Arginine 328 of the beta-subunit of the mitochondrial ATPase in yeast is essential for protein stability

• Published in: The Journal of biological chemistry 1988-04, Vol.263 (12), p.5634-5639
• Main Author: Mueller, D M
• Format: Article
• Language: English
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; ARGININA; ARGININE; Base Sequence; Biological and medical sciences; Catalysis; CHEMICOPHYSICAL PROPERTIES; DNA, Fungal - genetics; Enzymes and enzyme inhibitors; ENZYMIC ACTIVITY; Fundamental and applied biological sciences. Psychology; GTP Phosphohydrolases; Half-Life; HIDROLASAS; HYDROLASE; HYDROLASES; Kinetics; MITOCHONDRIA; Mitochondria - enzymology; MITOCHONDRIE; MITOCONDRIA; Molecular Sequence Data; Mutation; Phenylglyoxal - pharmacology; PLASTE; PLASTIDIOS; PLASTIDS; PROPIEDADES FISICO-QUIMICAS; PROPRIETE PHYSICO-CHIMIQUE; Protein Conformation; PROTEINAS; PROTEINE; PROTEINS; Proton-Translocating ATPases - antagonists & inhibitors; Proton-Translocating ATPases - genetics; Proton-Translocating ATPases - metabolism; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Structure-Activity Relationship; Transformation, Genetic
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)60612-X

The mitochondrial ATPase is rapidly inactivated by the arginine selective reagent phenylglyoxal. Recently, the purported major reacting residue has been reported for the chloroplast enzyme (Viale, A. M., and Vallejos, R. H. (1985) J. Biol. Chem. 260, 4958-4962) corresponding to Arg-328 in the beta-subunit of the yeast Saccharomyces cerevisiae mitochondrial ATPase, a highly conserved residue in the ATPase. This arginine residue was concluded to be in the active site of the ATPase and possibly involved in the binding of nucleotides. To test this hypothesis, site-directed mutagenesis of the yeast enzyme has been used to replace Arg-328 with alanine and lysine. The modified genes were transformed into a yeast strain, DMY111, which contained a null mutation in the gene coding for the beta-subunit of the ATPase. Both of the substitutions were functional in vivo as demonstrated by the ability of yeast transformants to grow on a nonfermentable carbon source. The water soluble F1-ATPase with Ala-328 and Lys-328 were extremely unstable, but could be stabilized with glycerol. The rate of enzymatic decay followed first order kinetics with half-lives of 1.1 and 4.0 min for the mutants with Ala-328 and Lys-328 in 10% and 5% glycerol, respectively, while the wild type enzyme was stable even in the absence of glycerol. Kinetic analysis of both ATPase and GTPase has been determined. The wild type enzyme had two observable apparent Km and Vmax values for ATPase which were 0.056 mM-1 and 67 units/min/mg and 0.140 mM-1 and 100 units/min/mg. The mutant enzyme containing Lys-328 showed similar kinetic values of 0.066 mM-1 and 23 units/min/mg and 0.300 mM-1 and 43 units/min/mg. The mutant enzyme containing Ala-328, however, only demonstrated a single site with values of 0.121 mM-1 and 45 units/min/mg. In contrast to ATPase activity, kinetic values for GTPase were nearly identical for the wild type and mutant enzymes. Opposite to predicted results, the mutant enzymes were more sensitive to the reagent phenylglyoxal. These results indicate that Arg-328 is important for protein stability, but not involved in catalysis.