Coenzyme B12-dependent diol dehydrase: Chemical modification with 2,3-butanedione and phenylglyoxal

The apoenzyme of diol dehydrase was inactivated by two arginine-specific reagents, 2,3-butanedione and phenylglyoxal, in borate buffer. In both cases, the inactivation followed pseudo-first-order kinetics. Kinetic data show that the incorporation of a single reagent molecule per active site of the enzyme is necessary for the complete inactivation. The modification with 2,3-butanedione was reversed by dilution of the reagent and borate concentrations (65% activity recovered). 1,2-Propanediol (substrate) partially protected the enzyme against inactivation. The holoenzyme was almost insensitive to 2,3-butanedione and phenylglyoxal, indicating that the essential arginine residue is prevented from the attack of these reagents either by direct blockage with the bound coenzyme or by an indirect conformational change caused by coenzyme binding. The inactivation of diol dehydrase by 2,3-butanedione did not result in dissociation of the enzyme into subunits. From these results, we concluded that the essential arginine residue is located at or in close proximity to the active site of diol dehydrase.     

A functional arginine residue in NADPH-dependent aldehyde reductase from pig kidney.

Pig kidney aldehyde reductase is inactivated by 2,3-butanedione, phenylglyoxal, methylglyoxal, and 1,2-cyclohexanedione. 2,3-Butanedione caused the most rapid loss in enzyme activity, the rate of loss being proportional to the concentration of 2,3-butanedione. Neither D-glyceraldehyde nor pyridine 3-aldehyde, both substrates for this broadly specific enzyme, protected the enzyme from inactivation but 1 mM NADPH or NADP completely prevented the loss of activity by 2,3-butanedione suggesting the involvement of arginine in the binding of cofactor. Nicotinamide mononucleotide (NMN) (reduced form) offered no protection to inactivation whereas ADP-ribose phosphate gave complete protection indicating that it is the latter portion of NADPH which interacts with the essential arginine. Both NMN and ADP-ribose phosphate are competitive inhibitors of aldehyde reductase with respect to NADPH. Butanedione-modified aldehyde reductase could still bind to a blue dextran-Sepharose 4B column suggesting that the modified arginine did not bind NADPH. This was confirmed by fluorescence spectra which showed that chemically modified aldehyde reductase caused the same blue shift of NADPH fluorescence as did native aldehyde reductase. Of additional interest was the quenching of NADPH fluorescence by aldehyde reductase which, with one exception, is in contrast to the fluorescence behavior of all other oxidoreductases.     

Evidence for an active site arginine in UDP-glucuronyltransferase

2,3-Butanedione inactivates the pure form of UDP-glucuronyltransferase used in these experiments (GT2P) (EC 2.4.1.17) purified from pig liver microsomes. The kinetics of the reaction indicates that 2,3-butanedione reacts with two amino acids that affect activity. A rapid, partial inactivation is followed by a slower rate of inactivation that leads eventually to completely inactive enzyme. UDP-glucuronic acid and glucuronic acid, as compared with UDP, are effective as protectors against the slow, secondary phase of inactivation; no ligand tested protected against the rapid phase of inactivation. The lipid environment of GT2P was a determinant of the pseudo-first order rate constant for the slow phase of inactivation, but did not affect the rate of the rapid phase of inactivation. The data suggest that GT2P contains an active site arginine that interacts with the -COO- at C-6 of the glucuronic acid moiety of UDP-glucuronic acid.     

Essential arginine residues in human liver arylsulfatase A.

Human liver arylsulfatase A was treated with arginine-specific reagents (diones), resulting in a loss of enzyme activitity with apparent first-order kinetics. Sulfite and borate—competitive inhibitors of the enzyme—provided complete protection from inactivation by phenylglyoxal. Sulfite and substrate each likewise protected against enzyme inactivation by 2,3-butanedione. A plot of pseudo-first-order rate constants of enzyme inactivation versus 2,3-butanedione concentrations suggests that an essential arginine residue is modified with a loss in function of the binding site or of the active site of the protein. Chemical analysis of the butanedione-treated sulfatase indicates that complete enzyme inactivation corresponds to a modification of only about 2 of the 20 arginine residues per enzyme subunit. Taken together, all of the results strongly suggest that arginine residues are essential for the activity of arylsulfatase A. An incidental discovery in this work is that borate ion is a competitive inhibitor of human arylsulfatase A with a K_i of 2.5 × 10^?4 M.     

Arginyl residues in the NADPH-binding sites of phenol hydroxylase

Phenol hydroxylase was inactivated by the arginine reagents 2,3-butanedione, 1,2-cyclohexanedione, and phenylglyoxal. The cosubstrate NADPH, as well as NADP⁺ and several analogues thereof, protected the enzyme against inactivation. Phenol did not protect the activity against any of the reagents used, nor did modification by 2,3-butanedione affect the binding of phenol. We propose the presence of arginyl residues in the binding sites for the adenosine phosphate part of NADPH.     

Binding of the human "electron transferring flavoprotein" (ETF) to the medium chain acyl-CoA dehydrogenase (MCAD) involves an arginine and histidine residue

The interaction between the "electron transferring flavoprotein" (ETF) and medium chain acyl-CoA dehydrogenase (MCAD) enables successful flavin to flavin electron transfer, crucial for the beta-oxidation of fatty acids. The exact biochemical determinants for ETF binding to MCAD are unknown. Here we show that binding of human ETF, to MCAD, was inhibited by 2,3-butanedione and diethylpyrocarbonate (DEPC) and reversed by incubation with free arginine and hydroxylamine respectively. Spectral analyses of native ETF vs modified ETF suggested that flavin binding was not affected and that the loss of ETF activity with MCAD involved modification of one ETF arginine residue and one ETF histidine residue respectively. MCAD and octanoyl-CoA protected ETF against inactivation by both 2,3-butanedione and DEPC indicating that the arginine and histidine residues are present in or around the MCAD binding site. Comparison of exposed arginine and histidine residues among different ETF species, however, indicates that arginine residues are highly conserved but that histidine residues are not. These results lead us to conclude that this single arginine residue is essential for the binding of ETF to MCAD, but that the single histidine residue, although involved, is not.     

Evidence for an essential arginine residue at the active site of Escherichia coli acetate kinase

Escherichia coli acetate kinase (ATP: acetate phosphotransferase, EC 2.7.2.1.) was inactivated in the presence of either 2,3-butanedione in borate buffer or phenylglyoxal in triethanolamine buffer. When incubated with 9.4 mM phenylglyoxal or 5.1 mM butanedione, the enzyme lost its activity with an apparent rate constant of inactivation of 0.079 min-1, respectively. The loss of enzymatic activity was concomitant with the loss of an arginine residue per active site. Phosphorylated substrates of acetate kinase, ATP, ADP and acetylphosphate as well as AMP markedly decreased the rate of inactivation by both phenylglyoxal and butanedione. Acetate neither provided any protection nor affected the protection rendered by the adenine nucleotides. However, it interfered with the protection afforded by acetylphosphate. These data suggest that an arginine residue is located at the active site of acetate kinase and is essential for its catalytic activity, probably as a binding site for the negatively charged phosphate group of the substrates.     

Inactivation of purine nucleoside phosphorylase by modification of arginine residues.

Treatment of purine nucleoside phosphorylase (EC 2.4.2.1), from either calf spleen or human erythrocytes, with 2,3-butanedione in borate buffer or with phenylglyoxal in Tris buffer markedly decreased the enzyme activity. At pH 8.0 in 60 min, 95% of the catalytic activity was destroyed upon treatment with 33 mM phenylglyoxal and 62% of the activity was lost with 33 mm 2,3-butanedione. Inorganic phosphate, ribose-1-phosphate, arsenate, and inosine when added prior to chemical modification all afforded protection from inactivation. No apparent decrease in enzyme catalytic activity was observed upon treatment with maleic anhydride, a lysine-specific reagent. Inactivation of electrophoretically homogeneous calf-spleen purine nucleoside phosphorylase by butanedione was accompanied by loss of arginine residues and of no other amino acid residues. A statistical analysis of the inactivation data vis-à-vis the fraction of arginines modified suggested that one essential arginine residue was being modified.     

The importance of arginine residues in the catalytic and regulatory functions of bovine-liver glutamate dehydrogenase.

Bovine liver glutamate dehydrogenase reacts rapidly with 2,3-butanedione to yield modified enzyme with 29% of its original maximum activity, but no change in its Michaelis constants for substrates and coenzymes. No significant reduction in the inactivation rate is produced by the addition of the allosteric activator ADP or inhibitor GTP, while partial protection against inactivation is provided by the coenzyme NAD+ or substrate 2-oxoglutarate when added separately. The most marked decrease in the rate of inactivation (about 10-fold) is provided by the combined addition of NAD+ and 2-oxoglutarate, suggesting that modification takes place in the region of the active site. Reaction with 2,3-butanedione also results in loss of the ability of the enzyme to be activated by ADP. Addition of ADP (but not NAD+, 2-oxoglutarate or GTP) to the incubation mixture protects markedly against the loss of activatability of ADP. It is concluded that 2,3-butanedione produces two distinguishable effects on glutamate dehydrogenase: a relatively specific modification of the regulatory ADP site and a distinct modification in the active center. Reaction of two arginyl residues per peptide chain appears to be responsible for disruption of the ADP activation property of the enzyme, while alteration of a maximum of five arginyl residues can be related to the reduction of maximum catalytic activity. Electrostatic interactions between the positively charged arginine groups and the negatively charged substrate, coenzyme and allosteric purine nucleotide may be important for the normal function of glutamate dehydrogenase.     

Inactivation of Escherichia coli L-threonine dehydrogenase by 2,3-butanedione. Evidence for a catalytically essential arginine residue

Incubation of homogeneous preparations of L-threonine dehydrogenase from Escherichia coli with 2,3-butanedione, 2,3-pentanedione, phenylglyoxal, or 1,2-cyclohexanedione causes a time- and concentration-dependent loss of enzymatic activity; plots of log percent activity remaining versus time are linear to greater than 90% inactivation, indicative of pseudo-first order inactivation kinetics. The reaction order with respect to the concentration of modifying reagent is approximately 1.0 in each case suggesting that the loss of catalytic activity is due to one molecule of modifier reacting with each active unit of enzyme. Controls establish that this inactivation is not due to modifier-induced dissociation or photoinduced nonspecific alteration of the dehydrogenase. Essentially the same Km but decreased Vmax values are obtained when partially inactivated enzyme is compared with native. NADH (25 mM) and NAD+ (70 mM) give full protection against inactivation whereas much higher concentrations (i.e. 150 mM) of L-threonine or L-threonine amide provide a maximum of 80-85% protection. Amino acid analyses coupled with quantitative sulfhydryl group determinations show that enzyme inactivated 95% by 2,3-butanedione loses 7.5 arginine residues (out of 16 total)/enzyme subunit with no significant change in other amino acid residues. In contrast, only 2.4 arginine residues/subunit are modified in the presence of 80 mM NAD+. Analysis of the course of modification and inactivation by the statistical method of Tsou (Tsou, C.-L. (1962) Sci. Sin. 11, 1535-1558) demonstrates that inactivation of threonine dehydrogenase correlates with the loss of 1 "essential" arginine residue/subunit which quite likely is located in the NAD+/NADH binding site.     

Evidence for an essential arginine in the flavoprotein nitroalkane oxidase

The flavoprotein nitroalkane oxidase from the fungus Fusarium oxysporum catalyzes the oxidative denitrification of primary or secondary nitroalkanes to yield the respective aldehydes or ketones, hydrogen peroxide and nitrite. The enzyme is inactivated in a time-dependent fashion upon treatment with the arginine-directed reagents phenylglyoxal, 2,3-butanedione, and cyclohexanedione. The inactivation shows first order kinetics with all reagents. Valerate, a competitive inhibitor of the enzyme, fully protects the enzyme from inactivation, indicating that modification is active site directed. The most rapid inactivation is seen with phenylglyoxal, with a k(inact) of 14.3 +/- 1.1 M(-1) min(-1) in phosphate buffer at pH 7.3 and 30 degrees C. The lack of increase in the enzymatic activity of the phenylglyoxal-inactivated enzyme after removing the unreacted reagent by gel filtration is consistent with inactivation being due to covalent modification of the enzyme. A possible role for an active site arginine in substrate binding is discussed.     

Characterization of an essential arginine residue in the plasma membrane H+-ATPase of Neurospora crassa

Treatment of the plasma membrane H+-ATPase of Neurospora crassa with the arginine-specific reagents phenylglyoxal or 2,3-butanedione at 30 degrees C, pH 7.0, leads to a marked inhibition of ATPase activity. MgATP, the physiological substrate of the enzyme, protects against inactivation. MgADP, a competitive inhibitor of ATPase activity with a measured Ki of 0.11 mM, also protects, yielding calculated KD values of 0.125 and 0.115 mM in the presence of phenylglyoxal and 2,3-butanedione, respectively. The excellent agreement between Ki and KD values makes it likely that MgADP exerts its protective effect by binding to the catalytic site of the enzyme. Loss of activity follows pseudo-first order kinetics with respect to phenylglyoxal and 2,3-butanedione concentration, and double log plots of pseudo-first order rate constants versus reagent concentration yield slopes of 0.999 (phenylglyoxal) and 0.885 (2,3-butanedione), suggesting that the modification of one reactive site/mol of H+-ATPase is sufficient for inactivation. This stoichiometry has been confirmed by direct measurements of the incorporation of [14C]phenylglyoxal. Taken together, the results support the notion that one arginine residue, either located at the catalytic site or shielded by a conformational change upon nucleotide binding, plays an essential role in Neurospora H+-ATPase activity.     

Evidence for the importance of arginine residues in pig kidney alkaline phosphatase.

The arginine-specific reagents 2,3-butanedione and phenylglyoxal inactivate pig kidney alkaline phosphatase. As inactivation proceeds there is a progressive fall in Vmax. of the enzyme, but no demonstrable change in the Km value for substrate. Pi, a competitive inhibitor, and AMP, a substrate of the enzyme, protect alkaline phosphatase against the arginine-specific reagents. These effects are explicable by the assumption that the enzyme contains an essential arginine residue at the active site. Protection is also afforded by the uncompetitive inhibitor NADH through a partially competive action against the reagents. Enzyme that has been exposed to the reagents has a decreased sensitivity to NADH inhibition. It is suggested that an arginine residue is important for NADH binding also, although this residue is distinct from that at the catalytic site. The protection given by NADH against loss of activity is indicative of the close proximity of the active and NADH sites.     

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.     

Essential arginyl residues in the H+-translocating ATPase of plasma membrane from the yeast Schizosaccharomyces pombe

The H+-translocating adenosine-5'-triphosphatase (ATPase) purified from the yeast Schizosaccharomyces pombe is inactivated upon incubation with the arginine modifier 2,3-butanedione. The inactivation of the enzyme is maximal at pH values above 8.5. The modified enzyme is reactivated when incubated in the absence of borate after removal of 2,3-butanedione. The extent of inactivation is half maximal at 10 mM 2,3-butanedione for an incubation of 30 min at 30 degrees C at pH 7.0. Under the same conditions, the time-dependence of inactivation is biphasic in a semi-logarithmic plot with half-lives of 10.9 min and 65.9 min. Incubation with 2,3-butanedione lowering markedly the maximal rate of ATPase activity does not modify the Km for MgATP. These data suggest that two classes of arginyl residues play essential role in the plasma membrane ATPase activity. Magnesium adenosine 5'-triphosphate (MgATP) and magnesium adenosine 5'-diphosphate (MgADP), the specific substrate and product, protect partially against enzyme inactivation by 2,3-butanedione. Free ATP or MgGTP which are not enzyme substrates do not protect. Free magnesium, another effector of enzyme activity, exhibits partial protection at magnesium concentrations up to 0.5 mM, while increased inactivation is observed at higher Mg2+ concentrations. These protections indicate either the existence of at least one reactive arginyl in the substrate binding site or a general change of enzyme conformation induced by MgATP, MgADP or free magnesium.     

Evidence for involvement of arginyl residue at the catalytic site of penicillin acylase from Escherichia coli

Incubation of penicillin acylase from Escherichia coli with phenylglyoxal or 2,3-butanedione results in enzyme inactivation. Both benzylpenicillin and phenylacetate protect the enzyme against the inactivation, indicating the presence of arginine at or near the catalytic site. The reactions follow pseudofirst order kinetics and the inactivation kinetics indicate the presence of a single essential arginine moiety.     

A single arginine residue is required for the interaction of the electron transferring flavoprotein (ETF) with three of its dehydrogenase partners

The interaction of several dehydrogenases with the electron transferring flavoprotein (ETF) is a crucial step required for the successful transfer of electrons into the electron transport chain. The exact determinants regarding the interaction of ETF with its dehydrogenase partners are still unknown. Chemical modification of ETF with arginine-specific reagents resulted in the loss, to varying degrees, of activity with medium chain acyl-coenzyme A dehydrogenase (MCAD). The kinetic profiles showed the inactivations followed pseudo-first-order kinetics for all reagents used. For activity with MCAD, maximum inactivation of ETF was accomplished by 2,3-butanedione (4% residual activity after 120 min) and it was shown that modification of one arginine residue was responsible for the inactivation. Almost 100% restoration of this ETF activity was achieved upon incubation with free arginine. However, the same 2,3-butanedione modified ETF only possessed decreased activity with dimethylglycine- (DMGDH, 44%) and sarcosine- (SDH, 27%) dehydrogenases unlike the abolition with MCAD. Full protection of ETF from arginine modification by 2,3-butanedione was achieved using substrate-protected DMGDH, MCAD and SDH respectively. Cross-protection studies of ETF with the three dehydrogenases implied use of the same single arginine residue in the binding of all three dehydrogenases. These results lead us to conclude that this single arginine residue is essential in the binding of the ETF to MCAD, but only contributes partially to the binding of ETF to SDH and DMGDH and thus, the determinants of the dehydrogenase binding sites overlap but are not identical.     

Evidence for an essential arginine residue at the active site of ATP citrate lyase from rat liver.

Rat liver ATP citrate lyase was inactivated by 2, 3-butanedione and phenylglyoxal. Phenylglyoxal caused the most rapid and complete inactivation of enzyme activity in 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid buffer, pH 8. Inactivation by both butanedione and phenylglyoxal was concentration-dependent and followed pseudo- first-order kinetics. Phenylglyoxal also decreased autophosphorylation (catalytic phosphate) of ATP citrate lyase. Inactivation by phenylglyoxal and butanedione was due to the modification of enzyme arginine residues: the modified enzyme failed to bind to CoA-agarose. The V declined as a function of inactivation, but the Km values were unaltered. The substrates, CoASH and CoASH plus citrate, protected the enzyme significantly against inactivation, but ATP provided little protection. Inactivation with excess reagent modified about eight arginine residues per monomer of enzyme. Citrate, CoASH and ATP protected two to three arginine residues from modification by phenylglyoxal. Analysis of the data by statistical methods suggested that the inactivation was due to modification of one essential arginine residue per monomer of lyase, which was modified 1.5 times more rapidly than were the other arginine residues. Our results suggest that this essential arginine residue is at the CoASH binding site.     

Chemical modification of arginine residues in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens: a kinetic and fluorescence study

The flavoprotein p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was modified by several arginine-specific reagents. Modifications by 2,3-butanedione led to the loss of activity of the enzyme, but the binding of p-hydroxybenzoate and NADPH to the enzyme was little or not at all affected. However the formation of the enzyme-substrate complex of the modified enzyme was accompanied by an increase of the fluorescence of protein-bound FAD, in contrast to that of native enzyme which leads to quenching of the fluorescence. Enzyme modified by phenylglyoxal did not bind p-hydroxybenzoate nor NADPH. Quantification and protection experiments showed that two arginine residues are essential and a model is described which accounts for the results. Modification by 4-hydroxy-3-nitrophenylglyoxal reduced the affinity of the enzyme for the substrate and NADPH. The ligands offered no protection against inactivation. From this it is concluded that one arginine residue is essential at some stage of the catalysis. This residue is not associated with the substrate- or NADPH-binding site of the enzyme. Time-resolved fluorescence studies showed that the average fluorescence lifetime and the mobility of protein-bound FAD are affected by modification of the enzyme.     

Inactivation of Escherichia coli 2-amino-3-ketobutyrate CoA ligase by phenylglyoxal and identification of an active-site arginine peptide.

Treatment of homogeneous preparations of 2-amino-3-ketobutyrate CoA ligase from Escherichia coli, a pyridoxal 5'-phosphate-dependent enzyme, with phenylglyoxal, 4-(oxyacetyl)phenoxyacetic acid, 2,3-butanedione, or 1,2-cyclohexanedione results in a time- and concentration-dependent loss of enzymatic activity. Phenylglyoxal in 50 mM phosphate buffer (pH 7.0) is the most effective modifier, causing > 95% inactivation within 20 min at 25 degrees C. Controls establish that this inactivation is not due to modifier-induced dissociation or photoinduced nonspecific alteration of the ligase. The substrate, acetyl CoA, or the coenzyme, pyridoxal 5'-phosphate, gives > 50% protection against inactivation. Enzyme partially inactivated by phenylglyoxal has the same Km value for glycine but the Vmax decreases in proportion to the observed level of inactivation. Whereas the native apoligase shows good recovery of activity with time in parallel with an increase in 428-nm absorptivity when incubated with pyridoxal 5'-phosphate, no such effects are seen with the phenylglyoxal-modified apoligase. Reaction of the enzyme with [14C]phenylglyoxal allowed for the isolation of a peptide which, by amino acid composition and sequencing data, was found to correspond to residues 349-378 in the intact enzyme. These results indicate that arginine residue-366 and/or residue-368 in the primary structure of E. coli 2-amino-3-ketobutyrate ligase is at the active site.