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.     

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.     

An essential arginyl residue in yeast hexokinase

The inactivation of yeast hexokinase A (ATP:d-hexose 6-phosphotransferase, EC 2.7.1.1) by phenylglyoxal obeys pseudo first-order kinetics. Formation of a reversible enzyme-reagent complex prior to modification is suggested by the observed saturation kinetics. Loss of activity correlates with the incorporation of 1 mol of [¹⁴C]phenylglyoxal per mol 50 000 dalton subunit. No significant conformational occurs concomitantly. Inactivation is attributable to modification of an arginyl residue. The pattern of protection by substrates and analogs favors an interaction of this essential residue with the terminal phosphoryl group of ATP or glucose 6-phosphate.     

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.     

A sensitive and reproducible procedure for the assay of arginyl-tRNA: Protein arginyl transferase

The activity of arginyl-tRNA: protein arginyl transferase was found to be enhanced four- to sevenfold by substituting bovine α-lactalbumin for bovine serum albumin, the standard acceptor protein used thus far. With α-lactalbumin as the acceptor protein in place of serum albumin, a sensitive and reproducible procedure for the transferase assay was established.     

An essential arginyl residue in yeast hexokinase.

The inactivation of yeast hexokinase A (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) by phenylglyoxal obeys pseudo first-order kinetics. Formation of a reversible enzyme-reagent complex prior to modification is suggested by the observed saturation kinetics. Loss of activity correlates with the incorporation of 1 mol of [14C]phenylglyoxal per mol 50 000 dalton subunit. No significant conformational change occurs concomitantly. Inactivation is attributable to modification of an arginyl residue. The pattern of protection by substrates and analogs favors an interaction of this essential residue with the terminal phosphoryl group of ATP or glucose 6-phosphate.     

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.