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STUDIES ON CHLOROALANINE AND THE ALANINE RACEMASES FROM E. COLI AND B. SUBTILIS
Abstract
Interaction of chloroalanine with alanine racemase isolated from B. subtilis indicates that the alpha-beta elimination of HCl from chloroalanine was not catalyzed by this enzyme. The sources of the elimination of HCl from chloroalanine were found to be the non-enzymatic formation of pyruvate from chloroalanine and the action of D-amino acid oxidase (unpublished work of L. Henderson). The non-enzymatic elimination of HCl from chloroalanine was found to be dependent upon pH, ionic strength of the buffer, concentration of pyridoxal phosphate, presence of glutathione, and the addition of pyridoxal phosphate and glutathione. However the formation of pyruvate via a non-enzymatic mechanism did not exceed 10% of the total chloroalanine initially present. The presence of a D-amino acid oxidase activity can be demonstrated to be present in isolated E. coli membrane vesicles along with a racemase activity. In view of the unpublished work of L. Henderson and inability of the B. subtilis racemase to catalyze the alpha-beta elimination of HCl from chloroalanine the interaction of the chloroalanine with the membrane vesicles was investigated under the same conditions used by L. Henderson. The distribution of pyruvate and chloropyruvate observed by Henderson with the hog D-amino acid oxidase was also observed (within experimental limits) with the membrane vesicles from E. coli. The source of the pyruvate and chloropyruvate was attributed to the oxidase and not the racemase. Experiments conducted previously in this laboratory demonstrated that alanine racemase would exchange the alpha proton of alanine with the deuterium of the solvent. The resonances of the alpha and beta protons of chloroalanine, under the assay conditions, could not be distinguished as a result of the pD dependence of the resonances. The pD dependence provided information from which the pKa's of the carboxyl proton could be estimated as about 1.5 as measured by the separation of the alpha and beta protons of chloroalanine. In the presence of B. subtilis alanine racemase no exchange of the alpha proton of chloroalanine could be demonstrated indicating that the chloroalanine could not act as a suicide inhibitor of the racemase.
Subject Area
Biochemistry
Recommended Citation
SCHREIBER, EVERETT CHARLES, "STUDIES ON CHLOROALANINE AND THE ALANINE RACEMASES FROM E. COLI AND B. SUBTILIS" (1980). ETD collection for University of Nebraska-Lincoln. AAI8110580.
https://digitalcommons.unl.edu/dissertations/AAI8110580