The use of tritium exchange to detect conformational differences between intermediates in catalysis by the enzyme...

Tritium-exchange experiments have been performed on enzyme forms related to obligatory catalytic intermediates in the rhodanese reaction: the free enzyme, E, and the sulfur-substituted enzyme, ES. Under the experimental conditions used, each form displayed tritium-exchange behavior that can be interpreted in terms of at least three classes of exchangeable sites. Class I with a t 1/2 approximately equal to 180 min and Class II with a t 1/2 approximately equal to 15 min were present in both E and ES. The conversion of ES to E has the effect of changing approx. 50 protons from the slow Class I behavior to exchange at a rate too fast to measure (Class III). These results are consistent with a conformational change on conversion of rhodanese from ES to E. The fact that a large number of protons are involved together with the very large change in exchange rate might indicate that extended areas of the protein change contact with the solvent, and would be compatible with a model for rhodanese catalysis that includes a coupled conformational change.