Date of Award

12-1969

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biochemistry

Research Advisor

Charles Walter

Committee

William Purcell Ronald Quintana Nathan Sloane William Jefferson

Abstract

The inducible, NAD-linked glycerol dehydrogenase (E.C. 1.1.1.6) of a guanine requiring mutant of A. aerogenes has been purified to homogeneity. The molecular weight of the pure enzyme was found to be 3.4 x 105 daltons. The sedimentation coefficient of the enzyme was 10.7 x 10-13 sec.-1. The diffusion constant was 3.07 x 10-7 cm2/sec.

The partial specific volume calculated from the amino acid composition was 0.72 ml/g. The following kinetic parameters were determined; Km glycerol, 2.4 x 10-3 m, Km NAD, 2.8 x 10-4 M, Km dihydroxyacetone, 5.1 x 10-4 M, Km NADH, 2.3 x 10-5 M.

During the course of the purification of the enzyme it was found that multiple peaks of enzyme activity were eluted from ion-exchange chromatography experiments. Ultracentrifugation experiments with the enzyme treated with urea and dithiothreitol, and with dithiothreitol and p- hydroxymercuribenzoate in the presence of guanidinium chloride indicated that the isolated enzyme is composed of subunits. The untreated enzyme, with a molecular weight of 3.4 x 105 daltons, is a hexamer of a species of molecular weight 5.6 x 104 daltons. This species in turn is a dimer of a species of molecular weight 2.8 x 104 daltons. Disulfide bonds between the smallest species are responsible for the stability of the dimer, while the untreated hexamer of the dimers is stabilized by ionic and hydrophobic forces Thirteen major peaks and one minor peak of enzyme activity were consistently eluted during ion-exchange experiments with the native enzyme. Polyacrylamide gel electrophoresis of the enzyme treated with dithiothreitol and urea indicated the presence of two species of protein. The amino acid composition of these two proteins and of the thirteen major peaks are consistent with the hypothesis that these two proteins represent the proteins of molecular weight 2.8 x 104 daltons observed in the ultracentrifuge and that the stiochiometry of the thirteen major peaks may be represented by the formula (A (12-n) Bn), n = 0, 1,..., 12, where A and B represent the two proteins separated by polyacrylamide gel electrophoresis.

DOI

10.21007/ptd.cghs.1969.0561

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