Abstract
5-Oxo-L-prolinase (L-pyroglutamate hydrolase, EC 3.5.2.9) from Alcaligenes sp. F-137 has been purified to homogeneity as judged by acrylamide gel electrophoresis and ultracentrifugation. The molecular weight of the enzyme was about 106, 000 and 123, 000 by gel filtration and sedimentation equilibrium, respectively. Upon disc electrophoresis in 0.1% sodium dodecyl sulfate the purified preparation migrates as a single band of molecular weight 126, 000. The sedimentation coefficient (so20, w) of the enzyme was 6.82 S by ultracentrifugation and its isoelectric point was pH 5.1 by isoelectric focusing. The enzyme catalyzed the hydrolysis of 5-oxo-L-proline to glutamate coupled with the hydrolysis of ATP to ADP and inorganic phosphate, stoichiometrically. K+ and Mg2+ were required for the enzyme reaction. Michaelis constants of the enzyme were 0.07mM for 5-oxo-L-proline and 0.32mM for ATP. The enzyme was maximally active at pH 7.8 and 50°C. The enzyme was inhibited by p-chloromercuribenzoate of N-ethylmaleimide.
