Properties of Chorismate Synthase in Neurospora crassa

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.