STUDIES OF BIOORGANIC STEREOCHEMISTRY, PART I: STEREOCHEMICAL PROBES OF CHEMICAL CARCINOGENESIS BY DIMETHYLNITROSAMINE; PART II: MECHANISTIC STUDIES OF PYRIDOXAL PHOSPHATE-CONTAINING AMINO ACID RACEMASES

SHU-JANE SHEN, Purdue University

Abstract

Part I: To study the steric course and mechanism of DNA alkylation by the carcinogen DMN and to obtain information on the intracellular environment in which this process takes place, R- and S-DMN were synthesized from chiral acetic acid. Conditions were worked out for the metabolic activation of labeled DMN in the presence of DNA to give alkylated DNA, as well as for the degradation of the alkylated DNA to give labeled 7-methylguanine and for the further stereospecific conversion of the methyl group into acetic acid for analysis of its configuration. Studies on the alkylation of model nucleophiles showed that the transfer of the methyl group from activated chiral R- and S-DMN to the solvent, water, proceeds with about 60% inversion and 40% racemization. Surprisingly, however, transfer of the methyl group to the sulfur of 3,4-dichlorobenzenethiol occurs with a high degree of retention of configuration. Several mechanisms are discussed to account for this unexpected observation. Part II: Amino acid racemization by PLP-containing enzymes requires abstraction of a proton from C(,(alpha)) on one face of the PLP-amino acid Schiff's base followed by addition of a proton on the opposite face. The two proton transfers may be mediated by two bases positioned on opposite sides of the complex or by a single base with reorientation of substrate relative to this base. Observation of internal return of H(,(alpha)) in the conversion of, e.g., L-alanine to D-alanine would strongly support a single base mechanism. This question was studied with three enzymes catalyzing alanine racemization. Observing conversion of L-{(alpha)-('2)H}alanine in H(,2)O or L-alanine in ('2)H(,2)O to the D isomer in the presence of D-amino acid acetyltransferase as trapping system to ensure near single-turnover conditions, it was found that tyrosine phenol-lyase operates with at least 6% internal return. The enzyme also catalyzes (beta)-hydrogen exchange. Amino acid racemase from P. striata shows internal return of between 0.75 and 10% superimposed on a solvent isotope effect K(,H)/K(,D) (TURNEQ) 4.1. Alanine racemase from E. coli shows a solvent isotope effect of about 2.3 and no internal return. Thus, for two of the three enzymes a single base mechanism is indicated, whereas the data for the third enzyme are compatible with either a single or two base mechanism.

Degree

Ph.D.

Subject Area

Biochemistry

COinS