Metal ion catalysis of the transamination reaction

Abstract

Part I of the thesis deals with the formation and transamination of Schiff's base complexes of copper, pyridoxal phosphate and glutamate, and the transamination of reaction mixtures containing copper, pyridoxamine phosphate and alpha-ketoglutaric acid.The formation of the complex of copper,, pyridoxal phosphate and glutamate was found to be first order in both pyridoxal phosphate and glutamate and zero order in copper. Spectrophotometric studies showed isosbestiT points when reaction mixtures were scanned over the range 20,000-35.000 cm-1 indicating that only one step is involved.At high concentrations of copper (16 mM) the initial reaction rate became slightly dependent upon the copper concentration, and the initial optical density of the reaction mixture departed from that of pyridoxal phosphate. These deviations are explained by the postulation of an intermediate carbinolamine complex. The complex formed from copper, pyridoxal phosphate and glutamate transmigrated to give pyridoxamine phosphate and a-ketoglutaric acid.The presence of metal ions appears to catalyse the transamination reaction, copper being the most active. If account was taken of the fact that in the absence of metal ions the Schiff's base of pyridoxal phosphate and glutamate is largely hydrolysed, however, the rate of transamination was found to be less in the presence of metal ions than in their absence. The transamination of reaction mixtures containing copper, pyridoxamine phosphate and a-ketoglutaric acid took place without significant formation of the Schiff's base complex, due to the unfavourable equilibrium constant for the formation of the Schiff's base in this system. Transamination was found to be very much more rapid than in the case of the copper complexes of the Schiff's base of pyridoxal phosphate and glutamate. The transamination of pyridoxamine phosphate and alpha-ketoglutaric acid is first order in a-ketoglutaric acid (tailing off at higher concentrations of alphaKG) and exhibits a rate maximum at a copper concentration of twice that of pyridoxamine phosphate. This maximum is justified mathematically by assuming that the concentration of a complex of copper and the Schiff's base from pyridoxamine phosphate and alpha-lcetoglutarate is very low, and that the complex accepts a further Cu2+ ion at high' concentrations of copper. The transamination of. pyridoxamine phosphate was found to be preceded by what at first appeared to be an induction period, further study of which indicated that the very small change in absorbance was caused by a rate limiting dehydration of the carbinolaraine of pyridoxamine phosphate and a-ketoglutarate. Low concentrations of carbinolaraine; Schiff's base complex; Schiff's base and carbinolamine complex account for the first order nature of the reaction. The non-zero values of these concentrations probably cause the deviation from first order. Part II of the thesis is concerned with the evaluation.of the stability constants of the simple and mixed complexes of pyridoxal phosphate, pyridoxamine phosphate, glutamate and alpha-ketoglutarate. The pK values of pyridoxal.phosphate, pyridoxamine phosphate and a-ketoglutaric acid (necessary for stability constant determinations) were found by means of a potentiometric titration method. The stability constants of copper and nickel with pyridoxal phosphate; copper, nickel, cobalt and zinc with pyridoxamine phosphate phosphate; and copper, nickel, cobalt and zinc with a-ketoglutaric acid were'also determined by a similar method. The stability constants as normally defined are shown to be meaningless where the complex can accept protons at p[ligand anion] values of 0.5 and 1.5 and results obtained by the usual procedures have been converted to more meaningful results when the pK values of the complex are known. A graphical method of determining the stability constants of the complexes of the Schiff's base of pyridoxal phosphate and glutamate has been developed. The method, relies on absorbance readings taken at one wavelength osly. A graph is plotted of Log(Stability Constant) against assumed values of the extinction coefficient of the complex The intersection of these lines for several sets of experimental conditions gives the required value of the stability constant. The equilibrium constant for the formation of the Schiff1s base of pyridoxal phosphate and glutamate, required in the above, was found by the usual graphical method at several pH values. A potentiometric titration method was used to evaluate the stability constants of complexes of the type MPpGg where P and G. represent pyridoxal (or pyridoxamine) phosphate and glutamate (or a-ketoglutarate) respectively, and where p and g can take values up to 2. A computer was used to solve the complex equations which were derived to describe the system.