Enzymes with Two Optimum pH Values

Andre Kimura Okamoto, Márcio Andre Miranda, Jose Mauro Granjeiro, Hiroshi Aoyama

Different chemical compounds were tested as buffers for the bovine milk acid phosphatase catalyzed reactions to elucidate the presence of an additional optimum pH value. Imidazole, histidine, alanine, glycine, malate, oxalate, succinate, citrate and acetate were studied as buffers, using p-nitrophenylphosphate or inorganic pyrophosphate as substrates. An optimum pH range between 4.5 and 6 was obtained for all the buffers studied. However, with glycine, and alanine, an additional pH optimum around 3 was observed. The specificity constant value (Vmax/Km), at pH 3, for glycine, was about twice the value obtained at pH 5 with acetate. The additional activity at pH 3 was also observed for other acid phosphatases. Our results suggest the existence of an inverse ratio between the negative character of the buffer and the reaction velocity.