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This paper reports the biochemical characterization of the flavoprotein L-aspartate oxidase from Escherichia coli. Modification of a previously published procedure allowed overexpression of the holoenzyme in an unproteolysed form. L-Aspartate oxidase is a monomer of 60 kDa containing 1 mol of non-covalently bound FAD/mol protein. A polarographic and two spectrophotometric coupled assays have been set up to monitor the enzymatic activity continuously. L-Aspartate oxidase was subjected to product inhibition since iminoaspartate, which results from the oxidation of L-aspartate, binds to the enzyme with a dissociation constant (Kd) equal to 1.4 μM. The enzyme binds FAD by a simple second-order process with Kd 0.67 μM. Site-directed mutagenesis of the residues E43, G44, S45, F47 and Y48 located in the putative binding site of the isoallossazinic portion of FAD reduces the affinity for the coenzyme. [ABSTRACT FROM AUTHOR]

The effect of the sulfhydryl reagent o-mercuriphenyl β-D-galactoside chloride (synthesis given) on β-galactosidase from Escherichia coli K 12 was investigated in view of the suggestion by Wallenfels and Malhotra in 1960 that cysteine participates in the catalytic mechanism of the enzyme. This reagent inactivated β-galactosidase in amounts smaller than 8 sulhydryl equivalents, corresponding to one tenth of the total amount of protein SH groups. No change in the ratio of hydrolase and transferase activities was found in the partially inactivated enzyme. Competitive inhibitors were partially protective and inactivation could be reversed with 2-mercaptoethanol. It was established that o-mercuriphenyl β-D-galactoside chloride could not be regarded as an active site directed reagent for β-galactosidase. The compound is enzymatically hydroiyzed and its reaction product o-mercnriphenyt chloride is a more effective inhibitor than the galactoside itself. The sedimentation behaviour of the inactivated enzyme was comparable to the native enzyme. Yet upon standing, the modified protein tended to dissociate into the monomer of molecular weight 130000 and to aggregate. The modified and inactivated enzyme still exhibited a strong binding capacity: 4 independent binding sites and a dissociation constant equal to 21 μM for the competitive inhibitor 3-nitro4-aminophenyl β-D-thiogalactoside were found by ultracentrifugation studies with absorption optics. Although some SH groups seem to be indirectly involved irt enzyme function, it is evident that their modification, resulting in inactivation, does not significantly reduce substrate binding in the tetramer and that no SH group is involved in the catalytic mechanism itself. [ABSTRACT FROM AUTHOR]