Studies on the Brewer's yeast D-glucose 6-phosphate dehydrogenase.

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Title Studies on the Brewer's yeast D-glucose 6-phosphate dehydrogenase.
Publication Type dissertation
School or College School of Medicine
Department Biochemistry
Author Wu, James, Tsai-Yuan.
Date 1972-08
Description The equilibrium binding between D-glucose 6-phosphate dehydrogenase and its individual substrates e.g., TPN+, D-glucose 6-phosphate and TPNH have been studied by such methods as equilibrium dialysis, gel-filtration, difference spectrophotometric titration, and fluorescence techniques. All the results have been plotted according to the procedure of Scatchard. The values of K'd (apparent dissociation constant) which have been determined in the present study are consistent with those values estimated previously by Roy and Kuby from steady-state kinetics. In the case of TPN+, linear Scatchard plots were obtained in the presence of EDTA, but concave-downward curves resulted in its previous conclusions drawn by Yue and Kuby from their ultracentrifugal titration study, viz., that EDTA prevents TPN+-induced enzyme dimerization and that only a single intrinsic equilibrium dissociation constant exists in that system. In the absence of EDTA, however, more than one binding constant is required to describe the entire binding curve. The extrapolated values of N(maximum number of moles of substrate bound per 1 mole of enzyme) from the present study show that there are 2 binding sites for either TPN+ D-glucose 6-phosphate, or TPNH, per enzyme monomer of molecular weight of 102,000 g mole[-1]. The binding of D-glucose 6-phosphate to the enzyme yielded linear Scatchard plots at relatively low substrate concentration even in the absence of EDTA; but an additional nonspecific binding site was observed in the presence of relatively high concentrations of D-glucose 6-phosphate, with lower K'd of approximately 1 x 10[-4] M. The total amino acid composition of D-glucose 6-phosphate dehydrogenase has been analyzed after acid hydrolysis for several time periods. Hydrolysis with barium hydroxide was used for tryptophan determination. Amino acid analysis after preformic acid oxidation or spectrophotometric measurements after reaction with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) have indicated there are only maximum of 4 sulfhydryl groups per enzyme "monomer" (or a maximum of 2 per subunit). No disulfide linkages were found in the enzyme molecule. DTNB was also found to inactivate D-glucose 6-phosphate dehydrogenase and to react with one sulfhydryl group per subunit. The inactivation proved to be slow and followed the pseudo first-order kinetics. The activity of the DTNB-inactivated enzyme could be fully recovered by incubating with dithioerythretol, which indicate that one sulfhydryl group per subunit is at or close to the active site of the enzyme. Furthermore, D-glucose 6-phosphate or TPN+ protects the enzyme from inactivation by DTNB. Equilibrium dissociation constants for TPN+ and glucose 6-phosphate can be derived from the pseudo-first order inactivation rate constants measured in the presence of varying concentrations of TPN+ or D-glucose 6-phosphate. The values are in good agreements with those values determined by the other methods. A procedure for the enzymatic preparation (from TPN+) and purification (by chromatography) of TPNH, from TPN+ of a very low salt concentration is described. TPNH sample relatively free from salt contaminants could also be obtained. The stability of TPNH has also been studied at pH values from pH 2 to 10 and at three different temperatures. The effects of phosphate and acetate concentrations on the rate of TPNH degradation were studied at constant pH values and at fixed ionic strengths. In contrast to the case for DPNH the reaction orders with respect to hydronium ion, phosphate or acetate concentration were all close to ca. 0.5 for the TPHN degradation but ca. 1 for DPNH, pointing to different mechanism for degradation of TPNH.
Type Text
Publisher University of Utah
Subject Dehydrogenases; Biochemistry
Subject MESH Yeasts; Amino Acids
Dissertation Institution University of Utah
Dissertation Name PhD
Language eng
Relation is Version of Digital reproduction of "Studies on the Brewer's yeast D-glucose 6-phosphate dehydrogenase." Spencer S. Eccles Health Sciences Library. Print version of "Studies on the Brewer's yeast D-glucose 6-phosphate dehydrogenase." available at J. Willard Marriott Library Special Collection. QR6.5 1972 .W8.
Rights Management © James Ysai-Yuan Wu.
Format application/pdf
Format Medium application/pdf
Identifier us-etd2,56
Source Original: University of Utah Spencer S. Eccles Health Sciences Library (no longer available).
Funding/Fellowship National Science Fundation Research Grant.
ARK ark:/87278/s6pn9m95
Setname ir_etd
ID 193819
Reference URL https://collections.lib.utah.edu/ark:/87278/s6pn9m95
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