INVESTIGATIONS OF 3-DEOXY-D-ARABINO - HEPTULOSONATE 7-PHOSPHATE SYNTHASE: I. IMMUNOLOGICAL STUDIES ON THE ISOZYMES FROM ESCHERICHIA COLI. II. PURIFICATION AND MOLECULAR CHARACTERIZATION OF ONE ISOZYME FROM CARROT (DAUCUS CAROTA)

JOANN ALEXIS SUZICH, Purdue University

Abstract

The immunological relationships between the isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) (DAHP synthase) from Escherichia coli were studied by immunodiffusion, immunoinactivation and immunoprecipitation from solution, and competitive enzyme-linked immunosorbent assay. Antibodies directed against the phenylalanine sensitive DAHP synthase inactivated the tyrosine sensitive isozyme in solution. The tyrosine sensitive DAHP synthase reacted with antibodies against the tryptophan sensitive isozyme in solution. No cross reactivity between the three DAHP synthases was detected by immunodiffusion or competitive enzyme-linked immunosorbent assay. It was concluded that there is little immunological relationship between the DAHP synthase isozymes from E. coli. No data was obtained to indicate that there was even a single antigenic site shared by all three enzymes. DAHP synthase activity was detected in extracts of cultured cells and whole root tissue of Daucus carota after partial purification by ammonium sulfate. Unlike the enzyme from E. coli, carrot DAHP synthase was not inhibited by the aromatic amino acids; rather, the enzyme was feedback activated by tryptophan and tyrosine. The physiological significance of feedback activation of DAHP synthase in relation to shikimate pathway regulation was discussed. Chromatography of carrot root extracts on phosphocellulose resolved three DAHP synthase activities. The three isozymes exhibited similar molecular properties. One of the carrot root isozymes, DAHP synthase III, was purified about 10,000-fold. DAHP synthase III sub-units represented the major protein component in the purest enzyme preparations analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme subunits were used to prepare anti-DAHP synthase III antibodies. Native DAHP synthase III is a dimer with an estimated molecular weight of 103,000. The subunit molecular weight was estimated to be 53,000. DAHP synthase III activity in highly purified preparations was stimulated by micromolar concentrations of tryptophan and Mn('2+). The isozyme was inhibited by 1,3-propanediol and activated by potassium phosphate. DAHP synthase III was shown to be a hysteretic enzyme. The enzyme responded slowly to the addition of substrates which resulted in a measurable lag in its catalytic activity. Preincubation of DAHP synthase III with tryptophan or Mn('2+) reduced the lag in the catalytic activity. The hysteretic behavior of the enzyme was entirely eliminated by preincubation with 1,3-propanediol.

Degree

Ph.D.

Subject Area

Biochemistry

COinS