334 Maundersvented by the inclusion of Mg 2+ in the reaction buffer, which stimu-
lates the monoesterase activity and almost completely inhibits
pyrophosphatase activity (23).
3.2. Temperature
The enzymic reaction occurs over a wide range of temperature, and
is usually employed within a range dependent on the substrate. The
rate of reaction at 37°C is twice that at 25°C (25).
3.3. pH
As its name implies, the enzyme is active and stable in mildly alka-
line solution (pH 7.5-9.5)(26). It is rapidly inactivated at acid pH (e.g.,
pH 5), because of the loss of Zn 2÷. Lost activity cannot be restored by
addition of zinc ions, but this inactivation is prevented by presence of
inorganic phosphate (27). The pH optimum of ClAP increases with
substrate concentration and decreases with increased ionic strength (1).
3.4. Cations
Zinc ions are essential for the activity of alkaline phosphatase. Co 2÷,
Mg 2+, Mn 2÷, or Hg 2+ have been reported to substitute for Zn 2÷ in some
cases (1,21). An unusual bacterial alkaline phosphatase (not E. coli
BAP) has been described that is inhibited by Zn 2+ ions (0.1 mM) and
instead requires 5 mM CaC12 for activity (28).
In the case of CLAP, Ca 2÷, Ni 2+, and Cd 2+ have minimal effect, and
Be 2÷ is inhibitory. Zinc itself has been reported to be inhibitory at high
concentrations (1), the kinetics of which are complex, and the inhibi-
tion is not observed in the presence of glycine (29). The inhibition can
also be overcome by the presence of magnesium (30). It has been
suggested that magnesium can stimulate the action of additional active
sites on the enzyme molecule (31).
3.5. Inhibitors
Inorganic orthophosphate, thiophosphate, and arsenate are strong
competitive inhibitors of alkaline phosphatase with low K i values (32)
ranging from 0.6-20 ~/depending on the buffer employed. Other
inhibitors of ClAP include borate, carbonate, pyrophosphate, iodoso-
benzoate, and iodoacetamide anions, and urea (1,33).
Metal-binding agents that complex with the zinc atoms can also inhibit,
including EDTA, cyanide, ct,t~'-dipyridyl, and 1,10-phenanthroline.