Abstract
Abstract Chorismate synthase was isolated from Neurospora crassa by diethylaminoethylcellulose chromatography, and two peaks of activity were obtained. The enzyme in the first peak had a sedimentation coefficient of ∼10 S and appeared to be an unstable, larger molecular form of the enzyme in the second peak, which was ∼8 S. Both forms of the enzyme required TPNH for activity, and the reactions catalyzed by both exhibited a lag which could be eliminated by prior incubation with the substrate 3-enolpyruvylshikimate 5-phosphate (ES-5-P). No other metabolic effectors were found, but diaphorase stimulated the reaction over 10-fold, suggesting that a flavin-like moiety is involved in the catalytic process. With diaphorase in the assay mixture, prior incubation with both ES-5-P and diaphorase was required to eliminate a remaining lag. The Michaelis constant for TPNH was ∼10 µm. The concentration of ES-5-P necessary to eliminate 50% of the lag was ∼0.1 mm. A chorismate synthaseless mutant, Arom-3, strain 87-5A, appears to carry out a portion of the chorismate synthase reaction but only in the presence of TPNH and diaphorase. A method is described for the preparation of ES-5-P from shikimate with greater than 90% yield. Previous yields were lower due to an inhibition by ADP of the ES-5-P synthetic activity catalyzed by the aromatic complex. Inhibition was eliminated by the addition of an ATP-regenerating system. These results suggest the possibility that the aromatic pathway of N. crassa is regulated by ADP.
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