Cosolutes Modify Alkaline Phosphatase Catalysis through Osmotic Stress and Crowding Mechanisms

Examining the effects
of different cosolutes on in vitro enzyme kinetics
yielded glimpses into their potential behavior when
functioning in their natural, complex, in vivo milieu.
Viewing cosolute in vitro influences on a model enzyme,
calf intestinal alkaline phosphatase, as a combination of competitive
and uncompetitive behaviors provided quantitative insights into their
effects on catalysis. Observed decreases in the apparent specificity
constant, Kasp, caused by the presence
of polyethylene glycols or betaine in the reaction solution, indicated
interference with enzyme–substrate complex formation. This
competitive inhibition appeared to be driven by osmotic stress. Dextran
6 K and sucrose strongly impeded the subsequent conversion of the
bound substrate into a free product, which was marked by sharp reductions
in Vmax, uncompetitive inhibition. For
the same step, smaller noncarbohydrate cosolutes, triethylene glycol,
polyethylene glycol 400, and betaine, also behaved as uncompetitive
inhibitors but to a lesser extent. However, polyethylene glycol 8000
and 20,000 were uncompetitive activators, increasing Vmax. Polyethylene glycol of molecular weight 1000 displayed
intermediate effects between these two groups of noncarbohydrate cosolutes.
These results suggested that crowding has a strong influence on free
product formation. The combination of competitive and uncompetitive
effects and mixed behaviors, caused by the cosolutes on calf intestinal
alkaline phosphatase kinetics, was consistent with the trends seen
in similar enzyme–cosolute studies. It is proposed that the
double-displacement mechanism of alkaline phosphatases, shared by
many other enzymes, could be the root of this general observation.