Essential arginine residues of creatine kinase from beef heart mitochondria

Creatine kinase from beef heart mitochondria is inactivated by 2,3-butanedione. The kinetics of inactivation of the mitochondrial enzyme is biphasic with a bend at a point corresponding to 50% inactivation. The inactivation rate constants of the first fast and the second slow phases of the reaction differ by one order of magnitude, thus suggesting the existence of two types of arginine residues, i.e. "fast" and "slow" ones, with different reactivities. The inactivation rate constant of the slow phase is very close to that for cytoplasmic creatine kinase. At saturating concentrations MgATP and MgADP afford complete protection of the slow phase of inactivation. It is assumed that the "slow" arginine is involved in the binding of metal-nucleotide substrates in the enzyme active center.     

Chemical modification of arginine residues of porcine muscle acylphosphatase

Acylphosphatase (acylphosphate phosphohydrolase, EC 3.6.1.7) from porcine skeletal muscle 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 also first order with respect to the reagent concentration. Among the competitive inhibitors for the enzyme examined, inorganic phosphate and ATP almost completely, and Cl- partially, protect the enzyme against the inactivation. The dissociation constants for inorganic phosphate and ATP determined from protection experiments by these inhibitors agree well with those from inhibition experiments by them. These results support the idea that the modification occurs at the phosphate-binding site. The amino-acid analysis reveals the lack of reaction at residues other than arginine. Circular dichroism spectra of the modified enzymes show that the inactivation seems not to be due to denaturation of the enzyme resulting from the modification of the non-essential arginine residues. The relationship between the loss of the enzyme activity and the number of arginine residues modified in the presence and absence of ATP shows that one arginine residue is possibly responsible for the inactivation of acylphosphatase.     

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.     

Study on the effect of some group-specific agents on clostridiopeptidase

The interaction of clostridiopeptidase of Clostridium histolyticum with EDC, TNM and MA, the specific reagents for COOH-groups, tyrosine and lysine residues was studied. It was shown that at pH 6.0 EDC inactivates the enzyme. The inactivation process follows the pseudo-first order kinetics and is described by a second order rate constant equal to 1 M-1 min-1. The synthetic substrate does not prevent, in practical terms, the enzyme inactivation by EDC. At pH 8.0 TNM modifies about 19 tyrosine residues in the clostridiopeptidase molecule which is accompanied by marked inhibition of the enzyme activity (down to 70-90%). In this case, the inactivation process is not described by simple pseudo-first order kinetics but is characterized by two steps (fast and slow) with second order rate constants of approximately 14 and 3.5 M-1 min-1, respectively. The synthetic substrate partly prevents the inactivation of the enzyme by TNM and protects 11 tyrosine residues. The MA-induced incorporation of 13 +/- 3 maleyl groups into the clostridiopeptidase molecule in partially prevented by the synthetic substrate with protects the enzyme against inactivation. The data obtained suggest that lysine residues are seemingly included into the active center of clostridiopeptidase, whereas tyrosine residues provide for the maintenance of active conformation of the enzyme.     

Calcium-ion-binding activity in human small-intestinal mucosal cytosol. Purification of two proteins and interrelationship of calcium-binding fractions.

Butane-2,3-dione inactivates the aspartyl proteinases from Penicillium roqueforti and Penicillium caseicolum, as well as pig pepsin, penicillopepsin and Rhizopus pepsin, at pH 6.0 in the presence of light but not in the dark. The inactivation is due to a photosensitized modification of tryptophan and tyrosine residues. In the dark none of the amino acid residues, not even arginine residues, is modified even after several days. In the light one arginine residue in pig pepsin is lost at a rate that is comparable with the rate of inactivation; however, the loss of the single arginine residue in the aspartyl proteinase of P. roqueforti and the second arginine residue of pig pepsin is slower than the loss of activity; penicillopepsin is devoid of arginine. Loss of most of the activity is accompanied by the following amino acid losses: P. roqueforti aspartyl proteinase, about two tryptophan and six tyrosine residues; penicillopepsin, about two tryptophan and three tyrosine residues; pig pepsin, about four tryptophan and most of the tyrosine residues. Modification of histidine residues was too slow to contribute to inactivation. None of the other residues, including half-cystine and methionine residues (when present), was modified even after prolonged incubation. The inactivation of P. roqueforti aspartyl proteinase and pig pepsin appears due to non-specific modification of several residues. With penicillopepsin, however, the reaction is more limited and initially affects only those tryptophan and tyrosine residues that lie in the active-site groove. In the presence of pepstatin the rate of inactivation is considerably diminished. After prolonged reaction a general structural breakdown occurs.     

Kinetics of inactivation of phytase (phy A) during modification of histidine residue by IAA and DEP

Chemical probing of histidine residues using specific modifiers, iodoacetic acid (IAA) and diethylpyrocarbonate (DEP) resulted in the inactivation of phytase (phy A). The kinetic theory of the substrate reaction during the modification of enzyme activity was applied to a study of the kinetics of the course of inactivation of phytase by IAA and DEP. The results suggested that histidine residues are involved in the active site of the enzyme. They also indicated that inactivation of the enzyme by IAA was via a complexing type inhibition, while the inhibition by DEP reaction involved a conformational change step before inactivation. The dissociation constant of the enzyme-inhibitor complex of IAA, the constant of the conformational change of DEP and the microscopic rate constants of two inhibitors were obtained.     

Role of domain 4 in sodium channel slow inactivation

Depolarization of sodium channels initiates at least three gating pathways: activation, fast inactivation, and slow inactivation. Little is known about the voltage sensors for slow inactivation, a process believed to be separate from fast inactivation. Covalent modification of a cysteine substituted for the third arginine (R1454) in the S4 segment of the fourth domain (R3C) with negatively charged methanethiosulfonate-ethylsulfonate (MTSES) or with positively charged methanethiosulfonate-ethyltrimethylammonium (MTSET) produces a marked slowing of the rate of fast inactivation. However, only MTSES modification produces substantial effects on the kinetics of slow inactivation. Rapid trains of depolarizations (2-20 Hz) cause a reduction of the peak current of mutant channels modified by MTSES, an effect not observed for wild-type or unmodified R3C channels, or for mutant channels modified by MTSET. The data suggest that MTSES modification of R3C enhances entry into a slow-inactivated state, and also that the effects on slow inactivation are independent of alterations of either activation or fast inactivation. This effect of MTSES is observed only for cysteine mutants within the middle of this S4 segment, and the data support a helical secondary structure of S4 in this region. Mutation of R1454 to the negatively charged residues aspartate or glutamate cannot reproduce the effects of MTSES modification, indicating that charge alone cannot account for these results. A long-chained derivative of MTSES has similar effects as MTSES, and can produce these effects on a residue that does not show use-dependent current reduction after modification by MTSES, suggesting that the sulfonate moiety can reach a critical site affecting slow inactivation. The effects of MTSES on R3C are partially counteracted by a point mutation (W408A) that inhibits slow inactivation. Our data suggest that a region near the midpoint of the S4 segment of domain 4 plays an important role in slow inactivation.     

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.     

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.     

Irreversible inactivation of human erythrocyte pyruvate kinase by 2,3-butanedione

Human erythrocyte pyruvate kinase was found to be irreversibly inactivated by butanedione in the dark. The second-order rate constants for inactivation at pH 8.0 and 25 degrees C were 2.14 and 2.74 M-1 min-1 in the absence and presence of 50 mM borate, respectively. The pH profile of the inactivation indicated the involvement of a residue with an apparent pK alpha of 8.1-8.3. ADP and phosphoenolpyruvate acted as partial inhibitors of the inactivation process. Certain details of the inactivation, spectral studies, and fluorometric determinations gave evidence for arginine as the only target residue. A total of 23 +/- 3 residues per subunit were modified within the period required for inactivation. In the same period the presence of 4 mM ADP reduced the extent of inactivation by 70% and the number of modified residues to 18 +/- 4. The number of the arginine residues protected by ADP from butanedione modification was 5.0 +/- 1.3 per subunit.     

Anion transport in red blood cells and arginine-specific reagents. Interaction between the substrate-binding site and the binding site of arginine-specific reagents

Phenylglyoxal is found to be a potent inhibitor of sulfate equilibrium exchange across the red blood cell membrane at both pH 7.4 and 8.0. The inactivation exhibits pseudo-first-order kinetics with a reaction order close to one at both pH 7.4 and 8. The rate constant of inactivation at 37 degrees C was found to be 0.12 min-1 at pH 7.4 and 0.19 min-1 at pH 8.0. Saturation kinetics are observed if the pseudo-first order rate constant of inhibition is measured as a function of phenylglyoxal concentration. Sulfate ions as well as chloride ions markedly decrease the rate of inactivation by phenylglyoxal at pH 7.4, suggesting that the modification occurs at or near to the binding site for chloride and sulfate. The decrease of the rate of inactivation produced at pH 8.0 by chloride ions is much higher than that produced by sulfate ions. Kinetic analysis of the protection experiments showed that the loaded transport site is unable to react with phenylglyoxal. From the data it is concluded that the modified amino acid(s) residues, presumably arginine, is (are) important for the binding of the substrate anion.     

The role of cysteinyl residues in the phosphorylase kinase activity as revealed by iodacetamide modification

In native nonactivated phosphorylase kinase [14C] iodacetamide interacts with 50 cysteinyl residues per enzyme molecule (alpha beta gamma delta)4. According to their reactivity towards iodacetamide these residues can be classified into 3 groups. The most reactive cysteinyl residues are involved in the enzyme activation caused by modification of SH-groups. The enzyme inhibition is biphasic. The fast and slow inactivation reactions follow the pseudo-first order kinetics. The rate of inactivation is increased by Ca2+. Mg-ATP effectively protects the enzyme against the inactivation and chemical modification of three SH-groups per protomer (apha beta gamma delta). The kinetics of inactivation and of the [14C] iodacetamide label incorporation demonstrate that two cysteinyl residues per enzyme protomer (alpha beta gamma delta) are essential for the enzyme activity. These residues are located near the ATP-binding site of the beta and gamma subunits of phosphorylase kinase.     

Interaction of synthetic NH2-terminal fragments of bacteriophage lambda cro protein with nucleic acids

Cu,Zn superoxide dismutase from baker's yeast, Saccharomyces cerevisiae, can be >98% inactivated by modification of one arginyl residue per subunit with phenylglyoxal. The loss of activity is not accompanied by loss of either Cu or Zn ions, suggesting that this arginine is essential for catalytic activity. 4-Hydroxy-3-nitrophenylglyoxal (HNPG), a chromophoric analogue of phenylglyoxal, also inactivates the yeast enzyme by modification of 1.0 arginine per subunit. The chromophoric properties of HNPG were utilized to identify Arg-143 as the essential arginine in yeast Cu,Zn superoxide dismutase.     

Role of an S4-S5 linker in sodium channel inactivation probed by mutagenesis and a peptide blocker

A pair of conserved methionine residues, located on the cytoplasmic linker between segments S4 and S5 in the fourth domain of human heart Na channels (hH1), plays a role in the kinetics and voltage dependence of inactivation. Substitution of these residues by either glutamine (M1651M1652/QQ) or alanine (MM/AA) increases the inactivation time constant (tau) at depolarized voltages, shifts steady-state inactivation (h infinity) in a depolarized direction, and decreases the time constant for recovery from inactivation. The data indicate that the mutations affect the rate constants for both binding and unbinding of a hypothetical inactivation particle from its binding site. Cytoplasmic application of the pentapeptide KIFMK in Na channels mutated to remove inactivation produces current decays resembling inactivation (Eaholtz, G., T. Scheuer, and W.A. Catterall. 1994. Neuron. 12: 1041-1048.). KIFMK produces a concentration-dependent, voltage-independent increase in the decay rate of MM/QQ and MM/AA currents at positive membrane potentials (Ki approximately 30 microM), while producing only a small increase in the decay rate of wild-type currents at a concentration of 200 microM. Although MM/QQ inactivates approximately 2.5-fold faster than MM/AA in the absence of peptide, the estimated rate constants for peptide block and unblock do not differ in these mutants. External Na+ ions antagonize the block by cytoplasmic KIFMK of MM/AA channels, but not the inactivation kinetics of this mutant in the absence of peptide. The effect of external [Na+] is interpreted as a voltage-dependent knock-off mechanism. The data provide evidence that KIFMK can only block channels when they are open and that peptide block does not mimic the inactivation process.     

Studies on inactivation of anion transport in human red blood cell membrane by reversibly and irreversibly acting arginine-specific reagents

Summary A chromophoric derivative of phenylglyoxal, 4-hydroxy-3-nitrophenylglyoxal (HNPG), known to be highly selective for modification of arginine residues in aqueous solution is found to be a potent inhibitor of anion transport across the red cell membrane. In contrast to the action of all other arginine-specific reagents used under the experimental conditions in this laboratory, the action of HNPG on sulfate transport is completely reversible. Hence, a kinetic analysis of its inhibitory effect on SO ₄ ^2– self-exchange could be performed. The effect of increasing chloride concentration on the inhibitory potency of HNPG is consistent with the concept that Cl^– and HNPG compete for the same site on the anion transporter. The IC₅₀ value for the inhibition of SO ₄ ^2– exchange with HNPG is about 0.13mm at pH 8.0 and 0.36mm at pH 7.4, and the Hill coefficient for the interaction between the transporter and the inhibitor is near one at both pH's. HNPG is able to protect the transport system against inhibition with the (under our experimental conditions) irreversibly acting arginine specific reagent, phenylglyoxal. Partial inactivation of the transport system with phenylglyoxal lowers the maximal rates of SO ₄ ^2– and chloride exchange but does not modify the apparentK _s for the substrate anions. Reversibly acting anion transport inhibitors known to interact with the DIDS binding site like salicylate, tetrathionate, APMB, DNDS, and flufenamate are able to protect the transport system against phenylglyoxalation. Other inhibitors like phloretin and phlorizin have no effect.     

Essential arginine residues in human liver arylsulfatase A.

Human liver arylsulfatase A was treated with arginine-specific reagents (diones), resulting in a loss of enzyme activitity with apparent first-order kinetics. Sulfite and borate—competitive inhibitors of the enzyme—provided complete protection from inactivation by phenylglyoxal. Sulfite and substrate each likewise protected against enzyme inactivation by 2,3-butanedione. A plot of pseudo-first-order rate constants of enzyme inactivation versus 2,3-butanedione concentrations suggests that an essential arginine residue is modified with a loss in function of the binding site or of the active site of the protein. Chemical analysis of the butanedione-treated sulfatase indicates that complete enzyme inactivation corresponds to a modification of only about 2 of the 20 arginine residues per enzyme subunit. Taken together, all of the results strongly suggest that arginine residues are essential for the activity of arylsulfatase A. An incidental discovery in this work is that borate ion is a competitive inhibitor of human arylsulfatase A with a K_i of 2.5 × 10^?4 M.     

Chemical modification of a functional arginine residue of rat liver glycine methyltransferase

Rat liver glycine methyltransferase is inactivated irreversibly by phenylglyoxal in potassium phosphate buffer. The inactivation obeys pseudo-first-order kinetics, and the apparent first-order rate constant for inactivation is linearly related to the reagent concentration. A second-order rate constant of 10.54 +/- 0.44 M-1 min-1 is obtained at pH 8.2 and 25 degrees C. Amino acid analysis shows that only arginine is modified upon treatment with phenylglyoxal. Sodium acetate, a competitive inhibitor with respect to glycine, affords complete protection in the presence of S-adenosylmethionine. Acetate alone has no effect on the rate of inactivation. The value of the dissociation constant for acetate determined from the protection experiment is in good agreement with that obtained by kinetic analysis. Comparison of the amount of [14C]phenylglyoxal incorporated into the protein and the number of arginine residues modified in the presence and absence of protecting ligands indicates that modification of one arginine residue per enzyme subunit eliminates the enzyme activity, and this residue is identified as Arg-175 by peptide analysis. The arginine-modified glycine methyltransferase appears to bind S-adenosylmethionine as the native enzyme does, as seen from quenching of the protein fluorescence by S-adenosylmethionine. These results suggest the requirement of Arg-175 in binding the carboxyl group of the substrate glycine.     

Evidence for an active site arginine in UDP-glucuronyltransferase

2,3-Butanedione inactivates the pure form of UDP-glucuronyltransferase used in these experiments (GT2P) (EC 2.4.1.17) purified from pig liver microsomes. The kinetics of the reaction indicates that 2,3-butanedione reacts with two amino acids that affect activity. A rapid, partial inactivation is followed by a slower rate of inactivation that leads eventually to completely inactive enzyme. UDP-glucuronic acid and glucuronic acid, as compared with UDP, are effective as protectors against the slow, secondary phase of inactivation; no ligand tested protected against the rapid phase of inactivation. The lipid environment of GT2P was a determinant of the pseudo-first order rate constant for the slow phase of inactivation, but did not affect the rate of the rapid phase of inactivation. The data suggest that GT2P contains an active site arginine that interacts with the -COO- at C-6 of the glucuronic acid moiety of UDP-glucuronic acid.     

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.     

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.