Masters Theses

Date of Award

6-1981

Degree Type

Thesis

Degree Name

Master of Science

Major Professor

Jorge E. Churchich

Committee Members

R. H. Feinberg, J. G. Joshi

Abstract

The inhibition kinetic patterns obtained when ATP and pyridoxal analogues are used as inhibitors of the reaction catalyzed by pyridoxal kinase are consistent with a rapid equilibrium random Bi-Bi, in which binary complexes, i.e., enzyme-ATP and enzyme-pyridoxal, are formed in kinetically significant amounts. Protein fluorescence quenching was used to determine the dissociation constant (KD = 25µM) of ATP-Zn bound to the nucleotide site of the kinase. The binding of ATP to the kinase induces a conformational change which is transmitted to other areas of the macromolecule.

Pyridoxaloxime, a competitive inhibitor of pyridoxal, was used as a probe of the pyridoxal binding site. It binds to the kinase with a KI = 2µM and displays a fluorescent decay time of 7.8 nanoseconds. Time emission anisotropy measurements yield a rotational correlation time for bound pyridoxaloxime of approximately 2 nanoseconds, which is considerably shorter than the rotational correlation time of the protein (Φ =38 nanoseconds). The fast rotation of pyridoxaloxime remains unaffected by the binding of ATP.

A rapid and simple method by which pig brain Pyridoxine-5-phosphate oxidase can be purified in high yield is described. m-Carboxyphenyl-pyridoxamine phosphate (CPPp), an excellent substrate for pyridoxine-5-phosphate oxidase, was used as a fluorescent prode for the catalytic site. The fluorescence enhancement detected upon mixing the apoenzyme with Cppp suggested the interaction of this synthetic substrate and protein. This result indicated that the substrate recognized its binding site even in the absence of the cofactor FMN.

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