A thermally induced reversible conformational transition of the tryptophan synthase beta2 subunit probed by the spectroscopic properties of pyridoxal phosphate and by enzymatic activity.
A reversible thermally induced conformational transition of the beta2 subunit of tryptophan synthase from Salmonella typhimurium has been detected by use of the pyridoxal 5'-phosphate coenzyme as a spectroscopic probe. Increasing the temperature converts the major form of pyridoxal 5'-phosphate bound to the beta2 subunit from a ketoenamine species with lambdamax at 410 nm to a enolimine species with lambdamax at 336 nm (Tm = approximately 43 degrees C) and results in loss of the circular dichroism signal at 410 nm and of fluorescence emission at 510 nm. The results indicate that increasing the temperature favors a conformer of the enzyme that binds pyridoxal 5'-phosphate in a more nonpolar environment and leads to loss of asymmetric pyridoxal 5'-phosphate binding. The internal aldimine between pyridoxal 5'-phosphate and the epsilon-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'-3H]pyridoxal 5'-phosphate. The thermal transition of the beta2 subunit below 60 degrees C produces reversible thermal inactivation (Ti = approximately 52 degrees C) and occurs at a much lower temperature than the major reversible unfolding at approximately 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 is the catalytically active form of the cofactor in the beta2 subunit and that the low temperature reversible conformational transition disturbs the active site and causes loss of catalytic activity.
Ahmed
S A
SA
Laboratory of Biochemical Pharmacology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
McPhie
P
P
Miles
E W
EW
D001894:Borohydrides / Q000737:chemistry; D002942:Circular Dichroism; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000737:chemistry; D012486:Salmonella typhimurium; D013050:Spectrometry, Fluorescence; D013057:Spectrum Analysis; D013696:Temperature; D013816:Thermodynamics; D014367:Tryptophan Synthase / Q000737:chemistry*
D001894:Borohydrides; D011732:Pyridoxal Phosphate; C025364:sodium borohydride; D014367:Tryptophan Synthase