PURIFICATION OF TRYPTOPHAN SYNTHASE FROM SACCHAROMYCES-CEREVISIAE AND PARTIAL ACTIVITY OF ITS NICKED SUBUNITS

Enzymes can be reconstituted with high yield after dissociation and deactivation in strong denaturants. This finding can be utilized to separate proteases and other impurities (with molecular weights differing under denaturing conditions from the subunit molecular weight of the enzyme to be purified) from the desired enzyme. Applying this approach to tryptophan synthase from yeast, a homogeneous preparation with a molecular weight of 154000 ± 5000 and a specific activity of 1200 ± 100 Yanofsky units/mg has been obtained. The latter value exceeds the previously reported maximum activity by a factor of 10, and characterizes the enzyme in its native state. The Km values for d‐glyceraldehyde 3‐phosphate and indole of the indole‐glycerolphosphate synthesis (reaction 2) and the pH optimum, as well as the Km values for l‐serine, indole, and pyridoxal 5′‐phosphate of the l‐tryptophan condensation (reaction 3) have been determined. At least two tryptophan‐synthase‐inactivating proteinases from yeast have been well characterized in the past. Therefore, the low activity of previous preparations may be assumed to be caused by proteolytic cleavage. To prove this hypothesis, tryptophan synthase was prepared following a conventional procedure. Using ultracentrifugation and gel chromatography under quasi‐physiological conditions, this enzyme has been found to be a homogeneous species of Mr= 75000 ± 3000 and s20,w= 3.3 ± 0.2 S, which is still capable of catalyzing both reactions 2 and 3 with a specific activity for the latter reaction of 120 Yanofsky units/mg. Comparing the Km values for all relevant ligands, only the Km value for pyridoxal 5′‐phosphate shows a marked difference with respect to the value observed for the native enzyme. At –18°C the nicked enzyme is stable over a period of several weeks. As measured by sedimentation equilibrium in 6M guanidine · HCl at pH 2.3, the molecule consists of two large polypeptide fragments, Mr,1= 22000 ± 3000 and Mr,2= 50000 ± 5000. These results suggest tryptophan synthase from yeast to be a dimer of two bifunctional subunits; obviously the specific proteinases preferentially attack a region forming a link between two domains of the enzyme corresponding to the α and β subunits of the Escherichia coli enzyme. Copyright © 1979, Wiley Blackwell. All rights reserved