Characterization of glycerol trinitrate reductase (NerA) and the catalytic role of active-site residues

Glycerol trinitrate reductase (NerA) from Agrobacterium radiobacter, a member of the old yellow enzyme (OYE) family of oxidoreductases, was expressed in and purified from Escherichia coli. Denaturation of pure enzyme liberated flavin mononucleotide (FMN), and spectra of NerA during reduction and reoxidation confirmed its catalytic involvement. Binding of FMN to apoenzyme to form the holoenzyme occurred with a dissociation constant of ca. 10(-7) M and with restoration of activity. The NerA-dependent reduction of glycerol trinitrate (GTN; nitroglycerin) by NADH followed ping-pong kinetics. A structural model of NerA based on the known coordinates of OYE showed that His-178, Asn-181, and Tyr-183 were close to FMN in the active site. The NerA mutation H178A produced mutant protein with bound FMN but no activity toward GTN. The N181A mutation produced protein that did not bind FMN and was isolated in partly degraded form. The mutation Y183F produced active protein with the same k(cat) as that of wild-type enzyme but with altered K(m) values for GTN and NADH, indicating a role for this residue in substrate binding. Correlation of the ratio of K(m)(GTN) to K(m)(NAD(P)H), with sequence differences for NerA and several other members of the OYE family of oxidoreductases that reduce GTN, indicated that Asn-181 and a second Asn-238 that lies close to Tyr-183 in the NerA model structure may influence substrate specificity.     

Biosynthesis of fosfomycin, re-examination and re-confirmation of a unique Fe(II)- and NAD(P)H-dependent epoxidation reaction

(S)-2-Hydroxypropylphosphonic acid epoxidase (HppE) catalyzes the epoxide ring closure of (S)-HPP to form fosfomycin, a clinically useful antibiotic. Early investigation showed that its activity can be reconstituted with Fe(II), FMN, NADH, and O2 and identified HppE as a new type of mononuclear non-heme iron-dependent oxygenase involving high-valent iron-oxo species in the catalysis. However, a recent study showed that the Zn(II)-reconstituted HppE is active, and HppE exhibits modest affinity for FMN. Thus, a new mechanism is proposed in which the active site-bound Fe2+ or Zn2+ serves as a Lewis acid to activate the 2-OH group of (S)-HPP and the epoxide ring is formed by the attack of the 2-OH group at C-1 coupled with the transfer of the C-1 hydrogen as a hydride ion to the bound FMN. To distinguish between these mechanistic discrepancies, we re-examined the bioautography assay, the basis for the alternative mechanism, and showed that Zn(II) cannot replace Fe(II) in the HppE reaction and NADH is indispensable. Moreover, we demonstrated that the proposed role for FMN as a hydride acceptor is inconsistent with the finding that FMN cannot bind to HppE in the presence of substrate. In addition, using a newly developed HPLC assay, we showed that several non-flavin electron mediators could replace FMN in the HppE-catalyzed epoxidation. Taken together, these results do not support the newly proposed "nucleophilic displacement-hydride transfer" mechanism but are fully consistent with the previously proposed iron-redox mechanism for HppE catalysis, which is unique within the mononuclear non-heme iron enzyme superfamily.     

Structure Determination and Functional Analysis of a Chromate Reductase from Gluconacetobacter hansenii

Environmental protection through biological mechanisms that aid in the reductive immobilization of toxic metals (e.g., chromate and uranyl) has been identified to involve specific NADH-dependent flavoproteins that promote cell viability. To understand the enzyme mechanisms responsible for metal reduction, the enzyme kinetics of a putative chromate reductase from Gluconacetobacter hansenii (Gh-ChrR) was measured and the crystal structure of the protein determined at 2.25 Å resolution. Gh-ChrR catalyzes the NADH-dependent reduction of chromate, ferricyanide, and uranyl anions under aerobic conditions. Kinetic measurements indicate that NADH acts as a substrate inhibitor; catalysis requires chromate binding prior to NADH association. The crystal structure of Gh-ChrR shows the protein is a homotetramer with one bound flavin mononucleotide (FMN) per subunit. A bound anion is visualized proximal to the FMN at the interface between adjacent subunits within a cationic pocket, which is positioned at an optimal distance for hydride transfer. Site-directed substitutions of residues proposed to involve in both NADH and metal anion binding (N85A or R101A) result in 90–95% reductions in enzyme efficiencies for NADH-dependent chromate reduction. In comparison site-directed substitution of a residue (S118A) participating in the coordination of FMN in the active site results in only modest (50%) reductions in catalytic efficiencies, consistent with the presence of a multitude of side chains that position the FMN in the active site. The proposed proximity relationships between metal anion binding site and enzyme cofactors is discussed in terms of rational design principles for the use of enzymes in chromate and uranyl bioremediation.     

Cooperativity of alpha- and beta-subunits of group II chaperonin from the hyperthermophilic archaeum Aeropyrum pernix K1

alpha- and beta-subunits (ApCpnA and ApCpnB) are group II chaperonins from the hyperthermophilic archaeum Aeropyrum pernix K1, specialized in preventing the aggregation and inactivation of substrate proteins under conditions of transient heat stress. In the present study, the cooperativity of alpha- and beta-subunits from the A. pernix K1 was investigated. The ApCpnA and ApCpnB chaperonin genes were overexpressed in E. coli Rosetta and Codonplus (DE3), respectively. Each of the recombinant alpha- and beta- subunits was purified to 92% and 94% by using anionexchange chromatography. The cooperative activity between purified alpha- and beta-subunits was examined using citrate synthase (CS), alcohol dehydrogenase (ADH), and malate dehydrogenase (MDH) as substrate proteins. The addition of both alpha- and beta-subunits could effectively protect CS and ADH from thermal aggregation and inactivation at 43 degreesC and 50 degreesC, respectively, and MDH from thermal inactivation at 80 degreesC and 85 degreesC. Moreover, in the presence of ATP, the protective effects of alpha- and beta-subunits on CS from thermal aggregation and inactivation, and ADH from thermal aggregation, were more enhanced, whereas cooperation between chaperonins and ATP in protection activity on ADH and MDH (at 85 degreesC) from thermal inactivation was not observed. Specifically, the presence of both alpha- and beta- subunits could effectively protect MDH from thermal inactivation at 80 degreesC in an ATP-dependent manner.     

Covalent modification of the inhibitor binding site(s) of Escherichia coli ADP-glucose synthetase: specific incorporation of the photoaffinity analogue 8-azidoadenosine 5'-monophosphate

The photoaffinity agent 8-azidoadenosine 5'-monophosphate (8-N3AMP) is an inhibitor site specific probe of the Escherichia coli ADP-glucose synthetase (ADPG synthetase). In the absence of light, 8-N3AMP exhibits the typical reversible allosteric kinetics of the physiological inhibitor AMP. In the presence of light (254 nm), the analogue specifically and covalently modifies the enzyme, and photoincorporation is linearly related to loss of catalytic activity up to at least 65% inactivation. The substrate ADPG provides nearly 100% protection from 8-N3AMP photoinactivation, while the substrate ATP provides approximately 50% protection and the inhibitor AMP, approximately 30% protection. These three adenylate allosteric effectors of E. coli ADPG synthetase also protect it from photoincorporation of 8-N3AMP. A structural overlap of the inhibitor and substrate binding sites is proposed which explains the protection data in light of the known binding and kinetic properties of this tetrameric enzyme.     

Structural and biochemical characterization of recombinant wild type and a C30A mutant of trimethylamine dehydrogenase from methylophilus methylotrophus (sp. W(3)A(1))

Trimethylamine dehydrogenase (TMADH) is an iron-sulfur flavoprotein that catalyzes the oxidative demethylation of trimethylamine to form dimethylamine and formaldehyde. It contains a unique flavin, in the form of a 6-S-cysteinyl FMN, which is bent by approximately 25 degrees along the N5-N10 axis of the flavin isoalloxazine ring. This unusual conformation is thought to modulate the properties of the flavin to facilitate catalysis, and has been postulated to be the result of covalent linkage to Cys-30 at the flavin C6 atom. We report here the crystal structures of recombinant wild-type and the C30A mutant TMADH enzymes, both determined at 2.2 A resolution. Combined crystallographic and NMR studies reveal the presence of inorganic phosphate in the FMN binding site in the deflavo fraction of both recombinant wild-type and C30A proteins. The presence of tightly bound inorganic phosphate in the recombinant enzymes explains the inability to reconstitute the deflavo forms of the recombinant wild-type and C30A enzymes that are generated in vivo. The active site structure and flavin conformation in C30A TMADH are identical to those in recombinant and native TMADH, thus revealing that, contrary to expectation, the 6-S-cysteinyl FMN link is not responsible for the 25 degrees butterfly bending along the N5-N10 axis of the flavin in TMADH. Computational quantum chemistry studies strongly support the proposed role of the butterfly bend in modulating the redox properties of the flavin. Solution studies reveal major differences in the kinetic behavior of the wild-type and C30A proteins. Computational studies reveal a hitherto, unrecognized, contribution made by the S(gamma) atom of Cys-30 to substrate binding, and a role for Cys-30 in the optimal geometrical alignment of substrate with the 6-S-cysteinyl FMN in the enzyme active site.     

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.     

Crystal structure of an aerobic FMN-dependent azoreductase (AzoA) from Enterococcus faecalis

The initial critical step of reduction of the azo bond during the metabolism of azo dyes is catalyzed by a group of NAD(P)H dependant enzymes called azoreductases. Although several azoreductases have been identified from microorganisms and partially characterized, very little is known about the structural basis for substrate specificity and the nature of catalysis. Enterococcus faecalis azoreductase A (AzoA) is a highly active azoreductase with a broad spectrum of substrate specificity and is capable of degrading a wide variety of azo dyes. Here, we report the crystal structure of the AzoA from E. faecalis determined at 2.07 A resolution with bound FMN ligand. Phases were obtained by single wavelength anomalous scattering of selenomethionine labeled protein crystals. The asymmetric unit consisted of two dimers with one FMN molecule bound to each monomer. The AzoA monomer takes a typical NAD(P)-binding Rossmann fold with a highly conserved FMN binding pocket. A salt bridge between Arg18 and Asp184 restricts the size of the flavin binding pocket such that only FMN can bind. A putative NADH binding site could be identified and a plausible mechanism for substrate reduction is proposed. Expression studies revealed azoA gene to be expressed constitutively in E. faecalis.     

Two-electron reduction of quinones by Enterobacter cloacae NAD(P)H:nitroreductase: quantitative structure-activity relationships

Enterobacter cloacae NAD(P)H:nitroreductase (NR; EC 1.6.99.7) catalyzes two-electron reduction of a series of quinoidal compounds according to a "ping-pong" scheme, with marked substrate inhibition by quinones. The steady-state catalytic constants (k(cat)) range from 0.1 to 1600s(-1), and bimolecular rate constants (k(cat)/K(m)) range from 10(3) to 10(8)M(-1)s(-1). Quinones, nitroaromatic compounds and competitive to NADH inhibitor dicumarol, quench the flavin mononucleotide (FMN) fluorescence of nitroreductase. The reactivity of NR with single-electron acceptors is consistent with an "outer-sphere" electron transfer model, taking into account high potential of FMN semiquinone/FMNH(-) couple and good solvent accessibility of FMN. However, the single-electron acceptor 1,1(')-dibenzyl-4,4(')-bipyridinium was far less reactive than quinones possessing similar single-electron reduction potentials (E(1)(7)). For all quinoidal compounds except 2-hydroxy-1,4-naphthoquinones, there existed parabolic correlations between the log of rate constants of quinone reduction and their E(1)(7) or hydride-transfer potential (E(7)(Q/QH(-))). Based on pH dependence of rate constants, a single-step hydride transfer seems to be a more feasible quinone reduction mechanism. The reactivities of 2-hydroxy-1,4-naphthoquinones were much higher than expected from their reduction potential. Most probably, their enhanced reactivity was determined by their binding at or close to the binding site of NADH and dicumarol, whereas other quinones used the alternative, currently unidentified binding site.     

Affinity labeling of Cys111 of glutathione S-transferase, isoenzyme 1-1, by S-(4-bromo-2,3-dioxobutyl)glutathione.

Incubation of S-(4-bromo-2,3-dioxobutyl)glutathione (S-BDB-G), a reactive analogue of glutathione, with the 1-1 isoenzyme of rat liver glutathione S-transferase at pH 6.5 and 25 degrees C results in a time-dependent inactivation of the enzyme. k(obs) exhibits a nonlinear dependence on S-BDB-G from 50 to 1200 microM, with a kmax of 0.111 min-1 and KI = 185 microM. The addition of 5 mM S-hexylglutathione, a competitive inhibitor with respect to glutathione, gives almost complete protection against inactivation by S-BDB-G. About 1.2 mol of [3H]S-BDB-G/mol of enzyme subunit is incorporated when the enzyme is 85% inactivated, whereas 0.33 mol of reagent/mol of subunit is incorporated in the presence of S-hexylglutathione when the enzyme has lost only 17% of its original activity. Modified enzyme, prepared by incubating glutathione S-transferase with [3H]S-BDB-G in the absence or in the presence of S-hexylglutathione, was reduced with sodium borohydride, reacted with N-ethylmaleimide, and digested with alpha-chymotrypsin. Analysis of the chymotryptic digests, fractionated by reverse-phase high-performance liquid chromatography, revealed Cys111 as the amino acid whose reaction with S-BDB-G correlates with enzyme inactivation. It is concluded that Cys111 lies within or near the hydrophobic substrate binding site of glutathione S-transferase, isoenzyme 1-1.     

The binding of Na(+) to apo-enolase permits the binding of substrate

Enolase from rabbit muscle (betabeta-enolase) is inactivated by NaClO(4). Enolase free of divalent cations is more susceptible to inactivation by NaClO(4) than is enolase in the presence of Mg(2+). We find that substrate protects apo-enolase against inactivation, indicating that substrate can bind to enolase in the absence of a divalent cation. This binding is not due to contamination by trace levels of divalent cations since (1) it occurs even in the presence of EDTA or EGTA and (2) metal analysis by ICP (inductively coupled plasma) mass spectrometry did not reveal sufficient contamination to account for the protection. The binding of PGA to apo-enolase did require Na(+). When TMAClO(4) was used instead of NaClO(4), there was no protection by PGA. Protection was restored when TMAClO(4) plus NaCl were used. The inactivation of apo-enolase by NaClO(4) is due to dissociation into inactive monomers. We conclude that Na(+) binds to apo-enolase, permitting substrate to then bind. Of the three known Me(2+) binding sites on enolase, we believe the most likely binding site for Na(+) is the carboxylate cluster of site 1, the highest affinity site of enolase.     

Chemical modification studies on alkaline phosphatase from pearl oyster (Pinctada fucata): a substrate reaction course analysis and involvement of essential arginine and lysine residues at the active site

Alkaline phosphatases (ALP, EC 3.1.3.1) are ubiquitous enzymes found in most species. ALP from a pearl oyster, Pinctada fucata (PALP), is presumably involved in nacreous biomineralization processes. Here, chemical modification was used to investigate the involvement of basic residues in the catalytic activity of PALP. The Tsou's plot analysis indicated that the inactivation of PALP by 2,4,6-trinitrobenzenesulfonic acid (TNBS) and phenylglyoxal (PG) is dependent upon modification of one essential lysine and one essential arginine residue, respectively. Substrate reaction course analysis showed that the TNBS and PG inactivation of PALP followed pseudo-first-order kinetics and the second-order inactivation constants for the enzyme with or without substrate binding were determined. It was found that binding substrate slowed the PG inactivation whereas had little effect on TNBS inactivation. Protection experiments showed that substrates and competitive inhibitors provided significant protection against PG inactivation, and the modified enzyme lost its ability to bind the specific affinity column. However, the TNBS-induced inactivation could not be prevented in presence of substrates or competitive inhibitors, and the modified enzyme retained the ability to bind the affinity column. In a conclusion, an arginine residue involved in substrate binding and a lysine residue involved in catalysis were present at the active site of PALP. This study will facilitate to illustrate the role ALP plays in pearl formation and the mechanism involved.     

A Switch between One- and Two-electron Chemistry of the Human Flavoprotein Iodotyrosine Deiodinase Is Controlled by Substrate

Reductive dehalogenation is not typical of aerobic organisms but plays a significant role in iodide homeostasis and thyroid activity. The flavoprotein iodotyrosine deiodinase (IYD) is responsible for iodide salvage by reductive deiodination of the iodotyrosine derivatives formed as byproducts of thyroid hormone biosynthesis. Heterologous expression of the human enzyme lacking its N-terminal membrane anchor has allowed for physical and biochemical studies to identify the role of substrate in controlling the active site geometry and flavin chemistry. Crystal structures of human IYD and its complex with 3-iodo-l-tyrosine illustrate the ability of the substrate to provide multiple interactions with the isoalloxazine system of FMN that are usually provided by protein side chains. Ligand binding acts to template the active site geometry and significantly stabilize the one-electron-reduced semiquinone form of FMN. The neutral form of this semiquinone is observed during reductive titration of IYD in the presence of the substrate analog 3-fluoro-l-tyrosine. In the absence of an active site ligand, only the oxidized and two-electron-reduced forms of FMN are detected. The pH dependence of IYD binding and turnover also supports the importance of direct coordination between substrate and FMN for productive catalysis.     

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     

Suicide inactivation of tyrosinase in its action on tetrahydropterines

Tetrahydrobiopterin (BH(4)), methyl-tetrahydropterin (MBH(4)) and dimethyl-tetrahydropterin (DMBH(4)) are oxidized by tyrosinase in a process during which the suicide inactivation of tyrosinase may occur. From the kinetic study of this process, [Formula: see text] (apparent maximum constant for the suicide inactivation), [Formula: see text] (Michaelis constant for the substrate) and r (number of turnovers that the enzyme makes before the inactivation) can be obtained. From the results obtained, it can be deduced that the velocity of the inactivation governed by ([Formula: see text]) and the potency of the same ([Formula: see text]) follow the order: BH(4) > MBH(4) > DMBH(4).     

A cysteine residue (cysteine-116) in the histidinol binding site of histidinol dehydrogenase

Salmonella typhimurium L-histidinol dehydrogenase (EC 1.1.1.23), a four-electron dehydrogenase, was inactivated by an active-site-directed modification reagent, 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl). The inactivation followed pseudo-first-order kinetics and was prevented by low concentrations of the substrate L-histidinol or by the competitive inhibitors histamine and imidazole. The observed rate saturation kinetics for inactivation suggest that NBD-Cl binds to the enzyme noncovalently before covalent inactivation occurs. The UV spectrum of the inactivated enzyme showed a peak at 420 nm, indicative of sulfhydryl modification. Stoichiometry experiments indicated that full inactivation was correlated with modification of 1.5 sulfhydryl groups per subunit of enzyme. By use of a substrate protection scheme, it was shown that 0.5 sulfhydryl per enzyme subunit was neither protected against NBD-Cl modification by L-histidinol nor essential for activity. Modification of the additional 1.0 sulfhydryl caused complete loss of enzyme activity and was prevented by L-histidinol. Pepsin digestion of NBD-modified enzyme was used to prepare labeled peptides under conditions that prevented migration of the NBD group. HPLC purification of the peptides was monitored at 420 nm, which is highly selective for NBD-labeled cysteine residues. By amino acid sequencing of the major peptides, it was shown that the reagent modified primarily Cys-116 and Cys-377 and that the presence of L-histidinol gave significant protection of Cys-116. The presence of a cysteine residue in the histidinol binding site is consistent with models in which formation and subsequent oxidation of a thiohemiacetal occurs as an intermediate step in the overall reaction.     

FAD is a preferred substrate and an inhibitor of Escherichia coli general NAD(P)H:flavin oxidoreductase

Escherichia coli general NAD(P)H:flavin oxidoreductase (Fre) does not have a bound flavin cofactor; its flavin substrates (riboflavin, FMN, and FAD) are believed to bind to it mainly through the isoalloxazine ring. This interaction was real for riboflavin and FMN, but not for FAD, which bound to Fre much tighter than FMN or riboflavin. Computer simulations of Fre.FAD and Fre.FMN complexes showed that FAD adopted an unusual bent conformation, allowing its ribityl side chain and ADP moiety to form an additional 3.28 H-bonds on average with amino acid residues located in the loop connecting Fbeta5 and Falpha1 of the flavin-binding domain and at the proposed NAD(P)H-binding site. Experimental data supported the overlapping binding sites of FAD and NAD(P)H. AMP, a known competitive inhibitor with respect to NAD(P)H, decreased the affinity of Fre for FAD. FAD behaved as a mixed-type inhibitor with respect to NADPH. The overlapped binding offers a plausible explanation for the large K(m) values of Fre for NADH and NADPH when FAD is the electron acceptor. Although Fre reduces FMN faster than it reduces FAD, it preferentially reduces FAD when both FMN and FAD are present. Our data suggest that FAD is a preferred substrate and an inhibitor, suppressing the activities of Fre at low NADH concentrations.     

Covalent modification of Escherichia coli ADPglucose synthetase with 8-azido substrate analogs

Two photoaffinity labeling agents, 8-azido-ATP and 8-azido-ADPglucose, are substrate site specific probes of the Escherichia coli ADPglucose synthetase. In the presence of light (254 nm), the analogs specifically and covalently modify the enzyme with concomitant loss of catalytic activity. The substrate ADPglucose completely protects the enzyme from covalent modification by these 8-azido analogs. ATP, another substrate, also provides nearly 100% protection from 8-azido-ATP inactivation but is less efficient in protection of inactivation by 8-azido-ADPglucose. In the absence of light, however, ADPglucose synthetase can utilize either 8-azido-ATP or 8-azido-ADPglucose as substrates.     

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.