Identification of essential arginyl residues in cytoplasmic malate dehydrogenase with butanedione.

The inactivation of cytoplasmic malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37) from porcine heart and the specific modification of arginyl residues have been found to occur when the enzyme is inhibited with the reagent butanedione in sodium borate buffer. The inactivation of the enzyme was found to follow pseudo-first order kinetics. This loss of enzymatic activity was concomitant with the modification of 4 arginyl residues per molecule of enzyme. All 4 residues could be made inaccessible to modification when a malate dehydrogenase-NADH-hydroxymalonate ternary complex was formed. Only 2 of the residues were protected by NADH alone and appear to be essential. Studies of the butanedione inactivation in sodium phosphate buffer and of reactivation of enzymatic activity, upon the removal of excess butanedione and borate, support the role of borate ion stabilization in the inactivation mechanism previously reported by Riordan (Riordan, J.F. (1970) Fed. Proc. 29, Abstr. 462; Riordan, J.F. (1973) Biochemistry 12, 3915-3923). Protection from inactivation was also provided by the competitive inhibitor AMP, while nicotinamide exhibited no effect. Such results suggest that the AMP moiety of the NADH molecule is of major importance in the ability of NADH to protect the enzyme. When fluorescence titrations were used to monitor the ability of cytoplasmic malate dehydrogenase to form a binary complex with NADH and to form a ternary complex with NADH and hydroxymalonate, only the formation of ternary complex seemed to be effected by arginine modification.     

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.     

Modification of arginyl residues in ferredoxin-NADP+ reductase from spinach leaves.

Reaction of spinach leaves ferredoxin-NADP+ reductase (NADPH:ferredoxin oxidoreductase, EC 1.6.7.1) with alpha-dicarbonyl compounds results in a biphasic loss of activity. The rapid phase yields modified enzyme with about 30% of the original activity, but no change in the Km for NADPH. Only partial protection against inactivation is provided by NADP+, NADPH and their analogs, whereas ferredoxin affords complete protection. The reductase inactivated to 30% of original activity shows a loss of about two arginyl residues, whereas only one residue is lost in the NADP+-protected enzymes. The data suggest that the integrity of at least two arginyl residues are requested for maximal activity of ferredoxin-NADP+ reductase: one residue being located near the NADP+-binding site, the other presumably situated in the ferredoxin-binding domain.     

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.     

Essential arginyl residues in yeast phosphoglyceromutase.

Inactivation of yeast phosphoglyceromutase (tetramer) with 1,2-cyclohexanedione correlates with the modification of six arginyl residues per mole of the enzyme. Protection experiments using 3-phosphoglycerate suggest that four arginyl residues (one residue per subunit) are involved in the binding of the substrate to the enzyme. The modified enzyme reversibly regained its activity upon incubation with hydroxylamine. The reactivity of lysyl residues which have been shown to be involved in the active site is markedly reduced in the enzyme inactivated with 1,2-cyclohexanedione, indicating that the lysyl and arginyl residues are in close proximity in the active site.     

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.     

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.     

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.     

4-Hydroxy-3-nitrophenylglyoxal. A chromophoric reagent for arginyl residues in proteins.

The chromophoric reagent, 4-hydroxy-3-nitrophenylglyoxal, is highly selective for the modification of arginine in aqueous solution at pH 7--9. The reagent also inactivates creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) in a manner analogous to that reported with phenylglyoxal.     

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.     

Functional consequences of modifying highly reactive arginyl residues of fructose 1,6-bisphosphatase. Loss of monovalent cation activation.

Modification of pig kidney fructose 1,6-bisphosphatase with 2,3-butanedione (in the presence of AMP) results in the loss of activation of the enzyme by monovalent cations. Under these conditions about 8 arginyl residues per mole of enzyme were modified. No other residues were modified. No loss of monovalent cation activation occurs when modification with 2,3-butanedione is carried out in the presence of AMP plus the substrate fructose 1,6-bisphosphate and 3.2 less arginyl residues were modified. Since fructose 1,6-bisphosphatase contains 4 subunits, it is suggested that one arginyl residue per subunit plays an essential role in monovalent cation activation of the enzyme. Studies on sulfhydryl group reactivity toward 5,5'-dithiobis(2-nitrobenzoic acid) explain the protection exerted by fructose 1,6-bisphosphate against the loss of monovalent cation activation in terms of an enzyme conformational change induced by substrate, which makes unreactive the essential arginyl residue. The results of the present paper, as well as previous evidence, are discussed in terms of the mechanism of monovalent cation activation of fructose 1,6-biphosphatase.     

Functional tyrosyl residues of carboxypeptidase A. The effect of protein structure on the reactivity of tyrosine-198

Coupling of bovine carboxypeptidase A with diazotized 5-amino-1H-tetrazole increases esterase activity, decreases peptidase activity slightly, and modifies one tyrosyl residue. Subsequent nitration of the azoenzyme has no further effect on esterase activity, decreases peptidase activity markedly, and modifies a second tyrosyl residue. Analysis of the azopeptides isolated from a chymotrypsin digest of the doubly modified enzyme by affinity, ion exchange, and high pressure liquid chromatography indicates that the principal residue modified by diazo-1H-tetrazole is Tyr-248. Analysis of the nitropeptides isolated by similar procedures indicates that nitration occurs mainly at Tyr-198. This residue becomes susceptible to modification only as a consequence of a conformational change that accompanies azo coupling of Tyr-248. These results describe a unique example of the influence of protein structure on the reactivity of functional amino acid residues and illustrate an important aspect of chemical modification of enzymes.     

Contribution of C-tail residues of potato carboxypeptidase inhibitor to the binding to carboxypeptidase A A mutagenesis analysis.

The role of each residue of the potato carboxypeptidase inhibitor (PCI) C-terminal tail, in the interaction with carboxypeptidase A (CPA), has been studied by the analysis of two main kinds of site-directed mutants: the point substitution of each C-terminal residue by glycine and the sequential deletions of the C-terminal residues. The mutant PCI-CPA interactions have been characterized by the measurement of their inhibition constant, Ki, in several cases, by their kinetic association and dissociation constants determined by presteady-state analysis, and by computational approaches. The role of Pro36 appears to be mainly the restriction of the mobility of the PCI C-tail. In addition, and unexpectedly, both Gly35 and Pro36 have been found to be important for folding of the protein core. Val38 has the greatest enthalpic contribution to the PCI-CPA interaction. Although Tyr37 has a minor contribution to the binding energy of the whole inhibitor, it has been found to be essential for the interaction with the enzyme following the cleavage of the C-terminal Gly39 by CPA. The energetic contribution of the PCI secondary binding site has been evaluated to be about half of the total free energy of dissociation of the PCI-CPA complex.     

Chemical modification of functional arginyl residues in beef kidney D-aspartate oxidase.

Chemical modification of beef kidney D-aspartate oxidase by phenylglyoxal is a biphasic process involving the transient formation of an enzymatic species with a decreased activity versus dicarboxylic substrates, an increased activity versus D-proline and a new activity versus other monocarboxylic D-amino acids which is absent in the native protein. Prolonged incubation with the modifier causes complete inactivation of the enzyme. The presence of the competitive inhibitor L-tartrate in the incubation mixture prevents enzyme inactivation. Kinetic and structural data suggest that complete loss of activity is paralleled by modification of eight arginine residues, of which two are critical for the specificity and the activity of the enzyme. We propose that the two essential arginine residues are located in the substrate binding site of D-aspartate oxidase.