Tryptophan synthase from yeast. Purification by affinity chromatography, physical properties.

Abstract

Tryptophan synthase was purified from Saccharomyces cerevisiae with the aid of affinity chromatography up to a specific activity of 525 units/g protein. Electrophoresis on polyacrylamide gel showed one main protein component. Gel filtration on Sephadex G‐150 indicates an average molecluar weight of 143000. Dissociation with dodecylsulfate followed by dodecyl‐sulfate‐acrylamide gel electrophoresis leads to the conclusion that the enzyme is probably composed of four subunits of equal size. The Km‐values with respect to its substrates indoleglycerol phosphate, indole and l‐serine were estimated. l‐Serine was shown to increase the Km‐value for indoleglycerol phosphate about 7‐fold. Several indole analogues inhibit the tryptophan synthase reaction (B) (i.e. indole + serine → tryptophan); indoleacrylic acid is the most effective. The pH‐optimum of tryptophan synthase is around 6.9 to 7.0. Sulfhydryl modification by 5,5′‐dithiobis(2‐nitrobenzoic acid) and p‐chloromercuriphenylsulfonic acid results in a large loss of tryptophan synthase activity, which cannot be prevented by the substrates l‐serine and indole. The tryptophan synthase inactivating system from yeast affects the catalysis of both reactions (A) (i.e. indoleglycerol phosphate → indole + glyceraldehyde 3‐phosphate) and (B).