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.     

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 that both arginine 102 and arginine 747 are involved in substrate binding to neutral endopeptidase (EC 3.4.24.11)

Neutral endopeptidase (EC 3.4.24.11, NEP) is a Zn-metallopeptidase involved in the degradation of biologically active peptides, notably the enkephalins and atrial natriuretic peptide. Recently, the structure of the active site of this enzyme has been probed by site-directed mutagenesis, and 4 amino acid residues have been identified, namely 2 histidines (His583 and His587), which act as zinc-binding ligands, a glutamate (Glu584) involved in catalysis, and an arginine residue (Arg102), suggested to participate in substrate binding. Site-directed mutagenesis has now been used to investigate the role of 4 other arginine residues (Arg408, Arg409, Arg659, and Arg747) that have been proposed as possible active site residues and to further analyze the role of Arg102. In each case, the arginine was replaced with a methionine, and both enzymatic activity and the IC50 values of several NEP inhibitors were measured for the mutated enzymes and compared to wild-type enzyme. The results suggest that 2 arginines, Arg102 and Arg747, could both be important for substrate and inhibitor binding. Arg747 seems to be positioned to interact with the carbonyl amide group of the P'1 residue and can be modified when the enzyme is treated with the arginine-specific reagents phenylglyoxal and butanedione. Arg102 could be positioned to interact with the free carboxyl group of a P'2 residue in some substrates and inhibitors and can be modified by phenylglyoxal but not by butanedione. The results could explain the dual dipeptidylcarboxypeptidase and endopeptidase nature of NEP.     

Nicotinamide-adenine dinucleotide inhibition of pig kidney alkaline phosphatase.

1. The interaction of NAD+, NADH and various nucleotide analogues with pig kidney alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum) EC 3.1.3.1) has been investigated by kinetic means. Some inhibitors act uncompetitively whereas others markedly increase the slopes of double reciprocal plots suggesting they have some affinity for the free enzyme. 2. The compounds seem to bind to alkaline phosphatase through interactions of their bases with a relatively non-specific region of the enzyme, although it is likely that for those nucleotides having some affinity for the free enzyme there is some attraction between the pyrophosphate backbone and the active site. 3. From studies of the effect of NAD+ and NADH on ATPase activity it was concluded that the substrate inhibition that is characteristic of the ATPase activity of alkaline phosphatase originates from binding of ATP to the site assumed to exist for NAD+ and NADH. The potentiation of NAD+-inhibition of ATPase activity by Mg-2+ is probably a result of the depletion of [ATP-4-] the true substrate. The depletion allows NAD+ to complete more effectively for the active site. 4. Binding of NADH is favoured by protonation of an enzymic group with a pK of approx. 9.0 belonging possibly to a tyrosine residue or a zinc hydrate. 5. A large entropy decrease was found to accompany the binding of NAD+ and NADH to alkaline phosphatase. This may be further evidence of an "induced-fit" mechanism previously suspected because of the synergistic inhibitory effects of adenosine and nicotinamide.     

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.     

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.     

Function of arginine-166 in the active site of Escherichia coli alkaline phosphatase

The function of arginine residue 166 in the active site of Escherichia coli alkaline phosphatase was investigated by site-directed mutagenesis. Two mutant versions of alkaline phosphatase, with either serine or alanine in the place of arginine at position 166, were generated by using a specially constructed M13 phage carrying the wild-type phoA gene. The mutant enzymes with serine and alanine at position 166 have very similar kinetic properties. Under conditions of no external phosphate acceptor, the kcat for the mutant enzymes decreases by approximately 30-fold while the Km increases by less than 2-fold. When kinetic measurements are carried out in the presence of a phosphate acceptor, 1.0 M Tris, the kcat for the mutant enzymes is reduced by less than 3-fold, while the Km increases by more than 50-fold. For both mutant enzymes, in either the absence or the presence of a phosphate acceptor, the catalytic efficiency as measured by the kcat/Km ratio decreases by approximately 50-fold as compared to the wild type. Measurements of the Ki for inorganic phosphate show an increase of approximately 50-fold for both mutants. Phenylglyoxal, which inactivates the wild-type enzyme, does not inactivate the Arg-166----Ala enzyme. This result indicates that Arg-166 is the same arginine residue that when chemically modified causes loss of activity [Daemen, F.J.M., & Riordan, J.F. (1974) Biochemistry 13, 2865-2871]. The data reported here suggest that although Arg-166 is important for activity is not essential. The analysis of the kinetic data also suggests that the loss of arginine-166 at the active site of alkaline phosphatase has two different effects on the enzyme. First, the binding of the substrate, and phosphate as a competitive inhibitor, is reduced; second, the rate of hydrolysis of the covalent phosphoenzyme may be diminished.     

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.     

Arginine residues at the active site of avian liver phosphoenolpyruvate carboxykinase

The presence of arginine at the active site of avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification followed by a characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione all irreversibly inhibit the enzyme with second-order rate constants of 3.42 M-1 min-1, 3.13 M-1 min-1 and 0.313 M-1 min-1, respectively. The substrates phosphoenolpyruvate, IDP, and the activator Mn2+ offer little to modest protection from inhibition. Either CO2 or CO2 in the presence of any of the other substrates elicited potent protection against modification. Protection by CO2 against modification by phenylglyoxal or 1,2-cyclohexanedione gave a biphasic pattern. Rapid loss in activity to 40-60% occurred, followed by a very slow loss. Kinetics of inhibition suggest that the modification of arginine is specific and leads to loss of enzymatic activity. Substrate protection studies indicate an arginine residue(s) at the CO2 site of phosphoenolpyruvate carboxykinase. Apparently no arginine residues are at the binding site of the phosphate-containing substrates. Partially inactive (40-60% activity) enzyme, formed in the presence of CO2, has a slight change of its kinetic constants, and no alteration of its binding parameters or secondary structure as demonstrated by kinetic, proton relaxation rate, and circular dichroism studies. Labeling of enzyme with [(7-)14C]phenylglyoxal in the presence of CO2 (40-60% activity) showed 2 mol of phenylglyoxal/enzyme or 1 arginine or cysteine residue modified. Labeling of phosphoenolpyruvate carboxykinase in the absence of CO2 yielded 6 mol of label/enzyme. Labeling results indicate that avian phosphoenolpyruvate carboxykinase has 2 or 3 reactive arginine residues out of a total of 52 and only 1 or 2 are located at the active site and are involved in CO2 binding and activation.     

Functional modification of an arginine residue on salicylate hydroxylase

Salicylate hydroxylase from Pseudomonas putida (EC 1.14.13.1, salicylate, NADH:oxygen oxidoreductase) is an FAD-containing monooxygenase, which catalyzes decarboxylative hydroxylation of salicylate to produce catechol in the presence of NADH and O2. By chemical treatment of the enzyme with dicarbonyl reagents, such as glyoxal, the original oxygenase activity was converted to the salicylate-dependent NADH-dehydrogenase activity with free FAD as electron acceptor. One of twenty arginine residues of this enzyme is concerned with this alteration of activity, as shown by the result of its modification at pH 6.9. This result is further supported by the isolation of one arginine-modified enzyme by chromatographic methods on DEAE-Sephadex, A-50 columns. It exhibits the dehydrogenase activity predominantly. This modified enzyme is spectrophotometrically and electrophoretically characterized by a minute conformational change around the active site, and kinetically by a 7-fold increase in an apparent Km for NADH and a decrease of more than 5-fold in an apparent Km for FAD as electron acceptor, with an apparent Vmax of 22 s-1 for the dehydrogenase activity. Flow kinetics also showed a marked decrease in the rate for oxygenation of the reduced enzyme-salicylate complex from 21 s-1 (native enzyme) to 3.3 s-1 (modified enzyme). These facts suggest that one arginine residue of the enzyme is responsible for the NADH binding site, and chemical modification of one arginine residue of the enzyme induces some conformational change around the active site to alter the catalytic activity from oxygenation to dehydrogenation.     

X-ray structure reveals a new class and provides insight into evolution of alkaline phosphatases

The alkaline phosphatase (AP) is a bi-metalloenzyme of potential applications in biotechnology and bioremediation, in which phosphate monoesters are nonspecifically hydrolysed under alkaline conditions to yield inorganic phosphate. The hydrolysis occurs through an enzyme intermediate in which the catalytic residue is phosphorylated. The reaction, which also requires a third metal ion, is proposed to proceed through a mechanism of in-line displacement involving a trigonal bipyramidal transition state. Stabilizing the transition state by bidentate hydrogen bonding has been suggested to be the reason for conservation of an arginine residue in the active site. We report here the first crystal structure of alkaline phosphatase purified from the bacterium Sphingomonas. sp. Strain BSAR-1 (SPAP). The crystal structure reveals many differences from other APs: 1) the catalytic residue is a threonine instead of serine, 2) there is no third metal ion binding pocket, and 3) the arginine residue forming bidentate hydrogen bonding is deleted in SPAP. A lysine and an aspargine residue, recruited together for the first time into the active site, bind the substrate phosphoryl group in a manner not observed before in any other AP. These and other structural features suggest that SPAP represents a new class of APs. Because of its direct contact with the substrate phosphoryl group, the lysine residue is proposed to play a significant role in catalysis. The structure is consistent with a mechanism of in-line displacement via a trigonal bipyramidal transition state. The structure provides important insights into evolutionary relationships between members of AP superfamily.     

Reaction of neutral endopeptidase 24.11 (enkephalinase) with arginine reagents

The effect of the arginine-specific reagents phenylglyoxal and butanedione on the activity of neutral endopeptidase 24.11 ("enkephalinase") was determined. Inactivation of the enzyme by butanedione is completely protected by methionine-enkephalin, but only partially protected by methionine-enkephalinamide. In contrast, phenylglyoxal inactivation of the enzyme exhibits saturation kinetics with a Kd of 20 mM. The enzyme is only partially protected against phenylglyoxal inactivation by both methionine-enkephalin and its amide, indicating that phenylglyoxal reacts at two sites. Reaction of the enzyme with phenylglyoxal in the presence of saturating methionine-enkephalin involves the direct reaction of the reagent with the enzyme-substrate complex. Enzyme treated with butanedione or with phenylglyoxal (at site 1) exhibits a 3-5 decrease in substrate binding with little change in kcat. In contrast, reaction with phenylglyoxal in the presence of saturating methionine-enkephalin shows little change in substrate binding but a 4-fold decrease in kcat. Enzyme inactivation involves the incorporation of approximately 1 mol of phenylglyoxal/enzyme subunit in the absence of methionine-enkephalin and approximately 2.5 mol of phenylglyoxal/enzyme subunit in the presence of saturating methionine-enkephalin. These results suggest that an arginine residue on the enzyme is involved in substrate binding.     

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.     

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.     

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.     

Chemical modification and composition of tetrameric isozyme K of alkaline phosphatase from harp seal intestinal mucosa

1. The carbohydrate content of isozyme K of alkaline phosphatase (EC 3.1.3.1) from harp seal intestinal mucosa was examined. The presence of N-acetylglucosamine, N-acetylgalactosamine and considerable amounts of mannose residues was shown. 2. The amino acid content of seal alkaline phosphatase was determined. A high extent of homology (85%) between bovine and seal alkaline phosphatases was demonstrated. 3. By chemical modification lysine, dicarboxylic acids, arginine and tyrosine residues of tetrameric seal alkaline phosphatase are located near or at the active site. By contrast, the modification of either thiol or imidazole groups resulted in no alterations of the enzyme activity. 4. It has been demonstrated that inorganic phosphate is an inhibitor of alkaline phosphatase and entirely prevents the enzyme inactivation with succinic anhydride.     

Kinetics of inhibition of alkaline phosphatase from green crab (Scylla serrata) by N-bromosuccinimide

The inactivation of alkaline phosphatase from green crab (Scylla serrata) by N-bromosuccinimide has been studied using the kinetic method of the substrate reaction during modification of enzyme activity previously described by Tsou [(1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436]. The results show that inactivation of the enzyme is a slow, reversible reaction. The microscopic rate constants for the reaction of the inactivator with free enzyme and the enzyme-substrate complex were determined. Comparison of these rate constants indicates that the presence of substrate offers marked protection of this enzyme against inactivation by N-bromosuccinimide. The above results suggest that the tryptophan residue is essential for activity and is situated at the active site of the enzyme.Abbreviations ALP alkaline phosphatase - PNPP p-nitrophenyl phosphate - NBS N-bromosuccinimide     

Chemical modification of bovine heart mitochondrial malate dehydrogenase. Selective modification of cysteine and histidine.

Bovine mitochondrial malate dehydrogenase (EC 1.1.1.37) was inactivated by the specific modifications of a single histidine residue upon reaction with iodoacetamide. NADH protected against this loss of activity and reaction with the histidine residue, suggesting that the histidine is at the NADH binding site. N-Ethylmaleimide also modified the enzyme by reacting with 1 sulfhydryl residue. The reaction rate with N-ethylmaleimide was increased by decreasing the pH from neutrality or by the addition of urea. NADH protected against the modification of the sulfhydryl group under all the conditions tested, again suggesting active site specificity for this inactivation. This enzyme has a subunit weight of 33,000 and is a dimer. The native malate dehydrogenase will bind only 1 mol of NADH and it is thus assumed that there is only a single active site per dimer.