Two tyrosines are supposed to be essential for the activity and to participate in the stabilization of Escherichia coli inorganic pyrophosphatase (PP(i)ase) against heat denaturation [Samejima, T., Tamagawa, Y., Kondo, Y., Hachimori, A., Kaji, H., Takeda, A. and Shiroya, Y. (1988) J. Biochem. (Tokyo) 103, 766-772]. To locate these two tyrosines in the amino acid sequence, we substituted all the eight tyrosines of E. coli PP(i)ase with phenylalanine and studied the properties of these YF mutant PP(i)ases.Interestingly, substitution of the tyrosines (Tyr51, Tyr55 and Tyr141) conserved with the amino acid sequence of yeast PP(i)ase [Lahti, R., Kolakowski, L. F., Heinonen, J., Vihinen, M., Pohjanoksa, K. and Cooperman, B. (1990) Biochim. Biophys. Acta 1038, 338-345] exerted the most drastic effects on the structure and activity of E. coli PP(i)ase. PP(i)ase variants YF51, YF55 and YF141 had 64%, 7% and 22% of the wild-type PP(i)ase activity, respectively. Furthermore, PP(i)ase variant YF141 had an increased sensitivity to heat denaturation, whereas mutant PP(i)ase YF55 displayed a profound conformational change, as demonstrated by the binding of the fluorescent dye 9-(diethylamino)-5H-benzo(alpha) phenoxazine-5-one (Nile red) that monitors the hydrophobicity of protein surfaces. None of the tyrosines of E. coli PP(i)ase seem to be essential for catalysis, but Tyr55 and Tyr141 are important for the structural integrity of E. coli PP(i)ase.
|Tidskrift||European Journal of Biochemistry|
|Status||Publicerad - 1991|