Role of arginyl residues in yeast hexokinase PII.

Yeast hexokinase PII is rapidly inactivated (assayed at pH 8.0) by either butanedione in borate buffer or phenylglyoxal, reagents which are highly selective for the modification of arginyl residues. MgATP alone offers no protection against inactivation, consistent with low affinity of hexokinase for this nucleotide in the absence of sugar. Glucose provides slight protection against inactivation, while the combined presence of glucose and MgATP gives significant protection, suggesting that modified arginyl residues may lie at the active site, possibly serving to bind the anionic polyphosphate of the nucleotide in the ternary enzyme:sugar:nucleotide complex. Extrapolation to complete inactivation suggests that inactivation by butanedione correlates with the modification of 4.2 arginyl residues per subunit, and complete protection against inactivation by the combined presence of glucose and MgATP correlates with the protection of 2 to 3 arginyl residues per subunit. When the modified enzyme is assayed at pH 6.5, significant activity remains. However, modification by butanedione in borate buffer abolishes the burst-type slow transient process, observed when the enzyme is assayed at pH 6.5, to such an extent that after extensive modification the kinetic assays are characterized by a lag-type slow transient process. But even after extensive modification, hexokinase PII still demonstrates negative cooperativity with MgATP and is still strongly activated by citrate when assayed at pH 6.5.     

Essential arginyl residues in fructose-1,6-bisphosphatase.

Modification of pig kidney fructose-1,6-bisphosphatase with 2,3-butanedione in borate buffer (pH 7.8) leads to the loss of the activation of the enzyme by monovalent cations, as well as to the loss of allosteric adenosine 5'-monophosphate (AMP) inhibition. In agreement with the results obtained for the butanedione modification of arginyl residues in other enzymes, the effects of modification can be reversed upon removal of excess butanedione and borate. Significant protection to the loss of K+ activation was afforded by the presence of the substrate fructose 1,6-bisphosphate, whereas AMP preferentially protected against the loss of AMP inhibition. The combination of both fructose 1,6-bisphosphate and AMP fully protected against the changes in enzyme properties on butanedione treatment. Under the latter conditions, one arginyl residue per mole of enzyme subunit was modified, whereas three arginyl residues were modified by butanedione under conditions leading to the loss of both potassium activation and AMP inhibition. Thus, the modification of two arginyl residues per subunit would appear to be responsible for the change in enzyme properties. The present results, as well as those of a previous report on the subject (Marcus, F. (1975), Biochemistry 14, 3916-3921) support the conclusion that one arginyl residue per subunit is essential for monovalent cation activation, and another arginyl residue is essential for AMP inhibition. A likely role of the latter residue could be its involvement in the binding of the phosphate group of AMP.     

Essential arginyl residues in yeast enolase.

Yeast enolase is rapidly inactivated by butanedione in borate buffer, complete inactivation correlating with the modification of 1. 8 arginyl residues per subunit. Protection against inactivation is provided by either an equilibrium mixture of substrates or inorganic phosphate, a competitive inhibitor of the enzyme. Complete protection by substrates correlates with the shielding of 1. 3 arginyl residues per subunit, while phosphate protects 1. 0 arginyl residue per subunit from modification.     

The role of arginyl residues in directing carboxymethylation of horse liver alcohol dehydrogenase.

The selective carboxymethylation by iodoacetate of Cys-46 in the active center of horse liver alcohol dehydrogenase has been shown to be mediated by interaction of the anionic reagent with the arginyl residue(s) previously shown to be responsible for binding NADH (L.G. Lange, J.F. Riordan, and B.L. Vallee (1974), Biochemistry 13, 4361). Thus, sequential and reversible chemical modification of arginine with butanedione and of cysteine with pmercuribenzoate demonstrate that the essential thiol groups are not affected by arginine modification. Importantly, the rate of incorporation of [14C]idoacetate into native horse liver alcohol dehydrogenase is ten times faster than that for the butanedione-modified enzyme. Moreover, as evidenced by peptide isolation, the radiolabel incorporated into the latter occurs at low levels in several different peptides as opposed to the single, strongly labeled CmCys-46 peptide obtained from the native enzyme. The demonstration that the arginyl residue(s) involved in coenzyme binding promotes enhanced reactivity of the active site thiol supports the general hypothesis that the spatial arrangement of structural features allowing expression of enzymatic function may also account for enhanced chemical reactivity of certain active site residues (B.L Vallee and J.F. Riordan (1969), Annu. Rev. Biochem. 38, 733).     

Phosphoglycerate mutase has essential arginyl residues

Phosphoglycerate mutase is inactivated by butanedione in borate buffer. Inactivation by 0.13 mM reagent correlates with the modification of one arginyl residue per subunit, and is prevented by either 2, 3-diphosphoglycerate or 3-phosphoglycerate. With 0.50 mM butanedione, inactivation is accompanied by the modification of three arginyl residues per subunit, two of which are protected by the combined presence of cofactor and substrate.     

Localization of arginyl residues modified with butanedione in glyceraldehyde-3-phosphate dehydrogenase from pig muscle

Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) from pig muscle was inactivated by incubation with butanedione in triethanolamine buffer, pH 8.3. The inactivation was reversible after short treatment with butanedione; it became irreversible after 12-15 h, with a concomitant loss of two arginyl residues per subunit. The modified enzyme was digested with TPCK-trypsin and the peptides were purified by chromatography and electrochromatography. Two new peptides were obtained as the result of modification. From their partially determined sequence the modified arginyl residues were identified as Arg-13 and Arg-231 in the primary structure of pig muscle enzyme.     

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.     

Comparative titration of arginyl residues in purified D-β-hydroxybutyrate apodehydrogenase and in the reconstituted phospholipid-enzyme complex

In order to titrate and understand the role of arginyl residues of D-β-hydroxybutyrate dehydrogenase, arginyl specific reagents: butanedione, 1,2-cyclohexanedione and phenylglyoxal were incubated with three different forms of the enzyme; native enzyme (inner mitochondrial membrane bound), purified apoenzyme (phospholipid -free) and phospholipid-enzyme complex (reconstituted active form).After complete inactivation of the enzyme by [¹⁴C]-phenylglyoxal, the number of modified arginyl residues was different: one with the lipid-free apoenzyme and three with the phospholipid-enzyme complex, suggesting a conformational change of the enzyme triggered by the presence of phospholipids.After exhaustive chemical modification either of the apoenzyme or of the phospholipid-enzyme complex with [¹⁴C]-phenylglyoxal, four arginyl residues were titrated indicating that these residues are located in the hydrophilic part of the enzyme, not interacting with phospholipids.Reconstituted enzyme inactivated by butanedione could no longer bind a pseudosubstrate (succinate) which indicates that an arginyl residue is involved in the enzyme-substrate complex formation.The values of second order rate constants of D-β-hydroxybutyrate dehydrogenase inactivation by butanedione and 1,2-cyclohexanedione were unchanged with the three enzyme forms, suggesting that phospholipids are not involved in the substrate binding mechanism.     

The role of arginine residues in the function of D-glyceraldehyde-3-phosphate dehydrogenase.

Inactivation of apo-glyceraldehyde-3-phosphate dehydrogenase from rat skeletal muscle in the presence of butanedione is the result of modification of one arginyl residue per subunit of the tetrameric enzyme molecule. The loss of activity follows pseudo-first-order kinetics. NAD+ increases the apparent first-order rate constant of inactivation. The effect of NAD+ on the enzyme inactivation is cooperative (Hill coefficient = 2.3--3.2). Glyceraldehyde 3-phosphate protected the holoenzyme against inactivation, decreasing the rate constant of the reaction. At saturating concentrations of substrate the protection was complete. The Hill plot demonstrates that the effect is cooperative. This suggests that subunit interactions in the tetrameric holoenzyme molecule may affect the reactivity of the essential arginyl residues. In contrast, glyceraldehyde 3-phosphate had no effect on the rate of inactivation of the apoenzyme in the presence of butanedione. 100 mM inorganic phosphate protected both the apoenzyme and holoenzyme against inactivation. The involvement of the microenvironment of the arginyl residues in the functionally important conformational changes of the enzyme is discussed.     

Essential arginyl residues in thymidylate synthetase.

Thymidylate synthetase from amethopterin-resistant $\text{Lactobacillus}$$\text{casei}$ is rapidly and completely inactivated by 2,3-butanedione in borate buffer, a reagent that is highly selective for the modification of arginyl residues. The reversible inactivation follows pseudo-first order kinetics and is enhanced by borate buffer. dUMP and dTMP afford significant protection against inactivation while (±)-5,10-methylenetetrahydrofolate and 7,8-dihydrofolate provide little protection. Unlike native enzyme, butanedione-modified thymidylate synthetase is incapable of interacting with 5-fluoro-2′-deoxyuridylate and 5,10-(+)-methylenetetrahydrofolate to form stable ternary complex. The results suggest that arginyl residues participate in the functional binding of dUMP.     

Evidence for an essential arginyl residue in bovine milk gamma-glutamyltransferase

Treatment of bovine milk gamma-glutamyltransferase with 2,3-butanedione in borate buffer markedly inactivates its gamma-glutamyltransferase activity. Inactivation is prevented by a combination of the gamma-glutamyl donor and acceptor substrates, glutathione, and glycylglycine, but less effectively by only one of them. Serine plus borate of maleate provides no protection against the inactivation. Amino acid analysis of the enzyme treated with butanedione in the presence and absence of the protecting substrate combination indicates that complete inactivation correlates with the modification of a single arginyl residue per molecule. The residue modified is associated with the smaller subunit of the two equal subunits which comprise the enzyme. The butanedione-treated enzyme retains a hydrolytic activity, another but less significant catalytic function of the enzyme. The results indicate that the arginyl residue is involved in recognizing the anionic moiety of the acceptor and in binding it to the acceptor site located on the smaller subunit of the enzyme.     

Selective chemical modification of arginine residues in mitochondrial malate dehydrogenase

Mitochondrial malate dehydrogenase (L-malate: NAD⁺ oxidoreductase, EC 1.1.1.37) from porcine heart exhibits a time dependent loss in enzymatic activity in the presence of the reagent butanedione. The inhibition occurs concomitant with the modification of 2.4 residues of arginine per molecular weight of 70,000. The presence of the reduced coenzyme, NADH, protects the enzyme from inhibition by butanedione and from modification of arginine residues, suggesting that the residues modified are located near the coenzyme binding site and hence at or near the enzymatic active center of this enzyme.     

An essential arginyl residue at the nucleotide binding site of creatine kinase.

Treatment of rabbit muscle creatine kinase (EC 2.4.3.2) with either butanedione in borate buffer or phenylglyoxal in Veronal buffer decreases enzymatic activity correlating with the modification of a single arginyl residue per subunit of the dimeric enzyme. Very little activity is lost when modification is performed in the presence of MgATP or MgADP. Nucleotide binding to the modified enzyme is virtually abolished as determined by ultraviolet difference spectroscopy. The data suggest that an arginyl residue plays an essential role in the enzymatic mechanism of creatine kinase, probably as a recognition site for the negatively charged oligophosphate moiety of the nucleotide.     

乙醇酸氧化酶必需精氨酸残基的化学修饰

丁二酮能使GAO迅速失活,其失活速度受介质pH和硼酸浓度的显著影响;其修饰反应具可逆性,当透析除去修饰剂和硼酸时,活性得到恢复。失活进程表现为假一级动力学。而计算表明,酶的每一活性中心单位与一分子丁二酮结合便可引起酶的失活。底物和竞争性抑制剂均能有效地保护酶免于失活。氨基酸分析表明,酶的失活是因为丁二酮修饰了精氨酸残基。丁二酮修饰GAO后使酶的K_m增大,而V_m没有变化。     

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.     

Inactivation of porcine heart cytoplasmic malate dehydrogenase by pyridoxal 5'-phosphate.

Pyridoxal 5'-phosphate (pyridoxal-5'-P) has been found to act as a bifunctional reagent during the inactivation of porcine heart cytoplasmic malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37). The biphasic kinetics and X-azolidine-like structure formed were similar to those observed for mitochondrial malate dehydrogenase (Wimmer, M.J., Mo, T., Sawyers, D.L., and Harrison, J.H. (1975) J. Biol. Chem. 250, 710-715). In the cytoplasmic enzyme, however, irreversible inactivation representing X-azolidine formation was found to be the dominant characteristic of the interaction with pyridoxal-5'-P. Spectral evidence indicated that at total inactivation 2 mol of pyridoxal-5'-P were incorporated per mol of enzyme or one pyridoxal-5'-P per enzymatic active site. The presence of NADH protected the enzyme from inactivation suggesting interaction of pyridoxal-5'-P at or near the enzymatic active centers of this enzyme. Fluorometric titrations indicated that pyridoxal-5'-P-inactivated enzyme failed to bind NADH or at least failed to bind NADH in the same fashion as native enzyme.     

Affinity labeling of arginyl residues at the catalytic region of estradiol 17 beta-dehydrogenase from human placenta by 16-oxoestrone

16-Oxoestrone inhibited competitively the activity of estradiol 17 beta-dehydrogenase from human placenta against estradiol in phosphate buffer (pH 7.2), suggesting reversible binding of 16-oxoestrone to the substrate-binding site. 16-Oxoestrone irreversible inactivated the estradiol 17 beta-dehydrogenase in borate buffer (pH 8.5) in a time-dependent manner, following pseudo-first-order kinetics. The rate constant (k3) obtained for the inactivation by 16-oxoestrone was 8.3 x 10(-4) s-1. The rate of inactivation was significantly decreased by addition of estrone, estradiol, estriol, NAD(H) and NADP+. Also, the rate was reduced markedly by 2'AMP, 5'ATP and 2',5' ADP, but not by NMN(H) and 3-pyridinealdehyde adeninediphospho nucleotide. The inactivation by 16-oxoestrone was neither prevented by sodium azide nor influenced by light. From these data, 16-oxoestrone, an alpha-dicarbonyl steroid, was suggested to inactive estradiol 17 beta-dehydrogenase by modification of arginyl residues located around the substrate-binding site of the enzyme. Biphasic inactivation of the enzyme by 16-oxoestrone was observed with an increase of modified arginyl residues. The first phase of the inactivation was regarded as an affinity labeling of the arginyl residues at or near the substrate-binding site of the enzyme. Stoichiometry of the inactivation indicated that two arginyl residues were essential for maintenance of the enzyme activity. The second phase was considered as chemical modification of the arginyl residues outside of the catalytic region of the enzyme.     

Identification of an essential lysine in porcine heart mitochondrial malate dehydrogenase.

The reversible inactivation of porcine heart mitochondrial malate dehydrogenase by pyridoxal 5'-phosphate yields an irreversible modification upon sodium borohydride reduction. A 200-fold molar excess of pyridoxal-5'-P over enzyme results in inactivation to the extent of 54%, and incorporation of 5.7 mol of inactivator per mol of enzyme. The same inactivation carried out in the presence of 80 mM coenzyme, NADH, produces malate dehydrogenase which is approximately 94% active and contains 4.6 mol of pyridoxal-5'-P per mol of enzyme. The incorporation difference between inactivated and protected samples suggests, for total inactivation, the modification of 2 residues per mol of enzyme (i.e. 1 residue per subunit, or 1 per enzymatic active site). This specificity was confirmed by the isolation of a single pyridoxyl-5'-P-labeled "difference peptide" obtained by comparison of the Dowex 1-X2 elution profiles of tryptic digests of protected and inactivated samples, respectively. Amino acid analysis of the peptide demonstrated the presence of N6-pyridoxyl-L-lysine (Lys(Pyx)), establishing the existence of an essential lysing residue in the active center of malate dehydrogenase. The amino acid sequence of the active center hexapeptide has been determined to be: H2NLys(Pyx)Pro-Gly-Met-Thr-Arg-COOH.     

Modification of an arginine residue essential for the activity of NAD-malic enzyme from Ascaris suum

Purified NAD-malic enzyme from Ascaris suum is rapidly inactivated by the arginine reagent, 2,3-butanedione, and this inactivation is facilitated by 30 mM borate. Determination of the inactivation rate as a function of butanedione concentration suggests a second-order process overall, which is first order in butanedione. A second-order rate constant of 0.6 M-1 s-1 at pH 9 is obtained for the butanedione reaction. The inactivation is reversed by removal of the excess reagent upon dialysis. The enzyme is protected against inactivation by saturating amounts of malate in the presence and absence of borate. The divalent metal Mg2+ affords protection in the presence of borate but has no effect in its absence. The nucleotide reactant NAD+ has no effect on the inactivation rate in either the presence or absence of borate. A dissociation constant of 24 mM is obtained for E:malate from the decrease in the inactivation rate as a function of malate concentration. An apparent Ki of 0.5 mM is obtained for oxalate (an inhibitor competitive vs malate) from E:Mg:oxalate while no significant binding is observed for oxalate using the butanedione modified enzyme. The pH dependence of the first-order rate of inactivation by butanedione gives a pKa of 9.4 +/- 0.1 for the residue(s) modified, and this pK is increased when NAD is bound. The arginine(s) modified is implicated in the binding of malate.