Trypsin-treated Neurospora tryptophan synthetase: I. Enzymic properties

Tryptic digests of highly purified native Neurospora crassa TSase‡ have been examined for retention of enzymic properties, number of bonds hydrolyzed, and fractionation properties of surviving activities. The kinetics of tryptic inactiva-tion of three reactions catalyzed by TSase were examined. Both reaction 1, the conversion of indole-3-glycerolphosphate plus L-serine to L-tryptophan plus D-glyceraldehyde-3-phosphate, and also reaction 2, the conversion of indole plus L-serine to L-tryptophan, decline exponentially over approximately a 100-fold range. Although reaction 3 has been regarded as the reversible splitting of indole-3-glycerol phosphate to indole plus D-glyceraldehyde-3-phosphate, the rates of tryptic inactivation for the forward (F) and reverse (R) reactions differ markedly from each other. Furthermore, both reaction 3F and 3R are more stable to tryptic treatment than reactions 1 and 2. Further analysis of reactions 3F and 3R confirms that the activities assayed are associated with tryptophan synthetase, but indicates that the formulation for this reaction is inadequate. A comparison of the kinetics of inactivation of reactions 1 and 2 and the loss of L-serine-pyridoxal phosphate stimulation of reaction 3R suggests that the hydrolysis of a single, critical peptide bond alters the ability of the enzyme to bind the two compounds. Titrimetric determinations of peptide bond hydrolysis show the alterations of the properties of TSase to be associated with the splitting of less than 5% of the total lysine and arginine peptide bonds. Sephadex fractionation and electrophoresis on acrylamide gel indicate that the surviving enzymic properties of TSase are associated with a molecule of nearly the size of the native enzyme, but with a different electrophoretic mobility.