Abstract
Alkaline phosphatase catalyzes the hydrolysis of phosphate ester bonds, which results in the removal of phosphate from many biologically relevant molecules (i.e. DNA, proteins, carbohydrates). This enzyme functions in a wide variety of organisms ranging from bacteria to mammals and has in vitro applications for genetic engineering. Numerous kinetic and crystal structure studies over the past fifty years have revealed many details of the alkaline phosphatase catalytic mechanism. However, few studies have investigated the solution‐phase conformational changes that accompany/drive this catalytic mechanism. Urea has long been used as a denaturant of proteins at concentrations above 4M. At low concentrations (0 to 3 M), urea can be used as a probe to detect changes in the amount of solution‐exposed amide surface. The activity of the alkaline phosphatase was examined at various, low concentrations of urea. We present the first quantitative analysis of solution‐phase conformational changes during the E. coli alkaline phosphatase mechanism.
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