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.     

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.     

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.     

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 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 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.     

Phosphorylation and dephosphorylation reactions of the red beet plasma membrane ATPase studied in the transient state

The reaction mechanism of the solubilized red beet (Beta vulgaris L.) plasma membrane ATPase was studied with a rapid quenching apparatus. Using a dual-labeled substrate ([γ-³²P]ATP and [5′,8-³H]ATP), the presteady-state time course of phosphoenzyme formation, phosphate liberation and ADP liberation was examined. The time course for both phosphoenzyme formation and ADP liberation showed a rapid, initial rise while the timecourse for phosphate liberation showed an initial lag. This indicated that ADP was released with formation of the phosphoenzyme while phosphate was released with phosphoenzyme breakdown. Phosphoenzyme formation was Mg²⁺-dependent and preincubation of the enzyme with free ATP followed by the addition of Mg²⁺ increased the rate of phosphoenzyme formation 2.3-fold. This implied that phosphoenzyme formation could result from a slow reaction of ATP binding followed by a more rapid reaction of phosphate group transfer. Phosphoenzyme formation was accelerated as the pH was decreased, and the relationship between pH and the apparent first-order rate constants for phosphoenzyme formation suggested the role of a histidyl residue in this process. Transient kinetics of phosphoenzyme breakdown confirmed the presence of two phosphoenzyme forms, and the discharge of the ADP-sensitive form by ADP correlated with ATP synthesis. Potassium chloride increased the rate of phosphoenzyme turnover and shifted the steady-state distribution of phosphoenzyme forms. From these results, a minimal catalytic mechanism is proposed for the red beet plasma membrane ATPase, and rate constants for several reaction steps are estimated.     

Modification of the Red Beet Plasma Membrane H-ATPase by Diethylpyrocarbonate

Incubation of the red beet (Beta vulgaris L.) plasma membrane H⁺-ATPase with micromolar concentrations of diethylpyrocarbonate (DEPC) resulted in inhibition of both ATP hydrolytic and proton pumping activity. Enzyme activity was restored when DEPC-modified protein was incubated with hydroxylamine, suggesting specific modification of histidine residues. Kinetic analyses of DEPC inhibition performed on both membrane-bound and solubilized enzyme preparations suggested the presence of at least one essential histidine moiety per active site. Inclusion of either ATP (substrate) or ADP (product and competitive inhibitor) in the modification medium reduced the amount of inhibition observed in the presence of DEPC. However, protection was not entirely effective in returning activity to noninhibited control values. These results suggest that the modified histidine does not reside directly in the ATP binding region of the enzyme, but is more likely involved in enzyme regulation through subtle conformational effects.     

Change in Target Molecular Size of the Red Beet Plasma Membrane ATPase during Solubilization and Reconstitution

The plasma membrane ATPase from red beet (Beta vulgaris L.) storage tissue associated with either native plasma membrane vesicles, a detergent-solubilized enzyme preparation or reconstituted liposomes was subjected to radiation inactivation analysis to determine if changes in target molecular size occurred with modification of its amphipathic environment. For each preparation of the enzyme, the decline in ATP hydrolytic activity with increasing dose of γ-ray radiation demonstrated a simple exponential profile indicating the presence of a single target size. Analysis of the radiation inactivation profiles for the plasma membrane associated, solubilized, and reconstituted enzyme revealed target molecular sizes of 225 kilodaltons (kD), 129 kD, and 218 kD, respectively. These results suggest that the plasma membrane associated and reconstituted ATPase preparations consist of enzyme present as a dimer of 100 kD subunits while the solubilized enzyme is present in the monomeric form. These results also indicate that the 100 kD catalytic subunit most likely represents the minimal unit of ATP hydrolytic activity.     

H-ATPase Activity from Storage Tissue of Beta vulgaris: III. Modulation of ATPase Activity by Reaction Substrates and Products

Two distinct membrane fractions containing H⁺-ATPase activity were prepared from red beet. One fraction contained a H⁺-ATPase activity that was inhibited by NO₃⁻ while the other contained a H⁺-ATPase inhibited by vanadate. We have previously proposed that these H⁺-ATPases are associated with tonoplast (NO₃⁻-sensitive) and plasma membrane (vanadate-sensitive), respectively. Both ATPase were examined to determine to what extent their activity was influenced by variations in the concentration of ATPase substrates and products. The substrate for both ATPase was MgATP²⁻, and Mg²⁺ concentrations in excess of ATP had only a slight inhibitory effect on either ATPase. Both ATPases were inhibited by free ATP (i.e. ATP concentrations in excess of Mg²⁺) and ADP but not by AMP. The plasma membrane ATPase was more sensitive than the tonoplast ATPase to free ATP and the tonoplast ATPase was more sensitive than the plasma membrane ATPase to ADP.

Inhibition of both ATPases by free ATP was complex. Inhibition of the plasma membrane ATPase by ADP was competitive whereas the tonoplast ATPase demonstrated a sigmoidal dependence on MgATP²⁻ in the presence of ADP. Inorganic phosphate moderately inhibited both ATPases in a noncompetitive manner.

Calcium inhibited the plasma membrane but not the tonoplast ATPase, apparently by a direct interaction with the ATPase rather than by disrupting the MgATP²⁻ complex.

The sensitivity of both ATPases to ADP suggests that under conditions of restricted energy supply H⁺-ATPase activity may be reduced by increases in ADP levels rather than by decreases in ATP levels per se. The sensitivity of both ATPases to ADP and free ATP suggests that modulation of cytoplasmic Mg²⁺ could modulate ATPase activity at both the tonoplast and plasma membrane.

     

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.     

Studies on the active site of the Neurospora crassa plasma membrane H+-ATPase with periodate-oxidized nucleotides

The Neurospora crassa plasma membrane H+-ATPase is inactivated by the periodate-oxidized nucleotides, oATP, oADP, and oAMP, with oAMP the most effective. Inhibition of the ATPase is essentially irreversible, because Sephadex G-50 column chromatography of the oAMP-treated ATPase does not result in a reversal of the inhibition. Inhibition of the ATPase by oAMP is protected against by the H+-ATPase substrate ATP, the product ADP, and the competitive inhibitors TNP (2',3'-O-(2,4,6-trinitrocyclohexadienylidine)-ATP and TNP-ADP, suggesting that oAMP inhibition occurs at the nucleotide binding site of the enzyme. The rate of inactivation of the ATPase by oAMP is only slightly affected by EDTA, indicating that the oAMP interaction with the nucleotide binding site of the H+-ATPase occurs in the absence of a divalent cation. The protection against oAMP inhibition by ADP is likewise unaffected by EDTA. The inhibition of the ATPase by oAMP is absolutely dependent on the presence of acidic phospholipids or acidic lysophospholipids known to be required for H+-ATPase activity, suggesting that these lipids either aid in the formation of the nucleotide binding site or render it accessible. Incubation of the ATPase with Mg2+ plus vanadate, which locks the enzyme in a conformation resembling the transition state of the enzyme dephosphorylation reaction, completely protects against inhibition by oAMP, suggesting that in this transition state conformation the nucleotide site either does not exist, or is inaccessible to oAMP. Labeling studies with [14C] oAMP indicate that the incorporation of 1 mol of oAMP is sufficient to cause complete inactivation of the ATPase.     

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.     

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.     

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.     

Reconstitution and rapid partial purification of the red beet plasma membrane ATPase

Red beet ( Beta vulgaris L., cv. Detroit Dark Red) plasma membrane ATPase solubilized from a deoxycholate-extracted plasma membrane fraction with Zwittergent 3–14 was reconstituted into liposomes. Detergent removal and reconstitution was carried out by column chromatography on Sephadex G-200 followed by centrifugation at 100 000 g for I h. Prior to reconstitution, optimal activity in the solubilized preparation was observed when dormant red beet tissue was used in the extraction/solubilization procedure. Following reconstitution into liposomes, ATP-dependent proton transport could be demonstrated by measuring the quenching of acridine orange fluorescence. Proton transport and ATPase activity in the reconstituted enzyme preparation were inhibited by orthovandate but stimulated by KNO₃. This stimulation most likely results from a reduction in the membrane potential generated during electrogenic proton transport by the reconstituted ATPase. The ATPase activity of the reconstituted ATPase was further characterized and found to have a pH optimum of 6.5 in the presence of both Mg²⁺ and K⁺. The activity was specific for ATP, insensitive to ouabain and azide but inhibited by N;N-dicyclohexylcarbodiimide and diethylstilbestrol. Stimulation of ATP hydrolytic activity occurred in the sequence: K⁺ Rb⁺ Na⁺ Cs⁺ Li⁺ and the kinetics of K⁺ stimulation of ATPase activity followed non-Michaelis-Menten kinetics as observed for both the membrane-bound and solubilized forms of the enzyme. Reconstitution of the plasma membrane ATPase from red beet allowed a substantial purification of the enzyme and resulted in the enrichment of a 100 kDa polypeptide representing the ATPase catalytic subunit.     

Role of the Plasma Membrane H+-ATPase in K+ Transport

The role of the plant plasma membrane H+-ATPase in K+ uptake was examined using red beet (Beta vulgaris L.) plasma membrane vesicles and a partially purified preparation of the red beet plasma membrane H+-ATPase reconstituted in proteoliposomes and planar bilayers. For plasma membrane vesicles, ATP-dependent K+ efflux was only partially inhibited by 100 [mu]M vanadate or 10 [mu]M carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. However, full inhibition of ATP-dependent K+ efflux by these reagents occurred when the red beet plasma membrane H+-ATPase was partially purified and reconstituted in proteoliposomes. When reconstituted in a planar bilayer membrane, the current/voltage relationship for the plasma membrane H+-ATPase showed little effect of K+ gradients imposed across the bilayer membrane. When taken together, the results of this study demonstrate that the plant plasma membrane H+-ATPase does not mediate direct K+ transport chemically linked to ATP hydrolysis. Rather, this enzyme provides a driving force for cellular K+ uptake by secondary mechanisms, such as K+ channels or H+/K+ symporters. Although the presence of a small, protonophore-insensitive component of ATP-dependent K+ transport in a plasma membrane fraction might be mediated by an ATP-activated K+ channel, the possibility of direct K+ transport by other ATPases (i.e. K+-ATPases) associated with either the plasma membrane or other cellular membranes cannot be ruled out.     

Effects of vanadate on the plasma membrane ATPase of red beet and corn

The effect of vanadate on the plant plasma membrane ATPase were investigated in plasma membrane fractions derived from corn roots (Zea mays L.) and red beets (Beta vulgaris L.). The K_i for vanadate inhibition of the plasma membrane ATPase from corn roots and red beets was between 6 and 15 micromolar vanadate. In both membrane fractions, 80% to 90% of the total ATPase was inhibited at vanadate concentrations below 100 micromolar. Vanadate inhibition was optimal at pH 6.5, enhanced by the presence of K⁺, and was partially reversed by 1 millimolar EDTA. The Mg:ATP kinetics for the plasma membrane ATPase were hyperbolic in both the absence and presence of vanadate. Vanadate decreased both the K_m and V_max of the red beet plasma membrane ATPase, indicating that vanadate inhibits the ATPase uncompetitively. These results indicate many similarities with respect to vanadate inhibition between the plant plasma membrane ATPase and other major iontranslocating ATPases from fungal and animal cells. The high sensitivity to vanadate reported here, however, differs from other reports of vanadate inhibition of the plant plasma membrane ATPase from corn, beets, and in some instances oats.     

Inhibition of chloroplast coupling factor by naphthylglyoxal

The trypsin-activated Ca²⁺ -ATPase of spinach chloroplast membranes was completely inhibited by treatment with naphthylglyoxal, a fluorescent compound that should bind covalently to arginine residues. The inhibition followed apparent first-order kinetics. The apparent order of reaction with respect to inhibitor concentration gave values near unity, suggesting that inactivation is a consequence of modifying one arginine residue per active site. Partial protection against naphthylglyoxal was afforded by ADP and ATP, with either less or no protection by other nucleotide bases. At inhibition levels less than complete, the K_m for ATP was not affected but the V_max of the enzyme was diminished. The light-dependent exchange of tightly bound nucleotides on the membrane-bound enzyme was not inhibited by naphthylglyoxal treatment, indicating significant retention of the conformational response of the enzyme to the membrane high-energy state. Using [³H]naphthylglyoxal, the extent of inhibition was a linear function of the amount of naphthylglyoxal bound up to 60% inhibition. The curves extrapolated to 2 mol naphthylglyoxal bound, associated with complete inhibition of ATPase. The radioactive naphthylglyoxal was distributed equally between α- and β-subunits.     

Chemical modification of sarcoplasmic reticulum. Stimulation of Ca2+ release.

Pretreatment of sarcoplasmic membranes with acetic or maleic anhydrides, which interact principally with amino groups, resulted in an inhibition of Ca2+ accumulation and ATPase activity. The presence of ATP, ADP or adenosine 5'-[beta, gamma-imido]triphosphate in the modification medium selectively protected against the inactivation of ATPase activity by the anhydride but did not protect against the inhibition of Ca2+ accumulation. Acetic anhydride modification in the presence of ATP appeared to increase specifically the permeability of the sarcoplasmic reticulum membrane to Ca2+ but not to sucrose, Tris, Na+ or Pi. The chemical modification stimulated a rapid release of Ca2+ from sarcoplasmic reticulum vesicles passively or actively loaded with calcium, from liposomes reconstituted with the partially purified ATPase fraction but not from those reconstituted with the purified ATPase. The inactivation of Ca2+ accumulation by acetic anhydride (in the presence of ATP) was rapid and strongly pH-dependent with an estimated pK value above 8.3 for the reactive group(s). The negatively charged reagents pyridoxal 5-phosphate and trinitrobenzene-sulphonate, which also interact with amino groups, did not stimulate Ca2+ release. Since these reagents do not penetrate the sarcoplasmic reticulum membranes, it is proposed that Ca2+ release is promoted by modification of internally located, positively charged amino group(s).