47] Active site of l-asparaginase: Reaction with diazo-4-oxonorvaline

Abstract

Publisher Summary L-Asparaginase from Escherichia coli catalyzes the conversion of the diazo ketone analog of L-asparagine, diazo-4-oxo-L-norvaline (DONV), to 5-hydroxy-4-oxo-L-norvaline and is also inactivated by covalent attachment of the analog to a region in the active site. In aqueous buffers the decomposition of DONV is so rapid, in comparison to the inactivation reaction, that labeling of the active site is not technically reasonable. If, however, L-asparaginase is permitted to react with DONV in the presence of 50% dimethyl sulfoxide (DMSO), there is virtually no catalytic decomposition of DONV, but a 400-fold increased rate of inactivation by DONV. The enzyme itself is stable in 50% DMSO for several hours at 25 ° in the absence of DONV, and at least 85% activity can be regained within a minute by dilution with aqueous buffers. The chapter discusses the synthesis of 5-DONV and its reaction conditions.