The effects of molecular crowding on the kinetics and small molecule inhibition of alkaline phosphatase

Abstract

Enzymes have adapted to function in complex environments crowded with many other solutes. To get a better understanding of in vivo crowding, we used polyethylene glycol (MW 8000) and dextran (MW 6000) as in vitro crowding agents and observed their effects both the kinetics of alkaline phosphatase-catalyzed para-nitrophenyl phosphate hydrolysis and the inhibition of this reaction by competitive and uncompetitive inhibitors. Reaction kinetics were followed using UV-visible spectrometry and the initial rate was analyzed using Michaelis-Menten kinetics to arrive at an apparent Vmax and Km for each reaction condition. We observed that polyethylene glycol increased Vmax while a similar amount of dextran strongly reduced Vmax. Crowding by these agents also significantly altered the effectiveness of small-molecule inhibitors and suggests that the action of drugs can be different going from “bench” research to “bedside” application.

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