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

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

Photochemical inactivation of human placental estradiol 17 beta-dehydrogenase in the presence of 2,3-butanedione

Estradiol 17 beta-dehydrogenase of human placenta was rapidly inactivated by 2,3-butanedione under u.v. light, and no protection against the inactivation was observed in the presence of sodium azide. Under ordinary laboratory illumination, the inactivation was biphasically progressed in time-dependent and concentration-dependent manners, while a partial protection from the inactivation was indicated by sodium azide. These results suggest that the inactivation mechanism of the dehydrogenase by 2,3-butanedione under laboratory illumination is different from that under u.v. light. Therefore, the inactivation under laboratory illumination proceeded by a reaction with excited singlet molecular oxygen (1 delta g or 1 sigma +g states), and that under u.v. light was caused by a reaction of substrate with triplet sensitizer. In the presence of NADP+, the inactivation of the enzyme by 2,3-butanedione was markedly reduced. The maximum protection by NADP+ was about 80% of the initial enzyme activity. Amino acid analysis of the enzyme treated with 2,3-butanedione under laboratory illumination showed that the modified enzyme contained considerably less of the following amino acids than the native enzyme: histidine, arginine, threonine, methionine, tyrosine and leucine. In addition, other dicarbonyl reagents, 1,4-dibromo-2,3-butanedione, 1-phenyl-1,2-propanedione, phenylglyoxal, 16-oxoestrone, 1,2-cyclohexanedione, 2,4-pentanedione and glyoxal were found to decrease the dehydrogenase activity in various degree.     

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.     

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.     

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.     

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.     

Steady-state kinetics and the inactivation by 2,3-butanedione of the energy-independent transhydrogenase of Escherichia coli cell membranes.

Kinetic measurements indicate that the energy-independent transhydrogenation of 3-acetylpyridine-NAD+ by NADPH in membranes of Escherichia coli follows a rapid equilibrium random bireactant mechanism. Each substrate, although reacting preferentially with its own binding site, is able to interact with the binding site of the other substrate to cause inhibition of enzyme activity. 5'-AMP (and ADP) and 2'-AMP interact with the NAD+- and NADP+-binding sites, respectively. Phenylglyoxal and 2,3-butanedione in borate buffer inhibit transhydrogenase activity presumably by reacting with arginyl residues. Protection against inhibition by 2,3-butanedione is afforded by NADP+, NAD+, and high concentrations of NADPH and NADH. Low concentrations of NADPH and NADH increase the rate of inhibition by 2,3-butanedione. Similar effects are observed for the inactivation of the transhydrogenase by tryptic digestion in the presence of these coenzymes. It is concluded that there are at least two conformations of the active site of the transhydrogenase which differ in the extent to which arginyl residues are accessible to exogenous agents such as trypsin and 2,3-butanedione. One conformation is induced by low concentrations of NADH and NADPH. Under these conditions the coenzymes could be reacting at the active site or at an allosteric site. The stimulation of transhydrogenase activity by low concentrations of the NADH is consistent with the latter possibility.     

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.     

Modification of arginine residues in calf intestinal alkaline phosphatase

Calf intestinal alkaline phosphatase is inactivated by 2,3-butanedione and phenylglyoxal. The reaction with either reagent results in a biphasic loss of enzymatic activity. Inactivation by 2,3-butanedione in borate buffer can be reversed after gel-filtration in Tris buffer but no enzyme reactivation is observed after phenylglyoxal treatment. Phosphate, ATP and NADH protect the enzyme from both compounds while no protection is displayed by L-phenylalanine. The selective chemical modification indicates that two differently reacting types of arginines are present in the active site domains of the dimeric enzyme.     

Breath gas metabolites and bacterial metagenomes from cystic fibrosis airways indicate active pH neutral 2,3-butanedione fermentation

The airways of cystic fibrosis (CF) patients are chronically colonized by patient-specific polymicrobial communities. The conditions and nutrients available in CF lungs affect the physiology and composition of the colonizing microbes. Recent work in bioreactors has shown that the fermentation product 2,3-butanediol mediates cross-feeding between some fermenting bacteria and Pseudomonas aeruginosa, and that this mechanism increases bacterial current production. To examine bacterial fermentation in the respiratory tract, breath gas metabolites were measured and several metagenomes were sequenced from CF and non-CF volunteers. 2,3-butanedione was produced in nearly all respiratory tracts. Elevated levels in one patient decreased during antibiotic treatment, and breath concentrations varied between CF patients at the same time point. Some patients had high enough levels of 2,3-butanedione to irreversibly damage lung tissue. Antibiotic therapy likely dictates the activities of 2,3-butanedione-producing microbes, which suggests a need for further study with larger sample size. Sputum microbiomes were dominated by P. aeruginosa, Streptococcus spp. and Rothia mucilaginosa, and revealed the potential for 2,3-butanedione biosynthesis. Genes encoding 2,3-butanedione biosynthesis were disproportionately abundant in Streptococcus spp, whereas genes for consumption of butanedione pathway products were encoded by P. aeruginosa and R. mucilaginosa. We propose a model where low oxygen conditions in CF lung lead to fermentation and a decrease in pH, triggering 2,3-butanedione fermentation to avoid lethal acidification. We hypothesize that this may also increase phenazine production by P. aeruginosa, increasing reactive oxygen species and providing additional electron acceptors to CF microbes.     

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.     

The role of various amino acid residues in the functioning of NADP-specific isocitrate dehydrogenase from bovine adrenal cortex cytoplasm

The modification of SH-groups in the native isocitrate dehydrogenase accessible to 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) is accompanied by the enzyme inactivation. Isocitrate rather than NADP and MnCl2 protects two SH-groups of the enzyme from modification by DTNB and attendant inactivation. The isocitrate dehydrogenase inactivation by DTNB obeys pseudofirst-order reaction kinetics. The number of DTNB-titrated sulphydryl groups does not change after the isocitrate dehydrogenase denaturation by sodium dodecyl sulphate. In the presence of manganese ions isocitrate and to a lesser extent NADP protect isocitrate dehydrogenase from the inactivation induced by 2,3-butanedione, a specific modifier of arginine residues. It has also been shown that the methylene blue-sensitized photoinactivation of the enzyme associated with the photooxidation of histidine residues decreases in the presence of NADP. These data provide evidence for an essential role of the SH-groups, arginine residues and, probably, histidine in the functioning of NADP-dependent isocitrate dehydrogenase from adrenal cortex.     

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.     

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

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

Modification of an essential arginine residue associated with the plasma membrane ATPase of red beet (Beta vulgaris L.) storage tissue

The dicarbonyl compounds, phenylgloxyl and 2,3-butanedione were used to demonstrate the presence of an essential arginine residue in the mechanism of the red beet (Beta vulgaris L.) plasma membrane ATPase. Treatment of the red beet ATPase with either of these reagents resulted in an inhibition of ATP hydrolytic activity protectable by the inclusion of either ATP or ADP during inhibitor incubation. Ligands of the ATP hydrolytic reaction also protected against phenylglyoxyl inhibition and affected the ability of ADP to protect against inhibition by this reagent. Kinetic analysis of 2,3-butanedione and phenylglyoxyl inhibition suggested the presence of a single arginine residue susceptible to attack by these reagents. As similar results with these arginine modification reagents were found for both the plasma membrane-associated and solubilized forms of the ATPase, it is apparent that the function of this arginyl moiety is not affected by detergent treatment and removal of the enzyme from the membrane.     

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

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

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.     

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

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

Presence of an essential arginyl residue in D-β-hydroxybutyrate dehydrogenase from mitochondrial inner membrane

D-β-hydroxybutyrate dehydrogenase, a lipid requiring enzyme, is rapidly and completely inactivated by phenylglyoxal, 2,3-butanedione and 1,2-cyclohexanedione. Inactivation, which occurs at the millimolar range, depends on the nature of buffer, borate ions are required to get enzyme inactivation by 2,3-butanedione. Most of the inactivation follows a pseudo first order kinetics, the stoichiometry being of one to one. Presence of NAD⁺ or methylmalonate (a substrate-like compound) prior addition of inhibitor does not affect inactivation, while methylmalonate in presence of NAD⁺ strongly protects against inactivation. Chemical modification of the enzyme does not affect K_D of NAD while K_M of β-hydroxybutyrate and Ki of methylmalonate (protecting agent) increase. In view of the high specificity of these inhibitors for arginyl residues of proteins, these results are in favour of the presence of at least one arginyl residue essential for enzyme activity and located in, or near the substrate binding site.