Abstract
A sensitive spectrophotometric method, which directly measures the formation of pyruvic oxime from pyruvate and NH
2
OH in the UV, is described for the detection of pyruvic oxime hydrolase activity in biochemical systems. The method was used in an attempt to detect pyruvic oxime hydrolase activity in cell-free extracts from an
Alcaligenes
sp. which can grow on pyruvic oxime. Although substantial pyruvic oxime + nitrite oxidative activity was detected in cells and cell-free extract of this bacterium, catalysis of pyruvic oxime formation was not observed within the error of the method (∼20% of the uncatalyzed rate constant of 1.9x10
−3
s
−1
at pH 7, 22°C). The rate of pyruvic oxime oxidation by cells and cell-free extract was at least 10
5
−10
6
times greater than the rate of its hydrolysis, thus implying that oxidation of pyruvic oxime need not require prior hydrolysis. The method would appear to be applicable to hydrolases directed toward a variety of oximes.