AbstractL-Difluoro-aspartic acid was prepared by the enzymic transamination of difluoro-oxaloacetate using aspartate aminotransferase (AAT). The purification and some physical properties of this novel compound sire reported. The chemical synthesis of D,L-difluoro-aspartic acid was attempted. The interaction of difluoro-aspartate with pig heart cytosolic AAT was studied. The observed potent inhibition by this compound in steady state kinetic experiments was interpreted as being due to the formation of an aldimine-AAT-difluoro-aspartate complex, an aminic-AAT-difluoro-aspartate complex and an enzyme-substrate-difluoro-aspartate ternary complex. The apparently aberrant formation of the latter two complexes is discussed. The interaction of difluoro-aspartate with the aldimine form of AAT was studied in detail. A complex of difluoro-aspartate with aldimine-AAT was detected and its dissociation constant determined by spectral titration. The spectrum of the complex was shown to be distinct from that of aldimine enzyme with an absorption maximum at 340nm the magnitude of which was insensitive to pH, and also a minor absorption band at ca 430nm. The pH variation of the dissociation constant was analysed. Stopped flow experiments provided lower bounds for the rate of formation and dissociation of the complex. Preliminary experimentation indicated the possibility of a very slow productive breakdown of the aldimine-AAT-difluoro-aspartate complex to yield an undentified keto- acid. The inefficiency of difluoro-aspartate as a substrate for AAT is discussed. Tentative conclusions about the molecular nature of the complex were drawn from the results of a circular dichroism study of the binding of difluoro-aspartate to aldimine-AAT and from an attempt to detect a difluoro- aspartate aldimine Schiff base by borohydride reduction. A molecular reaction scheme involving the tetrahedral transaldimination intermediate in equilibrium with a small amount of the difluoro-aspartate aldimine Schiff base is proposed. The pH variation of the kinetic parameters for the slow transamination reaction of difluoro-oxaloacetate with aminic-AAT was studied as a model for the pH variation of the transamination reaction. The observed invariance of Km and Vm with pH for the above reaction is discussed.
|Date of Award||1980|
Difluorinated analogues of oxaloacetate and aspartate as enzyme probes.
Dransfield, T. A. (Author). 1980
Student thesis: Doctoral Thesis › PhD