A THERMALLY-INDUCED REVERSIBLE CONFORMATIONAL TRANSITION OF THE TRYPTOPHAN SYNTHASE BETA(2) SUBUNIT PROBED BY THE SPECTROSCOPIC PROPERTIES OF PYRIDOXAL-PHOSPHATE AND BY ENZYMATIC-ACTIVITY

A reversible thermally induced conformational transition of the beta(2 ) subunit of tryptophan synthase from Salmonella typhimurium has been detected by use of the pyridoxal 5'-phosphate coenzyme as a spectrosco pic probe. Increasing the temperature converts the major form of pyrid oxal 5'-phosphate bound to the beta(2) subunit from a ketoenamine spec ies with lambda(max) at 410 nm to a enolimine species with lambda(max) at 336 nm (T-m = similar to 43 degrees C) and results in loss of the circular dichroism signal at 410 nm and of fluorescence emission at 51 0 nm, The results indicate that increasing the temperature favors a co nformer of the enzyme that binds pyridoxal 5'-phosphate in a more nonp olar environment and leads to loss of asymmetric pyridoxal 5'-phosphat e binding. The internal aldimine between pyridoxal 5'-phosphate and th e E-amino group of lysine 87 is not disrupted by increased temperature because sodium borohydride treatment of the enzyme at either 15 or 60 degrees C results in covalent attachment of [4'-H-3]pyridoxal 5'-phos phate. The thermal transition of the beta(2) subunit below 60 degrees C produces reversible thermal inactivation (T-i = similar to 52 degree s C) and occurs at a much lower temperature than the major reversible unfolding at similar to 80 degrees C (Remeta, D. P., Miles, E. W., and Ginsburg, A. (1995) Pure Appl. Chem. 67, 1859-1866). Our new results indicate that the 410 nm absorbing species of pyridoxal 5'-phosphate i s the catalytically active form of the cofactor in the beta(2) subunit and that the low temperature reversible conformational transition dis turbs the active site and causes loss of catalytic activity.