A determination of Ks, the substrate constant, for serum cholinesterase

Abstract

The hydrolysis of o-nitrophenyl butyrate by human serum cholinesterase was activated by alcohols and acetone. Lineweaver-Burk plots of the activities at. a number of butanol concentrations gave lines, which, on extrapolation into the lower left-hand quadrant, approximated a common point of intersection. The point of intersection could be used to determine Ks, k2e and k1e directly from the plot. It is suggested that this plot is typical of k3 activation. The activation was dependent on the chain length, methanol activating least and pentan-1-ol most. Butan-1-ol activated more than its stereoisomers. The hydrolysis of p-nitrophenyl butyrate was inhibited by butan-1-ol. Lineweaver-Burk plots of the activities at a number of butan-1-ol concentrations approximated a common point of intersection in the lower left-hand quadrant in a manner similar to the activation plot. This plot is probably typical of k3 inhibition. The human serum cholinesterase o-nitrophenyl butyrate Ks was 0.072 mM, compared with Km 0.139 mM, and the p-nitrophenyl butyrate Ks was 0.38 mM, compared with Km 0.80 mM. The serum cholinesterase Km of these substrates has therefore a mixed meaning, being both k3- and Ks-dependent. o-Nitro-phenyl butyrate hydrolysis by a partially purifed norse-serum cholinesterase preparation was also activated by butan-1-ol. o-Nitrophenyl acetate hydrolysis by human serum cholinesterase was activated by butan-1-ol and p-nitrophenyl acetate hydrolysis was inhibited. It is suggested that alcohols affect the activity of hydrolytic enzymes by changing some aspect of the environment on which k3 is dependent. The effect has been called "environmental" to distinguish it from specific inter-action. On the basis of an environmental effect alcohols may provide a useful method for determining the Ks of many hydrolytic enzymes.