Inactivation of ATP-dependent deoxyribonuclease of Micrococcus luteus by 2,3-butanedione

ATP-dependent deoxyribonuclease from Micrococcus luteus was purified to near homogeneity by a procedure involving gentle cell lysis, ammonium sulfate fractionation, TEAE-cellulose chromatography, Sephadex G-150 gel filtration and DNA-cellulose chromatography. Treatment of the enzyme with 2,3-butanedione, which binds specifically to arginyl residues, caused rapid loss of enzyme activities and the effect was enhanced by borate ion. The reaction obeyed first order kinetics with respect to the butanedione concentration, indicating that at least one functional arginyl residue is involved in the inactivation reaction. The enzyme was protected from inactivation by the presence of a low concentration of ATP, but not of ADP, AMP or adenosine. These results indicate that ATP-dependent deoxyribonuclease of Micrococcus luteus has functional arginyl residue(s) at an ATP-binding site.     

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.     

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.     

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.     

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.     

Identification of substrate and effector binding sites of phosphoenolpyruvate carboxylase from Crassula argentea. A possible role of phosphoenolpyruvate as substrate and activator

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) purified from leaves of the crassulacean acid metabolism plant (Crassula argentea) was chemically modified by the specific arginyl reagent 2,3-butanedione. Modification resulted in enzyme inactivation which followed pseudo first-order kinetics. Participation of arginyl residues involved in the binding of or response to both phosphoenolpyruvate and malate, respectively, was established. Inactivation and protection studies suggest the presence of three sites involved in the binding of the substrate, phosphoenolpyruvate, the activator, glucose 6-phosphate, and the inhibitor, malate. Studies using both fluorescence measurements of binding and steady-state kinetic methods indicate that phosphoenolpyruvate can bind both to the active site and to the activator site. Evidence for stimulation of the activity of phosphoenolpyruvate carboxylase upon the binding of substrate to the activation site was provided by kinetic studies using AMP, previously shown to be a specific ligand for the activation site.     

Arginine modification with butanedione inhibits the potassium ATPase of Streptococcus faecalis

The K+-ATPase of Streptococcus faecalis is inhibited by incubation with the arginine-modifying reagent 2,3-butanedione. The inactivation proceeds by pseudo - first order kinetics and a double-logarithmic plot of the pseudo - first order rate constants versus reagent concentrations yields a reaction order of 1.14 with respect to butanedione. Partially inactivated ATPase exhibits a decreased maximal velocity but the same affinity for ATP, as compared to the native enzyme. Butanedione modification is inhibited by adenine nucleotides. These results indicate the involvement of most likely one crucial arginyl residue in adenine nucleotide binding by the ATPase.     

Sulfhydryl groups of an extramitochondrial acetyl-CoA hydrolase from rat liver

An extramitochondrial acetyl-CoA hydrolase (EC 3.1.2.1) purified from rat liver was inactivated by heavy metal cations (Hg2+, Cu2+, Cd2+ and Zn2+), which are known to be highly reactive with sulfhydryl groups. Their order of potency for enzyme inactivation was Hg2+ greater than Cu2+ greater than Cd2+ greater than Zn2+. This enzyme was also inactivated by various sulfhydryl-blocking reagents such as p-hydroxymercuribenzoate (PHMB), N-ethylmaleimide (NEM), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and iodoacetate (IAA). DL-Dithiothreitol (DTT) reversed the inactivation of this enzyme by DTNB markedly, and that by PHMB slightly, but did not reverse the inactivations by NEM, DTNB and IAA. Benzoyl-CoA (a substrate-like competitive inhibitor) and ATP (an activator) greatly protected acetyl-CoA hydrolase from inactivation by PHMB, NEM, DTNB and IAA. These results suggest that the essential sulfhydryl groups are on or near the substrate binding site and nucleotide binding site. The enzyme contained about four sulfhydryl groups per mol of monomer, as estimated with DTNB. When the enzyme was denatured by 4 M guanidine-HCl, about seven sulfhydryl groups per mol of monomer reacted with DTNB. Two of the four sulfhydryl groups of the subunit of the native enzyme reacted with DTNB first without any significant inactivation of the enzyme, but its subsequent reaction with the other two sulfhydryl groups seemed to be involved in the inactivation process.     

Studies on the effects of coenzyme A-SH: acetyl coenzyme A, nicotinamide adenine dinucleotide: reduced nicotinamide adenine dinucleotide, and adenosine diphosphate: adenosine triphosphate ratios on the interconversion of active and inactive pyruvate dehydrogenase in isolated rat heart mitochondria.

The content of coenzyme A-SH (CoASH) and acetyl-CoA of suspensions of rat heart mitochondria was stabilized by the addition of DL-carnitine and acetyl-DL-carnitine, in the presence of the respiratory inhibitor rotenone. The mitochondrial content of NAD+ and NADH was similarly stabilized by the addition of acetoacetate and DL-3-hydroxybutyrate, and the content of ADP and ATP was imposed by the addition of these nucleotides to the mitochondrial suspension, in the presence of uncoupling agent and oligomycin, to inhibit ATPase. Under these conditions, mitochondrial CoASH/acetyl-CoA, NAD+/ NADH, and ADP/ATP ratios could be varied independently, and the effect on the interconversion of active and inactive pyruvate dehydrogenase could be studied. Decreases in both CoASH/acetyl-CoA and NAD+/NADH ratios were shown to be inhibitory to the steady state activity of pyruvate dehydrogenase, and this effect is described at three different ADP/ATP ratios and different concentrations of added MgCl2. A new steady state level of activity was achieved within 10 min of a change in either CoASH/acetyl-CoA or NAD+/NADH ratio; the rate of inactivation was much higher than the rate of reactivation under these conditions. Effects of CoASH/acetyl-CoA and NAD+/NADH may be additive but are still quantitatively lesser than the changes in activity of pyruvate dehydrogenase induced by changes in ADP/ATP ratio. The variation in activity of pyruvate dehydrogenase with ADP/ATP ratio is described in the absence of changes in the other two ratios, conditions which were not met in earlier studies which employed the oxidation of different substrates to generate changes in all three ratios.     

Regulation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria.

The mechanism by which fatty acid addition leads to the inactivation of pyruvate dehydrogenase in intact rat liver mitochondria was investigated. In all cases the fatty acid octanoate was added to mitochondria oxidizing succinate. Addition of fatty acid caused an inactivation of pyruvate dehydrogenase in mitochondria incubated under State 3 conditions (glucose plus hexokinase), in uncoupled, oligomycin-treated mitochondria, and in rotenone-menadione-treated mitochondria, but not in uncoupled mitochondria or in mitochondria incubated under State 4 conditions. A number of metabolic conditions were found in which pyruvate dehydrogenase was inactivated concomitant with an elevation in the ATP/ADP ratio. This is consistent with the inverse relationship between the ATP/ADP ratio and the pyruvate dehydrogenase activity proposed by various laboratories. However, in several other metabolic conditions pyruvate dehydrogenase was inactivated while the ATP/ADP ratio either was unchanged or even decreased. This observation implies that there are likely other regulatory factors involved in the fatty acid-mediated inactivation of pyruvate dehydrogenase. Incubation conditions in State 3 were found in which the ATP/ADP and the acetyl-CoA/CoASH ratios remained constant and the pyruvate dehydrogenase activity was correlated inversely with the NADH/NAD+ ratio. Other State 3 conditions were found in which the ATP/ADP and the NADH/NAD+ ratios remained constant while the pyruvate dehydrogenase activity was correlated inversely with the acetyl-CoA/CoASH ratio. Further evidence supporting these experiments with intact mitochondria was the observation that the pyruvate dehydrogenase kinase activity of a mitochondrial extract was stimulated strongly by acetyl-CoA and was inhibited by NAD+ and CoASH. In contrast to acetyl-CoA, octanoyl-CoA inhibited the kinase activity. These results indicate that the inactivation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria may be mediated through effects of the NADH/NAD+ ratio and the acetyl-CoA/CoASH ratio on the interconversion of the active and inactive forms of the enzyme complex catalyzed by pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase.     

Evidence for an essential arginine residue at the active site of Escherichia coli acetate kinase

Escherichia coli acetate kinase (ATP: acetate phosphotransferase, EC 2.7.2.1.) was inactivated in the presence of either 2,3-butanedione in borate buffer or phenylglyoxal in triethanolamine buffer. When incubated with 9.4 mM phenylglyoxal or 5.1 mM butanedione, the enzyme lost its activity with an apparent rate constant of inactivation of 0.079 min-1, respectively. The loss of enzymatic activity was concomitant with the loss of an arginine residue per active site. Phosphorylated substrates of acetate kinase, ATP, ADP and acetylphosphate as well as AMP markedly decreased the rate of inactivation by both phenylglyoxal and butanedione. Acetate neither provided any protection nor affected the protection rendered by the adenine nucleotides. However, it interfered with the protection afforded by acetylphosphate. These data suggest that an arginine residue is located at the active site of acetate kinase and is essential for its catalytic activity, probably as a binding site for the negatively charged phosphate group of the substrates.     

Characterization of an essential arginine residue in the plasma membrane H+-ATPase of Neurospora crassa

Treatment of the plasma membrane H+-ATPase of Neurospora crassa with the arginine-specific reagents phenylglyoxal or 2,3-butanedione at 30 degrees C, pH 7.0, leads to a marked inhibition of ATPase activity. MgATP, the physiological substrate of the enzyme, protects against inactivation. MgADP, a competitive inhibitor of ATPase activity with a measured Ki of 0.11 mM, also protects, yielding calculated KD values of 0.125 and 0.115 mM in the presence of phenylglyoxal and 2,3-butanedione, respectively. The excellent agreement between Ki and KD values makes it likely that MgADP exerts its protective effect by binding to the catalytic site of the enzyme. Loss of activity follows pseudo-first order kinetics with respect to phenylglyoxal and 2,3-butanedione concentration, and double log plots of pseudo-first order rate constants versus reagent concentration yield slopes of 0.999 (phenylglyoxal) and 0.885 (2,3-butanedione), suggesting that the modification of one reactive site/mol of H+-ATPase is sufficient for inactivation. This stoichiometry has been confirmed by direct measurements of the incorporation of [14C]phenylglyoxal. Taken together, the results support the notion that one arginine residue, either located at the catalytic site or shielded by a conformational change upon nucleotide binding, plays an essential role in Neurospora H+-ATPase activity.     

Properties of the Mg2+-induced low-affinity nucleotide binding site of (Na+ + K+)-activated ATPase

The Mg2+-induced low-affinity nucleotide binding by (Na+ + K+)-ATPase has been further investigated. Both heat treatment (50-65 degrees C) and treatment with N-ethylmaleimide reduce the binding capacity irreversibly without altering the Kd value. The rate constant of inactivation is about one-third of that for the high-affinity site and for the (Na+ + K+)-ATPase activity. Thermodynamic parameters (delta H degree and delta S degree) for the apparent affinity in the ATPase reaction (Km ATP) and for the true affinity in the binding of AdoPP[NH]P (Kd and Ki) differ greatly in sign and magnitude, indicating that one or more reaction steps following binding significantly contribute to the Km value, which thus is smaller than the Kd value. Ouabain does not affect the capacity of low-affinity nucleotide binding, but only increases the Kd value to an extent depending on the nucleotide used. GTP and CTP appear to be most sensitive, ATP and ADP intermediately sensitive and AdoPP[NH]P and AMP least sensitive to ouabain. Ouabain reduces the high-affinity nucleotide binding capacity without affecting the Kd value. The nucleotide specificity of the low-affinity binding site is the same for binding (competition with AdoPP[NH]P) and for the ATPase activity (competition with ATP): AdoPP[NH]P greater than ATP greater than ADP greater than AMP. The low-affinity nucleotide binding capacity is preserved in the ouabain-stabilized phosphorylated state, and the Kd value is not increased more than by ouabain alone. It is inferred that the low-affinity site is located on the enzyme, more specifically its alpha-subunit, and not on the surrounding phospholipids. It is situated outside the phosphorylation centre. The possible functional role of the low-affinity binding is discussed.     

Characterization of essential arginyl residues in sheep kidney (Na+ + K+) -ATPase.

Treatment of highly purified sheep kidney medulla (Na⁺ + K⁺)-ATPase with 2,3-butanedione results in a rapid inactivation of the enzyme. Contrary to a previous report using rabbit kidney enzyme (DePont et al., Biochim. Biophys. Acta (1977) 482, 213), the inactivation is biphasic under a variety of experimental conditions, with a rapid, initial inactivation which is followed by a slower loss of activity. The second, slower phase of the inhibition obeys pseudo-first order kinetics, with a second order rate constant for inhibition of 20 min^?1M^?1. ATP and ADP provide no protection in the initial phase of the inhibition, but protect the enzyme completely from the second phase of the inhibition. AMP, while less effective than ATP and ADP, provides a partial protection of the enzyme activity from inhibition by 2,3-butanedione. Inorganic phosphate provides partial protection in both phases of the inactivation. Adenosine alone is without effect, but adenosine plus inorganic phosphate provides a greater protection than phosphate alone. The results indicate that either (1) two or more active site residues or (2) a single arginine, experiencing different reactivities in two different active site conformations, are essential to (Na⁺ + K⁺)-ATPase activity.     

Evidence for an essential arginine residue at the active site of ATP citrate lyase from rat liver.

Rat liver ATP citrate lyase was inactivated by 2, 3-butanedione and phenylglyoxal. Phenylglyoxal caused the most rapid and complete inactivation of enzyme activity in 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid buffer, pH 8. Inactivation by both butanedione and phenylglyoxal was concentration-dependent and followed pseudo- first-order kinetics. Phenylglyoxal also decreased autophosphorylation (catalytic phosphate) of ATP citrate lyase. Inactivation by phenylglyoxal and butanedione was due to the modification of enzyme arginine residues: the modified enzyme failed to bind to CoA-agarose. The V declined as a function of inactivation, but the Km values were unaltered. The substrates, CoASH and CoASH plus citrate, protected the enzyme significantly against inactivation, but ATP provided little protection. Inactivation with excess reagent modified about eight arginine residues per monomer of enzyme. Citrate, CoASH and ATP protected two to three arginine residues from modification by phenylglyoxal. Analysis of the data by statistical methods suggested that the inactivation was due to modification of one essential arginine residue per monomer of lyase, which was modified 1.5 times more rapidly than were the other arginine residues. Our results suggest that this essential arginine residue is at the CoASH binding site.     

Arginyl and histidyl groups are essential for organic anion exchange in renal brush-border membrane vesicles

The effect of side chain modification on the organic anion exchanger in the renal brush-border membrane was examined to identify what amino acid residues constitute the substrate binding site. One histidyl-specific reagent, diethyl pyrocarbonate (DEPC), and 2 arginyl-specific reagents, phenylglyoxal and 2,3-butanedione, were tested for their effect on the specifically mediated transport of p-amino[3H]hippurate (PAH), a prototypic organic anion. The specifically mediated transport refers to the difference in the uptake of [3H]PAH in the absence and presence of a known competitive inhibitor, probenecid, and was examined in brush-border membrane vesicles isolated from the outer cortex of canine kidneys. The experiments were performed utilizing a rapid filtration assay. DEPC, phenylglyoxal, and 2,3-butanedione inactivated the specifically mediated PAH transport, i.e. probenecid inhibitable transport with IC50 values of 160, 710, and 1780 microM, respectively. The rates of PAH inactivation by DEPC and phenylglyoxal were suggestive of multiple pseudo first-order reaction kinetics and were consistent with a reaction mechanism whereby more than 1 arginyl or histidyl residue is inactivated. Furthermore, PAH (5 mM) did not affect the rate of phenylglyoxal inactivation. In contrast, PAH (5 mM) affected the rate of DEPC inactivation. The modification by DEPC was specific for histidyl residues since transport could be restored by treatment with hydroxylamine. The results demonstrate that histidyl and arginyl residues are essential for organic anion transport in brush-border membrane vesicles. We conclude that the histidyl residue constitutes the cationic binding site for the anionic substrate, whereas the arginyl residue(s) serves to guide the substrate to or away from the histidyl site.     

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.     

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.     

On the regulation of cold-labile cytosolic and of mitochondrial acetyl-CoA hydrolase in rat liver

The discovery of a cold-labile cytosolic acetyl-CoA hydrolase of high activity in rat liver by Prass et al. [(1980) J. Biol. Chem. 255, 5215-5223] has questioned the importance of mitochondrial acetyl-CoA hydrolase for the formation of free acetate [Grigat et al. (1979) Biochem. J. 177, 71-79] under physiological conditions. Therefore this problem has been reevaluated by comparing various properties of the two enzymes. Cold-labile cytosolic acetyl-CoA hydrolase bands with an apparent Mr of 68000 during SDS/polyacrylamide gel electrophoresis, while the native enzyme elutes in two peaks with apparent Mr of 136000 and 245000 during gel chromatography in the presence of 2 mM ATP. The mitochondrial enzyme elutes under the same conditions with an apparent Mr of 157000. Under conditions where the cold-labile enzyme binds strongly to DEAE-Bio-Gel and ATP-agarose, the mitochondrial enzyme remains unbound. The cold-labile enzyme can be activated 14-fold by ATP, half-maximal activation occurring already at 40 microM ATP. AdoPP[NH]P, AdoPP[CH2]P and GTP have a similar though weaker effect. ADP as well as GDP can completely inhibit the cold-labile enzyme with 50% inhibition occurring for both nucleotides at about 1.45 microM. The binding of ATP and ADP is competitive. Acetyl phosphate and pyrophosphate have no effect on the activity of the cold-labile enzyme. The mitochondrial acetyl-CoA hydrolase is not affected by these nucleotides. CoASH is a strong product inhibitor (approximately equal to 80% inhibition at 40 microM CoASH) of the cold-labile enzyme, but only a weak inhibitor of the mitochondrial enzyme. Under in vivo conditions the activity of the cold-labile cytosolic acetyl-CoA hydrolase can be no more than 7% of the activity calculated for mitochondrial acetyl-CoA hydrolase under the same conditions. Accordingly the mitochondrial enzyme seems to be mainly responsible for the formation of free acetate by the intact liver, especially in view of the fact that the substrate specificity of the mitochondrial enzyme is much higher (activity ratios acetyl-CoA/butyryl-CoA 4.99 and 1.16 for the mitochondrial and the cold-labile enzyme respectively). Alloxan diabetes neither increased the activity of the cold-labile enzyme nor that of the mitochondrial enzyme. No experimental support has been found yet for the hypothesis that the acetyl-CoA hydrolase activity of the cold-labile enzyme represents the side-activity of an acetyl-transferase.     

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

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