Purification of Tryptophan Synthase from Saccharomyces cerevisiae and Partial Activity of Its Nicked Subunits

Abstract

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 Jr 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 K , values for D-glyceraldehyde 3-phosphate and indole of the indole-glycerolphosphate synthesis (reaction 2 ) and the pH optimum, as well as the K,,, values for L-serine, indole, and pyridoxal5'-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 M , = 75000 f 3000 and = 3.3 k 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 K, values for all relevant ligands, only the K, 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, M,,I = 22000 k 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 ci and / 3 subunits of the Escherichia coli enzyme.Tryptophan synthase isolated from different microorganisms catalyzes the last step in tryptophan biosynthesis :

Tryptophan Synthase from Saccharomyces cerevisiae Is a Dimer of Two Polypeptide Chains of Mr 76000 Each

“…When this manuscript was completed we were informed that Bartholmes et al [48] had arrived at essentially the same conclusions as reported in this paper, using a different method for purifying tryptophan synthase from S. cerevisiae.…”

“…The maximum specific activity is estimated to be approximately 1200 units/mg protein, which is half as large as estimated for the maximum specific activity of tryptophan synthase from E. coli (2400 units/mg protein [39]). A specific activity of 1200 units/mg protein has been determined independently by Bartholmes et al [48]. The value of 660 units/mg protein for our final preparation (Table 1) indicates that the enzyme is not maximally active, perhaps by side chain amide hydrolysis or some other, uncontrolled chemical modification of the protein.…”

Pyrimidine biosynthesis in Saccharomyces cerevisiae: the ura2 cluster gene, its multifunctional enzyme product, and other structural or regulatory genes involved in de novo UMP synthesis

“…As a result of such difficulties, more sophisticated purification procedures have been developed and a great majority of multienzyme complexes have been shown to be synthetized as multifunctional proteins. But even in such conditions, the yield of the multifunctional protein species is low, for example, with tryptophan synthetase (Bartholmes et al 1979), meaning that part of the activity is likely to be carried by smaller species. We should not, however, take it for granted that the observed proteolysis is always artifactual; it may well have some physiological importance.…”

Studies on the association of the α and β2 subunits of the tryptophan synthase of Bacillus subtilis

“…These results were consistent with the idea that the tryptophan synthase of the more highly evolved sporeforming bacilli existed as an a@ species. Such a species might have represented an evolutionary intermediate toward the ap fusion product described in some of the lower eukaryotes (Tsai et al 1974;Dettwiler and Kirschner 1979;Bartholmes et al 1979). However, a more detailed examination of the factors affecting the association of the a and p subunits in B. subtilis determined the conditions necessary for the formation of higher molecular weight complexes.…”