Kinetic constants alkaline phosphatase

Neither the occurrence of a constant value of Vmax or a constant product ratio is sufficient proof of the presence of an intermediate. It was seen for alkaline phosphatase that a constant value for Vmax is an artifact, and also that there is no a priori reason why the attack of acceptors on a Michaelis complex should not also give constant product ratios. In order for partitioning experiments to provide a satisfactory proof of the presence of an intermediate, they must be linked with rate measurements. When the rate measurements are restricted to steady state kinetics, the most favorable situation is when the intermediate accumulates. If the kinetics of equations 7.5 to 7.7 hold, it may be concluded beyond a reasonable doubt that an intermediate occurs. The ideal situation is a combination of partitioning experiments with pre-steady state studies, as described for chymotrypsin and amides. 

The analysis of substrate dose-response curves for alkaline phosphatase (ALP), immobilized in methanol-containing polymerization mixture, revealed at least two kinetically different forms of the enzyme (27), The high affinity enzyme component had the Michaelis constant = 0.8 mM, which was also measured for the soluble ALP. About 90% of the enzyme activity, however, had an average of 7 mM. The pH maximum of immobilized ALP was about one pH unit higher than for the native enzyme. However, trypsin and acid phosphatase, prepared in the presence of PEG, demonstrated single form kinetics with close to that of the soluble trypsin (30),... 

---