3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase (tyrosine sensitive) was purified from Escherichia coli carrying the plasmid pKB45. Enzyme of high specific catalytic activity (70 IL/mg) was obtained from cells grown only to the early log phase. The purified protein contained Cu(ll) and showed an absorption band at 350 nm. Metal-free, catalytically inactive apoenzyme could be produced by dialysis against cyanide ion, and the holoenzyme could be reconstituted in terms of both catalytic activity and A350 by the binding of one Cu(II) ion per enzyme subunit. Zn(ll) also reactivated the apoenzyme to about 50% of the level seen with Cu(ll), although in this case no band appeared at 350 nm. In contrast to earlier reports that the enzyme contains substoichiometric levels of iron, insignificant amounts of iron were found in the isolated enzyme, and neither Fe(II) nor FE(Ill) regenerated either an absorption band at 350 nm or any catalytic activity from the apoenzyme. The evident preference of the enzyme as isolated for (Cu)II suggests that the synthase might naturally be a copper metalloenzyme.The first step of the shikimate pathway involves the condensation of phosphoenolpyruvate and erythrose 4-phosphate to produce the seven carbon keto acid, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP; Fig. 1). This reaction is catalyzed by DAHP synthase (EC 4.1.2.15), three isozyme forms of which exist in most microorganisms. Each isozyme is sensitive to feedback inhibition by one of the three aromatic amino acid products of the shikimate pathway (phenylalanine, tyrosine, or tryptophan), and this sensitivity is believed to represent an important element in the regulation of the metabolic sequence (6,20).One or more isozymes have been isolated from several sources, including Escherichia coli, Salmonella typhimurium, Bacillus subtilis, and Schizosaccharomyces pombe, but there is no real consensus on the properties even of the same isozyme from the same organism. For example, the activity of the tyrosine-sensitive enzyme from S. typhimurium has been found to depend upon the presence of Co(II) (14) and, later, to be independent of Co(II) and unaffected by the metal chelator EDTA (3). Similarly, the tyrosine-sensitive enzyme from E. coli has been found to be insensitive to Co(II) and to EDTA (18), whereas other reports suggest the opposite (4). The question of the metal requirement of DAHP synthase then appeared to have been resolved by the findings of Herrmann et al., who reported that both the phenylalanine-sensitive and the tyrosine-sensitive enzymes contain iron rather than cobalt (12). The bound iron appeared to be essential for the catalytic activity and, in the presence of one of the substrates (phosphoenolpyruvate), gave rise to an absorption band at 350 nm (12). These findings were made yet more interesting when amino acid sequence data became available for the tyrosine-sensitive enzyme, and it was found that there was some similarity between the sequence of residues 10 through 18 of this DAHP synthase and that of resid...