EFFECT OF PHENYL AND PHENOLIC ACIDS ON MEVALONATE-5-PHOSPHATE KINASE AND MEVALONATE-5-PYROPHOSPHATE DECARBOXYLASE OF THE RAT BRAIN

Abstract— Phenyl and phenolic acids are known to inhibit metabolism of mevalonate in rat brain. The site of inhibition has been found to be mevalonate-5-pyrophosphate decarboxylase. Phenolic acids also inhibited mevalonate-5-phosphate kinase on preincubation. The kinetics showed that p -coumaric acid and isoferulic acid were competing with substrates, mevalonate-5-phosphate or mevalonate-5-pyre phosphate, whereas others showed an uncompetitive type of inhibition. Chlorophenoxyisobutyrate, a hypocholesterolaemic drug, had no effect on these enzymes. An improved method for the synthesis of mevalonate-5-phosphate and mevalonate-5-pyrophosphate, labeled at carbon-1, is described.     

Studies on the diurnal rhythm of mevalonate metabolism by sterol and nonsterol pathways and of mevalonate-activating enzymes

Both in vivo and in vitro incorporation of mevalonic acid into nonsaponifiable lipids by 17-day-old chick liver and kidney did not show diurnal rhythm. Using 14CO2 production from MVA as an index of the shunt pathway not leading to sterols, we have demonstrated for the first time that there is no diurnal rhythm in this pathway. No significant differences were found in the specific activities of mevalonate kinase, mevalonate-5-phosphate kinase and mevalonate-5-pyrophosphate decarboxylase from chick liver and kidney throughout a period of 24 hr, using [1-14C]mevalonate as substrate. The absence of diurnal rhythm in the decarboxylase activity was corroborated by further experiments carried out using [2-14C]mevalonate-5-pyrophosphate as specific substrate of this enzyme.     

Purification and characterization of avian liver mevalonate-5-pyrophosphate decarboxylase

Mevalonate-5-pyrophosphate decarboxylase [ATP:5-diphosphomevalonate carboxy-lyase (dehydrating), EC 4.1.1.33] has been purified 5800 times from chicken liver and obtained in a stable and highly purified form. The protein is a dimer of molecular weight 85400 +/- 1941, and its subunits were not resolved by gel electrophoresis in denaturing conditions. The purified enzyme does not require the presence of SH-containing reagents for either activity or stability. The enzyme shows a high specificity for adenosine 5'-triphosphate (ATP) and requires for activity a divalent metal cation, Mg2+ being most effective. The optimum pH for the enzyme ranges from 4.0 to 6.5. Inhibitory effects for the enzyme activity were detected by citrate, phthalate, and phosphate. The isoelectric point, as determined by column chromatofocusing, is 4.8. The kinetics are hyperbolic for both substrates, showing a sequential mechanism; true Km values of 0.0141 mM and 0.504 mM have been obtained for mevalonate-5-pyrophosphate and ATP, respectively.     

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.     

An essential arginine residue at the binding site of pig kidney 3,4-dihydroxyphenylalanine decarboxylase

Pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase is inactivated by the arginine-specific reagent phenylglyoxal. Under these experimental conditions, the reaction follows pseudo-first-order kinetics with a second-order rate constant of 25 m-1 min-1. Holo- and apo-enzyme were inactivated at the same rate. However, inactivation seems to be related to modification of 1 and 2 arginyl residues per mol of holo- and apo-enzyme, respectively. Only one of these two residues was essential to decarboxylase activity of the enzyme. Phenylglyoxal-modified apo-Dopa decarboxylase retained the capacity to bind pyridoxal-P. Neither this reconstituted species nor the phenylglyoxal-modified holoenzyme were able to form Schiff base intermediates with aromatic amino acids in L and D forms. These data together with protection experiments suggest that the susceptible arginine residue in holoenzyme may somehow perturb the substrate binding site. However, unlike in other pyridoxal-P enzymes, this critical arginine in Dopa decarboxylase does not seem to behave as an anionic recognition site for the phosphate group of the coenzyme or the carboxy group of the substrate. It is speculated that this guanidyl group could function in hydrogen bonding of substrate side chain.     

Evidence of essential arginyl residues in rabbit muscle pyruvate kinase.

Rabbit muscle pyruvate kinase is inactivated by 2,3-butanedione in borate buffer. The inactivation follows pseudo-first-order kinetics with a calculated second-order rate constant of 4.6 m^?1 min^?1. The modification can be reversed with almost total recovery of activity by elimination of the butanedione and borate buffer, suggesting that only arginyl groups are modified; this result agrees with the loss of arginine detected by amino acid analysis of the modified enzyme. Using the kinetic data, it was estimated that the reaction of a single butanedione molecule per subunit of the enzyme is enough to completely inactivate the protein. The inactivation is partially prevented by phosphoenolpyruvate in the presence of K⁺ and Mg²⁺, but not by the competitive inhibitors lactate and bicarbonate. These findings point to an essential arginyl residue being located near the phosphate binding site of phosphoenolpyruvate.     

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.     

Role of mevalonate-5-pyrophosphate decarboxylase in the regulation of chick intestinal cholesterogenesis

The response to different dietary conditions of the enzymes responsible for the transformation of mevalonic acid to isopentenyl pyrophosphate has been studied for the first time in the small bowel of the chick to elucidate the role of these enzymes in the regulation of intestinal cholesterogenesis. Feeding a 2% cholesterol diet from hatching resulted in a small but significant inhibition of mevalonate-5-pyrophosphate decarboxylase, while mevalonate kinase and mevalonate-5-phosphate kinase remained unaltered. Similar results were obtained for the three enzymes when 13-day-old chicks fed a standard fat-free diet were switched to a 5% cholesterol diet. Starved chicks exhibited lower intestinal decarboxylase activity than chicks fed a standard diet, while refeeding resulted in levels of activity similar or slightly greater than controls. None of the enzymes effecting the conversion of mevalonate to isopentenyl pyrophosphate in the small intestine presented diurnal variations. Results obtained suggest that mevalonate-5-pyrophosphate decarboxylase may play a significant role in the regulation of cholesterol synthesis in the small intestine.     

Chemical modification of rabbit skeletal muscle phosphorylase kinase with phenylglyoxal

Nonactivated phosphorylase kinase from rabbit skeletal muscle is inactivated by treatment with phenylglyoxal. Under mild reaction conditions, a derivative that retains 10-15% of the pH 8.2 catalytic activity is obtained. The kinetics of inactivation profile, differential effects of modification on pH 6.8 and 8.2 catalytic activities, and the insensitiveness of the modified enzyme to activation by ADP reveal that the 10-15% of catalytic activity remaining is very likely due to intrinsic catalytic activity of the derivative rather than to the presence of unmodified enzyme molecules. The kinetic results also suggest that the inactivation is correlatable with the reaction of one molecule of the reagent with the enzyme without any prior binding of phenylglyoxal. The phenylglyoxal modification reduces the autophosphorylation rate of the kinase. Autophosphorylated phosphorylase kinase is inactivated by phenylglyoxal at a much slower rate than the inactivation of nonactivated kinase. Thus, phenylglyoxal modification influences the phosphorylation and vice versa. The modified enzyme can be reactivated by treatment with trypsin or by dissociation using chatropic salts. The activity of the phenylglyoxal-modified enzyme after trypsin digestion or dissociation with LiBr reaches the same level as that of the native enzyme digested with trypsin or treated with LiBr under identical conditions. The results suggest that the effect of modification is overcome by dissociation of the subunits of phosphorylase kinase and that the catalytic site is not modified under conditions when 85% of the pH 8.2 catalytic activity is lost. Among various nucleotides and metal ions tested, only ADP, with or without Mg2+, afforded effective protection against inactivation with phenylglyoxal. At pH 6.8, 1 mM ADP afforded complete protection against inactivation. Experiments with 14C-labeled phenylglyoxal revealed that ADP seemingly protects one residue from modification. This result is in agreement with the kinetic result that the inactivation seemingly is due to reaction of one molecule of the reagent with the enzyme. The results confirm the existence of a high-affinity ADP binding site on nonactivated phosphorylase kinase and suggest the involvement of a functional arginyl residue at or near the ADP binding site in the regulation of of pH 8.2 catalytic activity of the enzyme.     

The presence of functional arginine residues in phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) is completely inactivated by phenylglyoxal and 2,3-butanedione in borate buffer at pH 8.4, with pseudo-first-order kinetics and a second-order rate constant of 144 min-1 X M-1 and 21.6 min-1 X M-1, respectively. Phosphoenolpyruvate, ADP and Mn2+ (alone or in combination) protect the enzyme against inactivation, suggesting that the modification occurs at or near to the substrate-binding site. Almost complete restoration of activity was obtained when a sample of 2,3-butanedione-inactivated enzyme was freed of excess modifier and borate ions, suggesting that only arginyl groups are modified. The changes in the rate of inactivation in the presence of substrates and Mn2+ were used to determine the dissociation constants for enzyme-ligand complexes, and values of 23 +/- 3 microM, 168 +/- 44 microM and 244 +/- 54 microM were found for the dissociation constants for the enzyme-Mn2+, enzyme-ADP and enzyme-phosphoenolpyruvate complexes, respectively. Based on kinetic data, it is shown that 1 mol of reagent must combine per enzyme active unit in order to inactivate the enzyme. Complete inactivation of the carboxykinase can be correlated with the incorporation of 3-4 mol [7-14C]phenylglyoxal per mol of enzyme subunit. Assuming a stoichiometry of 1:1 between phenylglyoxal incorporation and arginine modification, our results suggest that the modification of only two of the three to four reactive arginine residues per phosphoenolpyruvate carboxykinase subunit is responsible for inactivation.     

Chemical modification of arginine residues of rat liver S-adenosylhomocysteinase

Rat liver S-adenosylhomocysteinase (EC 3.3.1.1) is inactivated by phenylglyoxal following pseudo-first order kinetics. The dependence of the apparent first order rate constant for inactivation on the phenylglyoxal concentration shows that the inactivation is second order in reagent. This fact together with the reversibility of inactivation upon removal of excess reagent and the lack of reaction at residues other than arginine as revealed by amino acid analysis and incorporation of phenylglyoxal into the protein indicate that the inactivation is due to the modification of arginine residue. The substrate adenosine largely but not completely protects the enzyme against inactivation. Although the modification of two arginine residues/subunit is required for complete inactivation, the relationship between loss of enzyme activity and the number of arginine residues modified, and the comparison of the numbers of phenylglyoxal incorporated into the enzyme in the presence and absence of adenosine indicate that one residue which reacts very rapidly with the reagent compared with the other is critical for activity. Although the phenylglyoxal treatment does not result in alteration of the molecular size of the enzyme or dissociation of the bound NAD+, the intrinsic protein fluorescence is largely lost upon modification. The equilibrium binding study shows that the modified enzyme apparently fails to bind adenosine.     

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.     

Mevalonate decarboxylation in lemon grass leaves

The activity of mevalonate-5-pyrophosphate (MVAPP) decarboxylase was assayed in the extracts of green leaves of lemon grass. The enzyme was found to be exclusively cytosolic, had a pH optimum of 6.0 and had a specific requirement for ATP; Mg²⁺ was required and Mn²⁺ could replace it partially. The phenolic compounds, p-coumaric acid, protocatechuic acid, ferulic acid and phloroglucinol carboxylic acid inhibited the activity.     

Modification of arginine residues in pyruvate kinase (author's transl)

Pyruvate kinase from pig heart is inactivated by the specific arginyl reagent phenylglyoxal. The loss of activity is caused by the reaction of a single molecule of phenylglyoxal per subunit of enzyme. During inactivation 3 - 6 arginyl residues are modified dependent on the concentration of phenylglyoxal used for modification. The solubility of the protein is reduced by the modification. ATP or phosphoenolpyruvate protect against inactivation. A single arginine is less subject to chemical modification in their presence. Therefore we assume that an arginine is essential at the substrate binding site. The activating ion K does not affectinactivation, where as Mg2 diminishes inactivation. Pyruvate kinase from rabbit muscle is modified by phenylglyoxal in a similar manner.     

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.     

Effect of arginine modifying reagents on pigeon liver fatty acid synthetase: evidence for the presence of essential arginine residues at the beta-ketoacyl reductase and enoyl-CoA reductase domain

Pigeon liver fatty acid synthetase was inactivated by arginine modifying reagent, phenylglyoxal and 2,3-butanedione. The inactivation of overall fatty acid synthetase was accompanied by the loss of beta-ketoacyl reductase and enoyl-CoA reductase activity. The inactivation followed a pseudo-first order kinetics and sum of the second order rate constants for the two reductase reactions equaled that for the synthetase reaction. Inactivation of all three activities was prevented by NADPH or its analogs 2',5'-ADP and 2'-AMP but not by the corresponding nucleotides containing the 5'-phosphate. These results suggest that binding of NADPH to fatty acid synthetase involves specific interaction of the 2'-phosphate with the guanidino group of arginine residues at the active site of the two reductases. pH-Dependent inactivation by phenylglyoxal indicated that a group with a pka 7.5 is involved in the loss of enzyme activity. Stoichiometric results showed that 4 out of 164 arginine residues per enzyme molecule were essential for the enzyme activity.     

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 arginyl residues in porphyrin binding to ferrochelatase

The role of cationic amino acid residues in the binding of porphyrin substrates by purified bovine ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) have been examined via chemical modification with camphorquinone-10-sulfonic acid, phenylglyoxal, butanedione, and trinitrobenzene sulfonate. The data obtained show that modification of arginyl, but not lysyl, residues results in the rapid inactivation of ferrochelatase. The 2,4-disulfonate deuteroporphyrin, which is a competitive inhibitor of mammalian ferrochelatase, protects the enzyme against inactivation. Ferrous iron has no protective effect. Reaction with radiolabeled phenylglyoxal shows that modification of 1 arginyl residue causes maximum inhibition of enzyme activity. The inactivation does not follow simple pseudo-first order reaction kinetics, but is distinctly biphasic in nature. Comparison of the enzyme kinetics for modified versus unmodified enzyme show that modification with camphorquinone-10-sulfonic acid has no effect on the Km for iron but does alter the Km for porphyrin.     

Changes in chick liver and brain mevalonate kinase, mevalonate-5-phosphate kinase and mevalonate-5-pyrophosphate decarboxylase during development

Phosphorylation and decarboxylation of mevalonate in chick liver and brain was investigated during early post hatching stages of development. In chick liver, both mevalonate kinase and mevalonate-5-phosphate kinase increased their activity from day 5 of age while pyrophosphate decarboxylase activity remained low during the first days after hatching, increased sharply up to day 9 of age, and remained practically unchanged thereafter. The developmental pattern obtained in brain shows a slight decrease in the phosphorylation and decarboxylation of mevalonate after the first week of postnatal development. Further studies were performed using the specific substrate of mevalonate-5-pyrophosphate decarboxylase, corroborating the results obtained using mevalonate as substrate. Changes in hepatic decarboxylase were more pronounced than those observed in mevalonate-phosphorylating enzymes, thus suggesting an important role for decarboxylase in the control of cholesterogenesis during postnatal development.     

Inactivation of glutamate dehydrogenase and glutamate synthase from Bacillus megaterium by phenylglyoxal, butane-2,3-dione and pyridoxal 5''-phosphate.

Reaction of phenylglyoxal with glutamate dehydrogenase (EC 1.4.1.4), but not with glutamate synthase (EC 2.6.1.53), from Bacillus megaterium resulted in complete loss of enzyme activity. NADPH alone or together with 2-oxoglutarate provided substantial protection from inactivation by phenylglyoxal. Some 2mol of [14C]Phenylglyoxal was incorporated/mol of subunit of glutamate dehydrogenase. Addition of 1mM-NADPH decreased incorporation by 0.7mol. The Ki for phenylglyoxal was 6.7mM and Ks for competition with NADPH was 0.5mM. Complete inactivation of glutamate dehydrogenase by butane-2,3-dione was estimated by extrapolation to result from the loss of 3 of the 19 arginine residues/subunit. NADPH, but not NADH, provided almost complete protection against inactivation. Butane-2,3-dione had only a slight inactivating effect on glutamate synthase. The data suggest that an essential arginine residue may be involved in the binding of NADPH to glutamate dehydrogenase. The enzymes were inactivated by pyridoxal 5'-phosphate and this inactivation increased 3--4-fold in the borate buffer. NADPH completely prevented inactivation by pyridoxal 5'-phosphate.