Woodward's reagent K. Substantial protection is afforded by binding of 1 mol of "conformational" metal ion/subunit. Inactivation is correlated with modification of 13 carboxyl groups/subunit in the absence of conformational metal ion and 17 in its presence. Ten tryptic peptides labeled by Woodward's reagent K can be isolated, mostly from the C-terminal half of the protein. The changes in reactivity of these peptides produced by conformational metal ion suggest direct coordination to Glu-181 together with a contraction of the protein.     

Chemical modification and labeling of glutamate residues at the stilbenedisulfonate site of human red blood cell band 3 protein

A new method has been developed for the chemical modification and labeling of carboxyl groups in proteins. Carboxyl groups are activated with Woodward's reagent K (N-ethyl-5-phenylisoxazolium 3'-sulfonate), and the adducts are reduced with [3H]BH4. The method has been applied to the anion transport protein of the human red blood cell (band 3). Woodward's reagent K is a reasonably potent inhibitor of band 3-mediated anion transport; a 5-min exposure of intact cells to 2 mM reagent at pH 6.5 produces 80% inhibition of transport. The inhibition is a consequence of modification of residues that can be protected by 4,4'-dinitrostilbene-2,2'-disulfonate. Treatment of intact cells with Woodward's reagent K followed by B3H4 causes extensive labeling of band 3, with minimal labeling of intracellular proteins such as spectrin. Proteolytic digestion of the labeled protein reveals that both the 60- and the 35-kDa chymotryptic fragments are labeled and that the labeling of each is inhibitable by stilbenedisulfonate. If the reduction is performed at neutral pH the major labeled product is the primary alcohol corresponding to the original carboxylic acid. Liquid chromatography of acid hydrolysates of labeled affinity-purified band 3 shows that glutamate but not aspartate residues have been converted into the hydroxyl derivative. This is the first demonstration of the conversion of a glutamate carboxyl group to an alcohol in a protein. The labeling experiments reveal that there are two glutamate residues that are sufficiently close to the stilbenedisulfonate site for their labeling to be blocked by 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate and 4,4'-dinitrostilbene-2,2'-disulfonate.     

Carboxyl residues in the active site of human phenol sulfotransferase (SULT1A1)

The carboxyl-specific amino acid modification reagent, Woodward's reagent K (WK), was utilized to characterize carboxyl residues (Asp and Glu) in the active site of human phenol sulfotransferase (SULT1A1). SULT1A1 was purified using the pMAL-c2 expression system in E. coli. WK inactivated SULT1A1 activity in a time- and concentration-dependent manner. The inactivation followed first-order kinetics relative to both SULT1A1 and WK. Both phenolic substrates and adenosine 3'-phosphate 5'-phosphosulfate (PAPS) protected against the inactivation, which suggests the carboxyl residue modification causing the inactivation took place within the active site of the enzyme. With partially inactivated SULT1A1, both V(max) and K(m) changed for PAPS, while for phenolic substrates, V(max) decreased and K(m) did not change significantly. A computer model of the three-dimensional structure of SULT1A1 was constructed based on the mouse estrogen sulfotransferase (mSULT1E1) X-ray crystal structure. According to the model, Glu83, Asp134, Glu246, and Asp263 are the residues likely responsible for the inactivation of SULT1A1 by WK. According to these results, five SULT1A1 mutants, E83A, D134A, E246A, D263A, and E151A, were generated (E151A as control mutant). Specific activity determination of the mutants demonstrated that E83A and D134A lost almost 100% of the catalytic activity. E246A and D263A also decreased SULT1A1 activity, while E151A did not change SULT1A1 catalytic activity significantly. This work demonstrates that carboxyl residues are present in the active site and are important for SULT1A1 catalytic activity. Glu83 and E134 are essential amino acids for SULT1A1 catalytic activity.     

Chemical modification of the active site of the NADP-linked glutamate dehydrogenase from Trypanosoma cruzi

The NADP-linked glutamate dehydrogenase (NADP-gluDH) purified from epimastigotes of the Tulahuén strain, Tul 2 stock, of Trypanosoma cruzi, was inhibited by Cibacron Blue FG3A, and inactivated by preincubation with phenylglyoxal or Woodward's Reagent K. The inhibition by Cibracron Blue FG3A, competitive towards NADPH with an apparent Ki of 20 microM, suggests that the enzyme presents the "dinucleotide fold" characteristic of most dehydrogenases and kinases. The inactivation of the NADP-gluDH by preincubation with phenylglyoxal, with a reaction order of 1, and the partial protection afforded by alpha-oxoglutarate, suggest the presence of one arginine residue in the active site of the enzyme, which might participate in the binding of alpha-oxoglutarate through interaction with one of the carboxyl groups of the substrate. The inactivation of the NADP-gluDH by preincubation with Woodward's Reagent K suggests the presence of a carboxyl group, from an aspartic or glutamic acid residue, at the active site, which might participate in the binding of the cationic substrate NH+4. The presence of NADPH during preincubation with the reagent increased the inactivation rate, which suggests that binding of the coenzyme increases the exposure of the reactive carboxyl group.     

Reaction of Woodward''s reagent K with D-xylose isomerases. Modification of an active site carboxylate residue.

D-Xylose isomerases from Streptomyces violaceoruber, Streptomyces sp., Lactobacillus xylosus, Lactobacillus brevis and Bacillus coagulans were rapidly inactivated by Woodward's reagent K. Second-order rate constants in the absence of ligands, at pH 6.0 and 25 degrees C, were 41, 36, 22, 95 and 26 M-1.min-1 respectively. Spectral analysis at 340 nm revealed that inactivation was correlated with modification of five, six, two, three and six carboxylate residues per monomer respectively. In the presence of protecting ligands, modification of one carboxylate group was prevented. The results support the idea of an active site glutamate or aspartate group that may contribute to the catalytic activity of all these D-xylose isomerases.     

Functionally important residues of glutamic acid in E. coli pyrophosphatase. I. Chemical modification and localization in the primary structure]

Inorganic pyrophosphatase of E. coli is rapidly and irreversibly inactivated by 5-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K). The appearance in the absorption spectrum of a maximum at 340 nm testifies to the formation of an enzyme enol ester with the inhibitor. The non-hydrolyzable substrate analog CaPP1 partly protects the enzyme from inactivation. A peptide has been isolated from a tryptic hydrolysate of inactivated enzyme which contains an amino acid residue whose modification is critical for the enzyme activity. This peptide corresponds to residues 95-104 of pyrophosphatase and contains four dicarboxylic acid residues. A peptide containing a modified glutamic acid residue was isolated from modified pyrophosphatase hydrolyzed by protease v8. This peptide represents a fragment of a tryptic modified peptide and has a Glu-Ala-Gly-Glu (residues 98-1C1) structure. It is concluded that inactivation of E. coli pyrophosphatase by Woodward's reagent K is a result of selective modification of Glu98, apparently by the most reactive dicarboxylic amino acid within the enzyme active center.     

Chemical modification of a beta-glucosidase from Schizophyllum commune: evidence for essential carboxyl groups

The beta-glucosidase from Schizophyllum commune was purified to homogeneity by a modified procedure that employed Con A-Sepharose. The participation of carboxyl groups in the mechanism of action of the enzyme was delineated through kinetic and chemical modification studies. The rates of beta-glucosidase-catalyzed hydrolysis of p-nitrophenyl-beta-D-glucoside were determined at 27 degrees C and 70 mM ionic strength over the pH range 3.0-8.0. The pH profile gave apparent pK values of 3.3 and 6.9 for the enzyme-substrate complex and 3.3 and 6.6 for the free enzyme. The enzyme is inactivated by Woodward's K reagent and various water-soluble carbodiimides; chemical reagents selective for carboxyl groups. Of these reagents, 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide iodide in the absence of added nucleophile was the most effective and a kinetic analysis of the modification indicated that one molecule of carbodiimide is required to bind to the beta-glucosidase for inactivation. Employing a tritiated derivative of the carbodiimide, 44 carboxyl groups in the enzyme were found to be labelled while the competitive inhibitor deoxynojirimycin protected three residues from modification. Treatment of the enzyme with tetranitromethane resulted in the modification of five tyrosine residues with approx. 28% diminution of enzymic activity. Titration of denatured enzyme with dithiobis(2-nitro-benzoic acid) indicated the absence of free thiol groups. Reaction of the enzyme with diethyl pyrocarbonate resulted in the modification of four histidine residues with the retention of 78% of the original enzymatic activity. The divalent transition metals Cu2+ and Hg2+ were found to be potent inhibitors of the enzyme, binding in an apparent irreversible manner.     

Reaction of alpha-mannosidase from Phaseolus vulgaris with group-specific reagents. Essential carboxyl groups.

When the pKm of alpha-mannosidase was determined at different pH values, the results indicated that ionizable groups with pK values of approx. 3.8 and 5.7 could be essential. Modification with carbodiimide or Woodward's Reagent K abolished the enzyme activity. The substrate analogue, alpha-methyl-D-mannoside, protected the enzyme against inactivation. Incorporation of a 14C-labeled nucleophile reagent in the presence or absence of the analogue suggested that 2--4 carboxyl groups were protected. Exchange studies indicated that the essential Zn2+ could be bound to such groups. There was no indication that hydroxyl groups, sulphydryl groups, guanidino groups or amino groups take part in the catalytic activity.     

Reconstruction of highly purified proton-translocating pyrophosphatase from Rhodospirillum rubrum

Using the freezing-thawing procedure, a highly purified preparation of PPase from R. rubrum chromatophore membranes was incorporated into soybean phospholipid liposomes. The activity of reconstituted PPase was increased in the presence of the uncoupler, FCCP, and the antibiotics, valinomycin (+KCl) and nigericin (+KCl). Oligomycin did not exert any inhibiting action, while imidodiphosphate and NaF significantly decreased the activity of the PPase incorporated into the liposomes. Preincubation of both PPase and ATPase prior to their incorporation into the liposomes did not affect the activity of the reconstituted enzyme. It was concluded that the PPase from R. rubrum chromatophores when incorporated into the liposomes may function as a proton pump independently of the ATPase.     

Functional carboxylic group in the active center of transketolase

Baker's yeast transketolase is rapidly inactivated in the presence of carboxylic group modifiers, i.e., 1-ethyl-3(3'-dimethylaminopropyl)-carbodiimide or Woodward's reagent K. This inactivation is due to modification of the carboxylic group in the enzyme active center. The essential groups localized in the two active centers of transketolase differ in the rate of modification; accordingly, the inactivation kinetics appears as biphasic. A complete loss of the enzyme activity occurs as a result of modification of one carboxylic group per enzyme active center. The pKa value of modifiable groups is equal to about 6.5. This modification decreases by two orders of magnitude the affinity of the substrate for the active center. The carboxylic groups are not directly involved in the interaction with the substrates; their modification does not significantly affect the coenzyme binding. It is supposed that these groups are responsible for the deprotonation of the second carbon in the thiamine pyrophosphate thiazolium ring.     

Purification and properties of a novel azide-sensitive ATPase of Exiguobacterium aurantiacum

Exiguobacterium aurantiacum BL77/1 possesses at least two distinct membrane-bound ATPases. One of them was solubilized with decanoyl N-methylglucamide, a non-ionic detergent, and purified by successive chromatography on DEAE-Sepharose and hydroxyapatite. The purified ATPase appears to consist of a single polypeptide component with an apparent molecular mass of 54 kDa. Among the triphosphates of various nucleosides tested, ATP was the best substrate. The enzyme exhibited a Km of 0.5 mM for ATP and a Vmax of 109 micromol ATP (mg protein)(-1) min(-1); the optimum pH for activity was near 6.5. The enzyme was sensitive to azide and inactivated by N,N'-dicyclohexylcarbodiimide. Analysis of the inhibition kinetics by N,N'-dicyclohexylcarbodiimide suggested that binding of the drug to a single carboxyl group per ATPase molecule is sufficient for inactivation.     

Studies on the mechanism of action of local anesthetics on proton translocating ATPase from Mycobacterium phlei

We have measured the inhibitory potencies of local anesthetics (procaine, lidocaine, tetracaine and dibucaine) on ATP-mediated H+-translocation, Ca2+-transport and ATPase activity in membrane vesicles from Mycobacterium phlei. Procaine and lidocaine up to 1 mM concentration did not inhibit ATP-dependent H+-translocation, Ca2+-transport and ATPase activity. However, tetracaine and dibucaine at 0.2 mM concentration caused dissipation of the proton gradient, measured by the reversal of the quenching of fluorescence of quinacrine, and inhibition of active Ca2+-transport. Tetracaine (1 mM) inhibited membrane-bound ATPase activity without affecting solubilized F1-ATPase activity. Studies show that these local anesthetics do not prevent the inactivation of F0-F1 ATPase by dicyclohexylcarbodiimide (DCCD). Binding of [14C]DCCD to F0-proteolipid component remained unchanged in the presence of tetracaine indicating that DCCD and tetracaine do not share common binding sites on the F0-proteolipid sector. The inhibition of H+-translocation and membrane-bound ATPase activity by tetracaine was substantially additive in the presence of vanadate.