Biodegradative Threonine Dehydratase by Glyoxylate"

A timeand concentration-dependent irreversible inactivation of the Escherichia coli biodegradative threonine dehydratase by glyoxylate is accompanied by enzyme aggregation, apparent covalent binding of 4 mol of glyoxylate/mol of aggregated species, and a displacement of the absorption maximum of the enzyme-bound pyridoxal phosphate from 413 to 388 nm (Park, L. S., and Datta, P. (1979) J Biol. Chem 264, 7927-7934). An examination of the visible spectrum of the inactive enzyme reisolated from the incubation mixture shows no absorption peak at 4 13 nm. Further, free pyridoxal phoshate is recovered from the incubation mixture as judged by its spectral characteristics and its ability to restore catalytic activity to an inactive preparation of apodehydratase. Direct fluorometric measurements indicate that the inactive enzyme has 2 mol of bound pyridoxal phosphate/mol of protein as compared to 4 mol/mol in the native enzyme. Interestingly, titration of apoenzyme with authentic pyridoxal phosphate restores full enzyme activity when approximately 2 mol of pyridoxal phosphates/mol of enzyme are added back, indicating that only half the total number of pyridoxal phosphates in the native enzyme are catalytically involved. In addition to the resolution of pyridoxal phosphate during enzyme inactivation, incubation of the enzyme with glyoxylate results in a time-dependent change in the circular dichroism spectra of the enzyme both in the 220and 275-nm regions. A comparison of the rates of changes in various parameters as a function of enzyme inactivation suggests that the glyoxylate-mediated loss in catalytic activity is due to changes in the conformation of the enzyme subunits accompanied by the resolution of 2 mol of enzymebound pyridoxal phosphate; aggregation of the protein and complete occupancy of all four sites on the aggregated species by glyoxylate follow the inactivation process.