KINETIC PROPERTIES OF COBALT ALKALINE PHOSPHATASE

The kinetics of cobalt alkaline phosphatase of Escherichia coli have been studied in an effort to elucidate the role of the metal atoms in the function of this enzyme. When assayed at pH 8.0, the enzymatic activity of the cobalt enzyme is about 12% of that of the native enzyme. In many respects, the native zinc and cobalt enzymes are similar. They hydrolyze a variety of phosphomonoesters at essentially similar rates. High ionic strength activates both metalloenzymes and phosphate, arsenate, and phenylphosphonate inhibit them competitively. However, the two metalloenzymes differ in their pH dependence, phosphotransferase activity, and rate-limiting step. For the cobalt enzyme, Fmai increases with pK values of 6.8 and 10.5. Ku increases with a pK of 9.6. This pH dependence differs qualitatively and quantitatively from that observed for the zinc enzyme, suggesting that the metal atom at the active site of alkaline phosphatase may function both in substrate binding and in catalysis. In contrast to the native enzyme, the cobalt enzyme lacks phosphotransferase activity. Assuming the currently accepted formation of a phosphoryl-enzyme intermediate, studies of the transient phase of the hydrolysis of phosphomonoesters suggest that dephosphorylation of the phosphoryl-enzyme is the rate-limiting step for cobalt phosphatase over the pH range from 4.5 to 11.0. This step is rate limiting for the native enzyme only at pH values less than 7. Dephosphorylation is slower for the cobalt enzyme than for native phosphatase, while the apparent rate for phosphorylation of the enzyme by substrate appears to be faster for the cobalt enzyme. © 1969, American Chemical Society. All rights reserved.