Many enzymes mould their structures to poise substrates in their active sites for chemical reaction such that conformational remodeling is necessary during each catalytic cycle. In both protein tyrosine phosphatase B, PtpB, from M. tuberculosis (a virulence factor of tuberculosis) and adenylate kinase, AK, from E. coli (a ubiquitous energy-balancing enzyme in cells), the conformational change involves large-amplitude rearrangements of the enzyme's lid domain. These domain movements have been followed in real time on their respective catalytic timescales using high-resolution single-molecule Förster resonance energy transfer (FRET) spectroscopy. It is shown quantitatively that both PtpB and AK are capable of dynamically sampling two distinct states that correlate well with those observed by x-ray crystallography. Surprisingly, the equilibrium favors the closed, active-site-forming configurations even in the absence of substrates, contrasting the widely accepted induced-fit picture. For AK, the experiments further showed that interaction with substrates restricts the spatial extent of conformational fluctuations rather than locking the enzyme into a compact state. Integrating these microscopic dynamics into macroscopic kinetics allows us to model AK's lid opening-coupled product release as the enzyme's rate-limiting step-direct evidence for conformation-gated enzymatic reactivity. To test the general principles garnered from these studies and to investigate the molecular mechanics underlying the transitions, we extend the studies to PtpB. The results will be discussed in the context of potential energy surface in an enzymatic cycle.
Host: David Talaga