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Date of Award

1981

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Graduate School

First Advisor

Michael J. Minch

First Committee Member

James W. Blankenship

Second Committee Member

Charles A. Matuszak

Third Committee Member

Paul H. Gross

Fourth Committee Member

Silvio Rodriguez

Abstract

An analysis of the 360 MHz ('1)H NMR spectra of the title compounds in ('2)H(,2O) is presented. The ('3)J values for the ribose vicinal protons of S-adenosyl-L-methionine are consistent with a predominantly C(,3')-exo conformation and with one highly favored gauche-anti conformation about the C(,4')-C(,5') bond. The corresponding ('3)J values for S-adenosyl-L-homocysteine imply a similar C(,3')-exo ribose ring conformation, but the orientation about the C(,4')-C(,5') bond is distributed between two gauche-anti rotamers. The methionine side chain of S-adenosyl-L-methionine has approximately equal populations of rotational isomers about the C(,(alpha))-C(,(beta)) and C(,(beta))-C(,(gamma)) bonds, whereas the side chain of S-adenosyl-L-homocysteine exhibits a conformational preference for the gauche-anti conformations about the C(,(alpha))-C(,(beta)) bond. ('1)H and ('13)C NMR spectra of commercially available samples of (-)S-adenosyl-L-methionine consistently available samples of (-)S-adenosyl-L-methionine consistently reveal the presence of a small amount of the (+) sulfonium diastereomer. This assignment was confirmed by the synthesis of both the ('1)H and ('13)C methyl derivatives of S-adenosyl-L-homocysteine. Arguments are presented to explain the failure of previous workers to detect (+)S-adenosyl-L-methionine in biological preparations. The possible biological significance of this finding is discussed with reference to the enzyme S-adenosyl-L-methionine: L-homocysteine S-methyltransferase which employs both (-)S-adenosyl-L-methionine and (+)S-adenosyl-L-methionine as methyl donors. An improved purification of S-Adenosyl-L-methionine: L-homocysteine S-methyltransferse (EC. 2.1.1.10) from Saccharomyces cerevisiae is reported. The enzyme was purified approximately 1500-fold by toluene extraction, ammonium sulfate precipitation, Sephadex G25 gel exclusion chromatography, DEAE Sephadex ion-exchange chromatography and affinity chromatography on L-methionine AH-Sepharose 4B. The procedure affords a 12-fold increase in yield and a 3-fold increase in purification over the previous fractionation scheme. It is hoped that the availability of a highly purified preparation of the enzyme will provide the basis for an investigation of enzymatic transmethylation in which the fate of the sulfonium diastereomers of S-adenosyl-L-methionine is directly observable by NMR spectroscopy.

Pages

72

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