L-ASPARTATE OXIDASE FROM ESCHERICHIA-COLI .1. CHARACTERIZATION OF COENZYME BINDING AND PRODUCT INHIBITION

This paper reports the biochemical characterization of the flavoprotei n L-aspartate oxidase from Escherichia coli. Modification of a previou sly published procedure allowed overexpression of the holoenzyme in an unproteolysed form. L-Aspartate oxidase is a monomer of 60 kDa contai ning 1 mol of non-covalently bound FAD/mol protein. A polarographic an d two spectrophotometric coupled assays have been set up to monitor th e enzymatic activity continuously. L-Aspartate oxidase was subjected t o product inhibition since iminoaspartate, which results from the oxid ation of L-aspartate, binds to the enzyme with a dissociation constant (K-d) equal to 1.4 mu M. The enzyme binds FAD by a simple second-orde r process with K-d 0.67 mu M. Site-directed mutagenesis of the residue s E43, G44, S45, F47 and Y48 located in the putative binding site of t he isoallossazinic portion of FAD reduces the affinity for the coenzym e.