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.     

UDP-glucose 4-epimerase from Saccharomyces fragilis. Presence of an essential arginine residue at the substrate-binding site of the enzyme

UDP-glucose 4-epimerase from Saccharomyces fragilis was inactivated by the arginine-specific reagents phenylglyoxal, 1,2-cyclohexanedione, and 2,3-butanedione following pseudo first order reaction kinetics. The reaction order with respect to phenylglyoxal was 1.8 and that with respect to the other two diones was close to unity. Protection afforded by substrate and competitive inhibitors against inactivation by phenylglyoxal and the reduced interaction of 1-anilinonaphthalene 8-sulfonic acid, a fluorescent probe for the substrate-binding region after phenylglyoxal modification, suggested the presence of an essential arginine residue at the substrate-binding region. Experiments with [7-14C]phenylglyoxal in the presence of UMP, a ligand known to interact at the substrate-binding region, showed that only the arginine residue at the active site could be modified by phenylglyoxal. The characteristic coenzyme fluorescence of the yeast enzyme was found to be enhanced three times in phenylglyoxal-inactivated enzyme suggesting the incorporation of the phenyl ring near the pyridine moiety of NAD.     

Evidence for an essential arginine residue in the substrate binding site of the mammalian succinate dehydrogenase

Phenylglyoxal and 2,3-butanedione rapidly inactivate membrane-bound or soluble bovine heart succinate dehydrogenase. The inhibition of the enzyme by these reagents is completely prevented by saturating concentration of malonate. The modification of the active site sulfhydryl group by p-chloromercuribenzoate decreases the rate of the enzyme inhibition by phenylglyoxal and abolishes the protective effect of malonate. Kinetic data suggest that the inactivation by phenylglyoxal results from the modification of an essential arginine residue(s) which interacts with dicarboxylate to form the primary enzyme-substrate complex.     

Essential arginines in mercuric reductase isolated from Yersinia enterocolitica 138A14.

The mercuric reductase from Yersinia enterocolitica 138A14 was inactivated by the arginine modifying reagents 2,3-butanedione and phenylglyoxal. The inactivation by 2,3-butanedione exhibited second order kinetics with rate constant of 32 min-1 M-1. In the case of phenylglyoxal, biphasic kinetics were observed. The oxidized coenzyme (NADP+) prevented inactivation of the enzyme by the alpha-dicarbonyl reagents, whereas the reduced coenzyme (NADPH) enhanced the inactivation rate. The loss of enzyme activity was related to the incorporation of [2-14C] phenylglyoxal; when two arginines per subunit were modified the enzyme was completely inactivated.     

Arginine-specific modification of rabbit muscle phosphoglucose isomerase: Differences in the inactivation by phenylglyoxal and butanedione and in the protection by substrate analogs

Rabbit muscle phosphoglucose isomerase was modified with phenylglyoxal or 2,3-butanedione, the reaction with either reagent resulting in loss of enzymatic activity in a biphasic mode. At slightly alkaline pH butanedione was found to be approximately six times as effective as phenylglyoxal. The inactivation process could not be significantly reversed by removal of the modifier. Competitive inhibitors of the enzyme protected partially against loss of enzyme activity by either modification. The only kind of amino acid residue affected was arginine. However, more than one arginine residue per enzyme subunit was found to be susceptible to modification by the dicarbonyl reagents. From protection experiments it was concluded (i) that both modifiers react specifically with an arginine in the phosphoglucose isomerase active site and nonspecifically with one or more arginine residues elsewhere in the enzyme molecule, (ii) that modification at either loci causes loss of catalytic activity, and (iii) that butanedione has a higher preference for active site arginine than for arginine residues outside of the catalytic center whereas the opposite is true for phenylglyoxal.     

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.     

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.     

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.     

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.     

Inactivation of carbonyl reductase from human brain by phenylglyoxal and 2,3-butanedione: a comparison with aldehyde reductase and aldose reductase

Aldehyde reductase (alcohol:NADP+ oxidoreductase, EC 1.1.1.2), aldose reductase (alditol:NAD(P)+ 1-oxidoreductase, EC 1.1.1.21) and carbonyl reductase (secondary-alcohol:NADP+ oxidoreductase, EC 1.1.1.184) constitute the enzyme family of the aldo-keto reductases, a classification based on similar physicochemical properties and substrate specificities. The present study was undertaken in order to obtain information about the structural relationships between the three enzymes. Treatment of human aldehyde and carbonyl reductase with phenylglyoxal and 2,3-butanedione caused a complete and irreversible loss of enzyme activity, the rate of loss being proportional to the concentration of the dicarbonyl reagents. The inactivation of aldehyde reductase followed pseudo-first-order kinetics, whereas carbonyl reductase showed a more complex behavior, consistent with protein modification cooperativity. NADP+ partially prevented the loss of activity of both enzymes, and an even better protection of aldehyde reductase was afforded by the combination of coenzyme and substrate. Aldose reductase was partially inactivated by phenylglyoxal, but insensitive to 2,3-butanedione. The degree of inactivation with respect to the phenylglyoxal concentration showed saturation behavior. NADP+ partially protected the enzyme at low phenylglyoxal concentrations (0.5 mM), but showed no effect at high concentrations (5 mM). These findings suggest the presence of an essential arginine residue in the substrate-binding domain of aldehyde reductase and the coenzyme-binding site of carbonyl reductase. The effect of phenylglyoxal on aldose reductase may be explained by the modification of a reactive thiol or lysine rather than an arginine residue.     

Detection of essential arginine in bacterial peptidyl dipeptidase-4: arginine is not the anion binding site

Peptidyl dipeptidase-4 from Pseudomonas maltophilia was modified with the arginine reagents p-hydroxyphenylglyoxal and 2,3-butanedione. The enzyme was inactivated in a pseudo-first-order manner by p-hydroxyphenylglyoxal with a half-time of 72 min. Inactivation by 2,3-butanedione was biphasic with a rapid phase followed by a slower inactivation to less than 10% activity within 24h. The competitive inhibitor thiorphan protected against inactivation by phydroxyphenylglyoxal and by 2,3-butanedione also but to a lesser degree. Inhibitory anions chloride and phosphate did not protect against inactivation by either reagent. These data support the conclusion that an active site arginine is essential for substrate hydrolysis. Furthermore, arginine is not the binding site for the inhibitors chloride and phosphate.     

Essential arginine residues in the nitrate uptake system from corn seedling roots

Three dicarbonyl reagents were used to demonstrate the presence of an essential arginine residue in the NO₃⁻ uptake system from corn seedling roots (Zea mays L., Golden Cross Bantam). Incubation of corn seedlings with 2,3-butanedione (0.125-1.0 millimolar) and 1,2-cyclohexanedione (0.5-4.0 millimolar) in the presence of borate or with phenylglyoxal (0.25-2.0 millimolar) at pH 7.0 and 30°C resulted in a time-dependent loss of NO₃⁻ uptake following pseudo-first-order kinetics. Second-order rate constants obtained from slopes of linear plots of pseudo-first-order rate constants versus reagent concentrations were 1.67 × 10⁻², 0.68 × 10⁻², and 1.00 × 10⁻² millimolar per minute for 2,3-butanedione, 1,2-cyclohexanedione, and phenylglyoxal, respectively, indicating the faster rate of inactivation with 2,3-butanedione at equimolar concentration. Double log plots of pseudo-first-order rate constants versus reagent concentrations yielded slope values of 1.031 (2,3-butanedione), 1.004 (1,2-cyclohexanedione), and 1.067 (phenylglyoxal), respectively, suggesting the modification of a single arginine residue. The effectiveness of the dicarbonyl reagents appeared to increase with increasing medium pH from 5.5 to 8.0. Unaltered K_m and decreased V_max in the presence of reagents indicate the inactivation of the modified carriers with unaltered properties. The results thus obtained indicate that the NO₃⁻ transport system possesses at least one essential arginine residue.     

Involvement of an essential arginyl residue in the coupling activity of Rhodospirillum rubrum chromatophores.

The arginine reagents phenylglyoxal and 2,3-butanedione in borate buffer completely inhibited photophosphorylation and Mg-ATPase of Rhodospirillum rubrum chromatophores. The inactivation rates followed apparent first order kinetics. Oxidative phospho-rylation and the light-dependent ATP-P_i exchange reactions ofR. rubrum chromatophores and the Ca-ATPase activity of the soluble coupling factor were similarly inhibited by 2,3-butanedione in borate buffer. The apparent order of reaction with respect to inhibitor concentrations for all these reactions gave values of near 1 suggesting that inactivation was the consequence of modifying one arginine per active site. ATP synthesis and hydrolysis by R. rubrum chromatophores were strongly protected against inactivation by ADP and ATP, respectively, and by other nucleotides that are substrates of the reactions but not by the products. Similarly, the Ca-ATPase of the soluble coupling factor was protected by ATP but not by ADP. Inactivation of chromatophores reactions by butanedione in borate buffer was more rapid in the light than in the dark. The results suggest that the catalytic sites for ATP synthesis and hydrolysis on the chromatophore coupling factor are different and both contain an essential arginine.     

Essential Arginyl Residue at the Active Site of Pyrophosphate:Fructose 6-Phosphate 1-Phosphotransferase from Potato (Solanum tuberosum) Tuber

The aim of this work was to test the proposal that the active site of pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP) contains an essential arginyl residue. Enzyme activity was inhibited equally in the glycolytic and gluconeogenic directions by arginine-modifying reagents. The second-order rate constants for 2,3-butanedione and phenylglyoxal were 13.1 [plus or minus] 0.45 and 55.3 [plus or minus] 1.3 M-1 min-1, respectively. The corresponding values for the kinetic order of inactivation by these modifying reagents were 0.84 [plus or minus] 0.049 for 2,3-butanedione and 0.89 [plus or minus] 0.052 for phenylglyoxal. The substrates, fructose 6-phosphate and pyrophosphate, and a range of substrate analogs protected the enzyme from inactivation by 2,3-butanedione. These data suggest that modification of no more than one arginyl residue at, or close to, the active site is required to inhibit the enzyme. This result supports the proposal that the active site of PFP in plants is equivalent to that of the bacterial ATP-phosphofructokinase (S.M. Carlisle, S.D. Blakeley, S.M. Hemmingsen, S.J. Trevanion, T. Hiyoshi, N.J. Kruger, and D.T. Dennis [1990] J Biol Chem 265: 18366-18371).     

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.     

Essential Arginyl Residues in the Plasma Membrane H-ATPase from Vigna radiata L. (Mung Bean) Roots

Proton-translocating ATPase (H⁺-ATPase) was purified from mung bean (Vigna radiata L.) roots. Treatment of this enzyme with the arginine-specific reagent 2,3-butanedione in the presence of borate at 37°C (pH 7.0), caused a marked decrease in its activity. Under this condition, half-maximal inhibition was brought about by 20 millimolar 2,3-butanedione at 12 minutes. MgATP and MgADP, the physiological substrate and competitive inhibitor of the ATPase, respectively, provided partial protection against inactivation. Loss of activity followed pseudo-first order kinetics with respect to 2,3-butanedione concentration, and double log plots of pseudo-first order rate constants versus reagent concentration gave a curve with a slope of 0.984. Thus, inactivation may possibly result from reaction of one arginine residue at each active site of the enzyme. The results obtained from the present study indicate that at least one arginyl residue performs an essential function in the plasma membrane H⁺-ATPase, probably at the catalytic site.     

Inactivation of three monohydroxybenzoate mono-oxygenases from Rhodococcus erythropolis: The role of an arginine residue in the substrate-binding domain of p-hydroxybenzoate 3-hydroxylase

Abstract p-Hydroxybenzoate 3-hydroxylase from Rhodococcus erythropolis was inactivated by 2,3-butanedione (BD), phenylglyoxal (PGO), and other chemical reagents. p -Hydroxybenzoate and NADH protected the enzyme from inactivation by BD. Judging from the amino acid composition of BD-treated enzyme in the presence and absence of p -hydroxybenzoate, one essential arginine residue in substrate-binding domain of the enzyme was shown to be essential to the binding of p -hydrozybenzoate to the enzyme. Salicylate 5-hydroxylase and m -hydroxybenzoate 6-hydroxylase from R. erythropolis were hardly inactivated. Neither of these two enzymes was considered to have a functional arginine residue required for interaction with the substrate.     

Inhibition of the mitochondrial tricarboxylate carrier by arginine-specific reagents

The effect of arginine-specific reagents on the activity of the partially purified and reconstituted tricarboxylate carrier of the inner mitochondrial membrane has been studied. It has been found that 1,2-cyclohexanedione, 2,3-butanedione, phenylglyoxal and phenylglyoxal derivatives inhibit the reconstituted citrate/citrate exchange activity. The inhibitory potency of the phenylglyoxal derivatives increases with increasing hydrophilic character of the molecule. Citrate protects the tricarboxylate carrier against inactivation caused by the arginine-specific reagents. Other tricarboxylates, which are not substrates of the carrier, have no protective effect. The results indicate that at least one essential arginine residue is located at the substrate-binding site of the tricarboxylate carrier and that the vicinity of the essential arginine(s) has a hydrophilic character.     

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 rat liver microsomal glutathione transferase defines residues of importance for catalytic function.

Amino acid residues that are essential for the activity of rat liver microsomal glutathione transferase have been identified using chemical modification with various group-selective reagents. The enzyme reconstituted into phosphatidylcholine liposomes does not require stabilization with glutathione for activity (in contrast with the purified enzyme in detergent) and can thus be used for modification of active-site residues. Protection by the product analogue and inhibitor S-hexylglutathione was used as a criterion for specificity. It was shown that the histidine-selective reagent diethylpyrocarbonate inactivated the enzyme and that S-hexylglutathione partially protected against this inactivation. All three histidine residues in microsomal glutathione transferase could be modified, albeit at different rates. Inactivation of 90% of enzyme activity was achieved within the time period required for modification of the most reactive histidine, indicating the functional importance of this residue in catalysis. The arginine-selective reagents phenylglyoxal and 2,3-butanedione inhibited the enzyme, but the latter with very low efficiency; therefore no definitive assignment of arginine as essential for the activity of microsomal glutathione transferase can be made. The amino-group-selective reagents 2,4,6-trinitrobenzenesulphonate and pyridoxal 5'-phosphate inactivated the enzyme. Thus histidine residues and amino groups are suggested to be present in the active site of the microsomal glutathione transferase.