Enzymic properties of a mutant tryptophan synthase from Neurospora crassa

Abstract

Tryptophan synthase ( l-serine hydro-lyase (adding indole), EC 4.2.1.20) obtained from Neurospora crassa strain td 201, a tryptophan auxotroph, is only capable of catalyzing the conversion of indole and serine to tryptophan. The enzyme is largely resolved from its cofactor, pyridoxal phosphate (pyridoxal- P); hence the enzymic activity in the crude extract is practically undetectable unless exogeneous pyridoxal- P is added. The stoichiometry of the cofactor has been determined by spectrophotometric titration with purified apoenzyme; about 1.5 moles of pyridoxal- P are bound to 1 mole of enzyme. Two types of binding of pyridoxal- P are present in the enzyme; one absorbs at 325 nm ( λ max emission = 385 nm) and the other absorbs at 410 nm ( λ max emission = 495 nm). These two forms are not interconvertible by varying the pH. The holoenzyme can be easily resolved by passage through a hydroxylapatite column. The resulting apoenzyme is inactive but it can be reactivated by pyridoxal- P. NaBH 4 reduction leads to irreversible inactivation of the enzyme. The enzyme is very sensitive to some endogenous proteases present in the Neurospora extract. Phenylmethanesulfonyl fluoride can prevent to some extent the spontaneous inactivation of the enzyme. Highly purified enzyme has been found to be fairly stable. Therefore, the apparent lability of this enzyme is not an intrinsic property. The catalytic activity of purified enzyme can be stimulated by proteins such as serum albumin. This phenomenon has been interpreted in terms of the “intermolecular cooperativity” which might exist in vivo between this enzyme and the other enzymic components of the tryptophan pathway. In this paper, an improved assay method for enzymic activity is also described.