Title

A study of coenzyme binding in pyruvate decarboxylase from brewer's yeast /|cby John H. Wittorf

Abstract

The synthesis of a new thiamine analogue, 2'-hydroxythiamine, is reported. Kinetic studies with thiamine pyrophosphate analogues and apopyruvate decarboxylase (EC 4.1.1.1) from brewer's yeast, gave the values of 2.3 X 10^-5 M as the K_m for thiamine pyrophosphate and 2.0 X 10^-5 M as the K_m for 2'-ethylthiamine pyrophosphate. The V_max for the latter was 14% that of thiamine pyrophosphate. Inhibitor constants, K_i, were determined for the following competitive inhibitors of thiamine pyrophosphate with the apoenzyme. All values are given for the pyrophosphate esters: tetrahydrothiamine, 0.65 X 10^-5 M; oxythiamine, 2.0 X 10^-5 M; 2'-n-butylthiamine, 4.5 X 10^-5 M; 2'-methoxythiamine, 7.0 X 10^-5 M; pyrithiamine, 7.8 X 10^-5; thiochrome, 15. X 10^-5 M; 2'-desmethylthiamine, 22. X 10^-5 M; 2'-hydroxythiamine, 38. X 10^-5 M. None of the inhibitors exhibited coenzyme activity. A hydrophobic interaction of the 2'-methyl group of thiamine pyrophosphate with the apoenzyme is suggested from these studies. The formation of a fluorescent complex at pH 6.7 between apopyruvate decarboxylase and thiochrome pyrophosphate was detected and found to be dependent upon Mg(II) ions. A similar complex between thiochrome and the apoenzyme could not be detected, demonstrating the importance of the pyrophosphate function in binding to the protein. The shift in the fluorescence emission spectrum of thiochrome pyrophosphate toward lower wavelengths upon complex formation with the apoenzyme, coincided with the behavior of thiochrome in solvents of decreasing dielectric constant. This latter observation suggests involvement of the thiochrome pyrophosphate with a hydrophobic region of the enzyme. A study of the pH dependency of the enzyme-coenzyme complex indicated considerable recombination of apoenzyme and coenzyme at alkaline pH, where dissociation of the coenzyme usually takes place. A rationale for the interpretation of the pH-behavior of the enzyme-coenzyme complex is offered. An amino acid analysis of a highly purified sample of pyruvate decarboxylase, considered to be essentially homogeneous, is reported. Assuming a molecular weight of 175,000 for the enzyme, a total of 1317 amino acid residues were calculated, of which 52.1% fall into the non-polar category. the half-cystine content was calculated as 10.3%, and the proline content, as 4.6%. The specific volume was calculated as 0.737 ml per g. A single low-angle X-ray diffraction study gave a value of 35.5 ± 1.5 A for the radius of gyration of pyruvate decarboxylase. Assuming a spherical shape, a diameter of 91.6 ± 4.0 A was calculated.

Degree

PhD

College and Department

Chemistry and Biochemistry

Rights

http://lib.byu.edu/about/copyright/

Date Submitted

1968-08-01

Document Type

Dissertation

Handle

http://hdl.lib.byu.edu/1877/Letd696

Keywords

Yeast, Enzymes, Pyruvate decarboxylase

Language

English

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