Inhibition of pigeon liver fatty acid synthetase by specific modification of lysine residues with 2,4,6-trinitrobenzenesulphonic acid.

Pigeon liver fatty acid synthetase was inactivated irreversibly by 2,4,6-trinitrobenzenesulphonic acid (TNBS). Biphasic inactivation of the enzyme was observed with the inhibitor. NADPH provided protection to the enzyme against inactivation by TNBS and the extent of protection increased with NADPH concentration indicating that the essential lysine residues are present at the NADPH binding site. The stoichiometric results with TNBS showed that 4 mol of lysine residues are modified per mole of fatty acid synthetase upon complete inactivation. The rapid reaction of two amino groups per enzyme molecule led to the loss of 60% of the enzyme activity. These approaches suggested that two lysine residues present at the active site are essential for the enzymatic activity of fatty acid synthetase.

Effect of arginine modifying reagents on pigeon liver fatty acid synthetase: evidence for the presence of essential arginine residues at the beta-ketoacyl reductase and enoyl-CoA reductase domain.

Pigeon liver fatty acid synthetase was inactivated by arginine modifying reagent, phenylglyoxal and 2,3-butanedione. The inactivation of overall fatty acid synthetase was accompanied by the loss of beta-ketoacyl reductase and enoyl-CoA reductase activity. The inactivation followed a pseudo-first order kinetics and sum of the second order rate constants for the two reductase reactions equaled that for the synthetase reaction. Inactivation of all three activities was prevented by NADPH or its analogs 2',5'-ADP and 2'-AMP but not by the corresponding nucleotides containing the 5'-phosphate. These results suggest that binding of NADPH to fatty acid synthetase involves specific interaction of the 2'-phosphate with the guanidino group of arginine residues at the active site of the two reductases. pH-Dependent inactivation by phenylglyoxal indicated that a group with a pka 7.5 is involved in the loss of enzyme activity. Stoichiometric results showed that 4 out of 164 arginine residues per enzyme molecule were essential for the enzyme activity.

Nature of o-phthalaldehyde reaction with pigeon liver fatty acid synthetase.

Pigeon liver fatty acid synthetase (FAS) was inactivated irreversibly by stoichiometric concentration of o-phthalaldehyde exhibiting a bimolecular kinetic process. FAS-o-phthalaldehyde adduct gave a characteristic absorption maxima at 337 nm. Moreover this derivative showed fluorescence emission maxima at 412 nm when excited at 337 nm. These results were consistent with isoindole ring formation in which the -SH group of cysteine and epsilon-NH2 group of lysine participate in the reaction. The inactivation is caused by the reaction of the phosphopantetheine -SH group since it is protected by either acetyl- or malonyl-CoA. The enzyme incubated with iodoacetamide followed by o-phthalaldehyde showed no change in fluorescence intensity but decrease in intensity was found in the treatment of 2,4,6-trinitrobenzenesulphonic acid (TNBS), a lysine specific reagent with the enzyme prior to o-phthalaldehyde addition. As o-phthalaldehyde did not inhibit enoyl-CoA reductase activity, so nonessential lysine is involved in the o-phthalaldehyde reaction. Double inhibition experiments showed that 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), a thiol specific reagent, binds to the same cysteine which is also involved in the o-phthalaldehyde reaction. Stoichiometric results indicated that 2 moles of o-phthalaldehyde were incorporated per mole of enzyme molecule upon complete inactivation.

Studies on the inactivation of Leuconostoc mesenteroides NRRL B-512F dextransucrase by o-phthalaldehyde: evidence for the presence of an essential lysine residue at the active site.

The kinetics of inactivation of Leuconostoc mesenteroides NRRL B-512F dextransucrase by o-phthalaldehyde showed that the reaction followed pseudo-first order reaction. The loss of enzyme activity was concomitant with an increase in fluorescence at 417 nm indicating that the inhibition involved the reaction of an epsilon-amino and a thiol group of the enzyme leading to the formation of an isoindole derivative. The stoichiometry of inactivation showed that one isoindole derivative was formed per enzyme molecule. The substrates sucrose and glucose provided protection against o-phthalaldehyde inactivation which was also corroborated by fluorescence studies. Dextransucrase was not inactivated by 5,5'-dithiobis(2-nitrobenzoic acid), showing that the cysteine present in close proximity to the lysine is not essential for enzyme activity. Denaturation of dextransucrase by urea or heat treatment prior to o-phthalaldehyde addition resulted in a decrease of fluorescence intensity indicating that the native conformation of the enzyme is essential for isoindole derivative formation. These results established that a lysine residue is present at the active site and is essential for the activity of dextransucrase.

Inactivation of enoyl-CoA reductase in pigeon liver fatty acid synthetase by pyridoxal 5'-phosphate: evidence for the presence of one lysine residue at the active site.

Pigeon liver fatty acid synthetase (FAS) was rapidly inactivated by pyridoxal 5'-phosphate (PLP). Assays of the partial activities of the PLP-treated synthetase showed that only the enoyl-CoA reductase was decreased significantly. The inactivation of both the overall activity and enoyl-CoA reductase activity of FAS by PLP could be reversed by dialysis or dilution but not by reduction with sodium borohydride. Malonyl-CoA and acetyl-CoA did not protect the enzyme, whereas NADPH provided 68% protection against PLP-inactivation indicating that PLP modified lysine residues present at or near the co-enzyme binding site. PLP-treated enzyme after reduction with sodium borohydride exhibited fluorescence with a maximum at 397 nm (irradiation at 325 nm). Stoichiometric analysis showed that modification of four lysine residues per enzyme molecule resulted in complete inactivation of the overall and enoyl-CoA reductase activities of FAS. NADPH prevented the inactivation by protecting two of these lysine residues from modification, suggesting the presence of two essential lysine residues per enzyme molecule. These results are consistent with the hypothesis that each subunit of the enzyme contains an enoyl-CoA reductase domain in which a lysine residue, at or near the active site, interacts with NADPH.

Chemical modification of dextransucrase from Leuconostoc mesenteroides NRRL B-512F by pyridoxal 5'-phosphate: evidence for the presence of an essential lysine residue at the active site.

The treatment of Leuconostoc mesenteroides NRRL B-512F dextransucrase with lysine specific reagent, pyridoxal 5'-phosphate (PLP) at pH 5.2 and 30 degrees C resulted in the loss of enzyme activity. The inactivation by PLP could be reversed completely by dilution or dialysis. Sucrose as well as acceptor substrates, glucose and dextran protected the enzyme against inactivation by PLP. A statistical, kinetic analysis of the inactivation by PLP showed that one lysine residue is essential for the enzyme activity. All these results showed that one lysine residue present at the active is essential for the activity of dextransucrase.

Modification of an essential amino group of glutathione reductase from yeast by pyridoxal 5'-phosphate.

Yeast glutathione reductase is inactivated by pyridoxal 5'-phosphate (PLP). The reactivation of the enzyme by dilution as well as a characteristic absorption peak at 325 nm exhibited by NaBH4-reduced-PLP modified enzyme show that the inactivation is due to the specific modification of the epsilon-amino group of lysine residue. The maximum of 70% inactivation was observed at 7mM PLP and the equilibrium was reached within 3 min. Kinetic and equilibrium analysis of inactivation data derived at different PLP concentrations showed that a noncovalent intermediate is formed prior to inactivation. From the studies on the effect of pH on the inactivation rate, the pKa of epsilon-amino group of the reactive lysine residue was calculated to be 7.3. Among various protecting agents tried, only NADP was found to be effective. The apparent stoichiometry of the reaction was one to one as the incorporation of 0.65 mole PLP/mole of enzyme led to 70% inactivation at saturating PLP concentration.

Evidence for the essential histidine at the NADPH binding site of enoyl-CoA reductase domain of pigeon liver fatty acid synthetase.

Pigeon liver fatty acid synthetase was inactivated by stoichiometric concentrations of diethylpyrocarbonate (DEP). The second order rate constant for the loss of synthetase activity was similar to the value for enoyl-CoA reductase indicating that ethoxyformylation destroys the ability of the enzyme to reduce the unsaturated acyl intermediate, without significant effect on beta-ketoacyl reductase activity. NADPH provided protection to the enzyme against inactivation by DEP indicating that essential histidine residues are present at the active site. DEP-modified enzyme showed a characteristic absorption maxima at 240 nm confirming the formation of ethoxyformic histidine. The reversal of inactivation by hydroxylamine and a pKa value of 7.0 obtained from the pH-rate profile for inactivation again confirmed the specificity of DEP for histidine. Stoichiometric results showed that two moles of histidine residue per mole of enzyme are essential for the activity of FAS.

Effect of certain nutrients on the production of dextransucrase from Leuconostoc mesenteroides NRRL B-512F.

The effects of certain nutrients on dextransucrase (sucrose: 1,6-alpha-d-glucan 6-alpha-d-glucosyltransferase EC 2.4.1.5) production from Leuconostoc mesenteroides NRRL B-512F were studied. An increase in concentration of sucrose to 4% in the enzyme production medium resulted in the increase of activity of dextransucrase. Higher enzyme yields were obtained at low yeast extract and high phosphate concentrations. The presence of peptone and beef extract in the medium in addition to 2% yeast extract resulted in an enhanced production of dextransucrase. The enzyme activity increased by 30% by both peptone and beef extract. Addition of Tween 80 to the medium enhanced the production of enzyme and the activity was increased by 25%. Magnesium ions stimulated the activity marginally. Sodium fluoride enhanced the activity of dextransucrase by 25%.

Inactivation of yeast glutathione reductase by O-phthalaldehyde.

Yeast glutathione reductase was inactivated by the bifunctional reagent, o-phthalaldehyde. The initial rate of inactivation followed pseudo-first order kinetics. Fluorescence spectral properties of modified enzyme indicated the formation of an isoindole derivative from cysteine and lyaine residues present in close proximity as shown by typical fluorescence emission and excitation maximum at 410 nm and 337 nm, respectively. The fluorescence spectral studies with o-phthalaldehyde in the presence and absence of N-ethylmaleimide indicated that both the inhibitors react with the same cysteine residue, which is non-essential for enzyme activity. The coenzyme NADPH did not protect the enzyme against the o-phthalaldehyde reaction while oxidised glutathione prevented o-phthalaldehyde inactivation. This could be due to reaction of the amino group of glutathione with o-phthalaldehyde. Stoichiometry of the reaction showed that the formation of approximately 2 isoindole derivatives per subunit of glutathione reductase is accompanied by 75% loss of activity. The results suggest that o-phthalaldehyde binds to non-essential cysteine and lysine residues present in close proximity which results in conformational changes leading to enzyme inactivation.

Involvement of a lysine residue in the inactivation of Leuconostoc mesenteroides NRRL B-512F dextransucrase by o-phthalaldehyde.

Leuconostoc mesenteroides NRRL B-512F dextransucrase was rapidly and irreversibly inactivated by o-phthalaldehyde. The dextransucrase-o-phthalaldehyde adduct showed a characteristic fluorescence maxima at 417 nm when excited at 337 nm. These results were consistent with the isoindole derivative formation in which the sulfhydryl group of cysteine and epsilon-amino group of lysine participate in the reaction. The stoichiometric determinations gave one isoindole derivative per enzyme molecule upon complete inactivation by o-phthalaldehyde. The enzyme showed no inhibition on treatment with thiol specific reagents. This indicated that cysteine is present in close proximity of the lysine and is involved in the isoindole derivative formation but is not participating in the catalysis. These results established for the first time that one lysine residue present at the active site is required for the activity of dextransucrase.

2,4,6-Trinitrobenzenesulphonic acid as a probe for lysine at the active site of dextransucrase from Leuconostoc mesenteroides NRRL B-512F.

Modification of Leuconostoc mesenteroides NRRL B-512F dextransucrase with 2,4,6-trinitrobenzenesulphonic acid (TNBS) at pH 5.2 and 30 degrees C resulted in the loss of enzyme activity. The kinetic profiles of inactivation showed that the reaction followed pseudo-first order reaction. Absorption spectra of TNBS modified enzyme gave characteristic maxima at 367 nm. The inactivation could not be reversed by dilution or dialysis. The substrate sucrose, provided protection to the enzyme against inactivation by TNBS, indicating that the essential residues are present at or near the active site. The stoichiometric results indicated that four mol of lysine are modified per mol of dextransucrase upon complete inactivation. However, more than 50% of the activity loss was accompanied by modification of 1 lysine residue. All these approaches suggested that one lysine residue present near or at the active site is essential for the enzymatic activity of dextransucrase.

Essential lysine residue in glutathione reductase: chemical modification by pyridoxal 5'-phosphate.

Yeast glutathione reductase was inactivated by pyridoxal 5'-phosphate. The inhibition was reversed by dilution. The enzyme-pyridoxal 5'-phosphate complex on reduction with sodium borohydride gave a characteristic absorption maximum at 325 nm and fluorescence maximum at 395 nm when exciated at 325 nm. These results were consistent with the reaction of epsilon-amino group of lysine residue of the enzyme with pyridoxal 5'-phosphate. The enzyme was protected against pyridoxal 5'-phosphate inhibition by NADP indicating thereby that the essential lysine residues are present during the NADP binding site.

Inactivation of Leuconostoc mesenteroids NRRL B-512F dextransucrase by specific modification of lysine residues with pyridoxal-5'-phosphate.

Dextransucrase from Leuconostoc mesenteroides NRRL B-512F was inactivated by pyridoxal-5'-phosphate (PLP). The inactivation was reversible in as much as the loss of enzyme activity was completely reversed by prolonged dialysis. PLP-modified dextransucrase after reduction with sodium borohydride showed a characteristic fluorescence emission maximum at 397 nm when excited at 325 nm. The stoichiometric results indicated that four lysine residues are modified by PLP under the experimental conditions. These results established for the first time that lysine residues are essential for the activity of dextransucrase.

Reactivity of essential cysteine and lysine residues present at the catalytic domain of pig heart mitochondrial malate dehydrogenase.

Pig heart mitochondrial malate dehydrogenase was inactivated very rapidly by omicron-phthalaldehyde as compared to 5,5'-dithio bis(2-nitrobenzoic acid) and pyridoxal 5'-phosphate. The omicron-phthalaldehyde reaction followed pseudo first order kinetics, and a second order rate constant of 38 M-1 S-1 was obtained. Cysteine and lysine residues participating in the omicron-phthalaldehyde reaction are located in the NADH binding region of malate dehydrogenase as shown by protection experiments. The decrease in the rate of inactivation in the presence of NADH was used to determine the dissociation constant of the enzyme-NADH complex. pH dependent studies and molar transition energy calculations of the omicron-phthalaldehyde-inactivated enzyme have indicated that cysteine and lysine residues involved in the isoindole derivative formation are located in a hydrophobic environment at the coenzyme binding site.

Investigation of the nature of o-phthalaldehyde reaction with octopine dehydrogenase.

The effect of o-phthalaldehyde on octopine dehydrogenase inactivation has been studied. o-Phthalaldehyde binds to the proximal cysteine and lysine residues of the enzyme leading to the formation of isoindole derivative. Double inhibition studies with o-phthalaldehyde and p-chloromercuricphenyl sulfonic acid have indicated that o-phthalaldehyde does not bind to the functional cysteine present at the active site. Protection experiments have shown that L-arginine prevented o-phthalaldehyde inactivation. This could be only due to the reaction of the amino group of L-arginine with o-phthalaldehyde as per the mechanism proposed elsewhere since L-arginine cannot bind to the enzyme prior to NADH. Other substrates such as pyruvate oR NADH could not prevent the o-phthalaldehyde reaction with the enzyme. Fluorescence spectral studies demonstrated that in the presence of externally added amino acid no isoindole derivative formation occurs. However, a characteristic isoindole derivative is formed in the presence of beta-mercaptoethanol although the enzyme does not lose its activity. This indicated that o-phthalaldehyde can bind with lysine of the enzyme and thiol of externally added beta-mercaptoethanol. Pyridoxal 5'-phosphate, a lysine specific reagent also binds to the enzyme giving the characteristic absorption and fluroescence peak at 325 nm and 395 nm respectively. However, no loss of enzyme activity was observed. On the basis of these experiments we would suggest that o-phthalaldehyde binds to non-essential cysteine and lysine residues present in close proximity which results in conformational changes leading to enzyme inactivation.

Evidence for proximal cysteine and lysine residues present at the nucleotide domain of rabbit muscle creatine kinase.

Inactivation of rabbit muscle creatine kinase by o-phthalaldehyde was investigated. The loss of enzyme activity was concomitant with the increase in fluorescence intensity at 410 nm. The modified enzyme showed a characteristic absorption peak at 336 nm. These evidences suggested that the mechanisms of inhibition of creatine kinase by o-phthalaldehyde involves binding of the thiol and the epsilon-amino group of enzyme leading to the formation of isoindole derivative. None of the substrates, except Mg-ATP, provided protection against o-phthalaldehyde inhibition. This was corroborated by fluorescence studies. Double inhibition experiments showed that p-chloromercuricphenyl sulphonic acid, a thiol-specific reagent, binds to the same cysteine which is also involved in the o-phthalaldehyde reaction. Stoichiometric results indicated that 2 mol of o-phthalaldehyde were incorporated per mole of enzyme molecule upon complete inactivation. Denaturation of creatine kinase by urea or heat treatment prior to o-phthalaldehyde addition resulted in the decrease of fluorescence intensity indicating that native conformation of the enzyme is essential for isoindole derivative formation.

Chemical modification of octopine dehydrogenase by thiol-specific reagents: evidence for the presence of an essential cysteine at the catalytic site.

Thiol-specific reagents, p-chloromercuricphenyl sulfonic acid (PCMS), 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and N-ethyl-maleimide (NEM), incubated with octopine dehydrogenase resulted in the loss of catalytic activity. The kinetic profile of PCMS inactivated enzyme showed that the reaction followed pseudo-first-order kinetics during the initial phase of inactivation. The reversal of enzyme activity was obtained by thiol-containing reagents. The loss of enzyme activity was prevented only by NADH and not by other substrates. The dissociation constant for NADH calculated from the decrease in the enzyme inactivation rate was 45 microM. Cyanolysis of the DTNB-modified enzyme with KCN led to the release of 5-thio-2-nitrobenzoate (TNB) accompanied by the formation of thio-cyano enzyme. By correlating the enzyme activity with the formation of thio-cyano derivative it was found that no activity was recovered after KCN treatment. These evidences clearly established the critical involvement of the thiol group in catalysis. Double inhibition studies with PCMS and NEM on octopine dehydrogenase showed that the inactivating reagents bind to the same functional thiol present at the catalytic center. pH-dependent inactivation by PCMS indicated that a group with a pKa value of 7.4 is involved in the loss of enzyme activity. These approaches suggested that at least one thiol group present at the coenzyme-binding domain is essential for the catalytic reaction of octopine dehydrogenase.

Involvement of different cysteines in the inactivation of octopine dehydrogenase by p-chloromercuricphenyl sulfonic acid and o-phthalaldehyde.

Inactivation of octopine dehydrogenase by p-chloromercuricphenyl sulfonic acid (PCMS) and o-phthalaldehyde have been investigated. The activity loss due to the PCMS was faster than o-phthalaldehyde. PCMS associated inhibition was reversed by dithiothreitol completely which was not observed with o-phthalaldehyde inactivated enzyme. Fluorescence spectra of o-phthalaldehyde modified enzyme showed the formation of isoindole derivative with characteristic emission maximum at 410 nm. This derivative formation essentially involves cross-linking of proximal cysteine and lysine residues. Protection and selective reversible reaction studies have established that NADH prevents the enzyme against PCMS inactivation and the essential cysteine present at the NADH binding site is not involved in the o-phthalaldehyde reaction.

Active site mapping studies of malate dehydrogenase : identification of essential amino acid residues by o-phthalaldehyde.

Mitochondrial malate dehydrogenase (MDH) was found to be rapidly inactivated by o-phthalaldehyde. MDH-o-phthalaldehyde adduct gives a characteristic absorption maximum at 337 nm. Moreover, this derivative shows fluorescence emission maxima at 405 nm when excited at 337 nm and 280 nm. These results were consistent with isoindole ring formation in which the -SH group of cysteine and epsilon-NH2 group of lysine participate in the reaction. The enzyme was found to be protected against o-phthalaldehyde inactivation by NADH, indicating that the essential residues are present at or near the coenzyme binding site. Stoichiometric results indicate that 4 isoindole derivatives are formed per enzyme molecule upon complete inactivation. However, 90% of the activity loss was accompanied by the formation of 2 moles of isoindole per mole of the enzyme. These approaches give consistent evidence that two cysteines along with two lysines in close proximity are essential for the enzymatic activity.