UREA-INDUCED UNFOLDING OF THE ALPHA-SUBUNIT OF TRYPTOPHAN SYNTHASE - ONE-DIMENSIONAL PROTON NMR EVIDENCE FOR RESIDUAL STRUCTURE NEAR HISTIDINE-92 AT HIGH DENATURANT CONCENTRATION

The urea-induced unfolding reaction of the alpha subunit of tryptophan synthase was monitored by examining the chemical shifts and peak area s of the C(epsilon) protons of the four histidine residues with ID NMR spectroscopy. In a native base-line region defined by tyrosine absorb ance and far-UV circular dichroism spectroscopy, histidine-146 appears to undergo a rapid, local unfolding reaction at increasing denaturant concentrations. As the native form is converted to a previously detec ted stable intermediate between 2 and 3 M urea [Matthews, C. R., & Cri santi, M. M. (1981) Biochemistry 20, 784], histidines-92 and -146 in t he amino folding unit (residues 1-188) and histidines-195 and -244 in the carboxy folding unit (residues 189-268) all experience a change in their environments which is slow on the NMR time scale. The subsequen t conversion of this intermediate to a newly detected, stable, partial ly folded form populated at 5 M urea appears to have no effect on any of the histidines at 25-degrees-C when an intermolecular association p rocess involving His-244 is taken into account. Strikingly, a slow exc hange process involving only His-92 is observed to begin at 5 M urea w here the unfolding transitions monitored by absorbance or far-UV circu lar dichroism spectroscopy are essentially complete. This residual ter tiary structure unfolds in a cooperative fashion as the urea concentra tion is increased to 8 M.