On the mechanism of enzyme action. LXII. Acetylation of alkaline phosphatase

Abstract

Alkaline phosphatase, which is inactivated by treatment with ketene, nitrous acid, formaldehyde, or phenyl isocyanate, forms an active acetylation product when acetylated with acetic anhydride in sodium acetate buffer as well as in several others which were employed. The pH optimum of alkaline phosphatase is shifted toward the alkaline side by acetylation with acetic anhydride as in the case of the active acetylation product of acid phosphatase. These results are comparable to those obtained with trypsin thereby indicating that this shift is a basic phenomenon. Zinc ions stabilize alkaline phosphatase in glycine buffer, magnesium ions activate the enzyme and decrease its stability, while when both zinc and magnesium ions are present in the incubating glycine buffer media the enzyme is further activated and is stabilized. Acetylation stabilizes the enzyme under all conditions in alkaline media, most strikingly in glycine buffer with no ions added. The stability imparted to the enzyme by acetylation and by zinc ions is additive. This is similar to the additive effect on stability of trypsin by calcium ions and acetylation. In acidic media, the acetylated enzyme is less stable than the native enzyme, which possesses a maximum stability zone around pH 4. The parallelism with the effect of acetylation on trypsin is thus further extended, and it is suggested that the stabilization of enzymes in the alkaline pH region, and decreased stability in acidic media is a general effect of acetylation, and applicable also to other enzymes.