Multiple intermediates, diverse conformations, and cooperative conformational changes underlie the catalytic hydride...

It has become increasingly clear that protein motions play an essential role in enzyme catalysis. However, exactly how these motions are related to an enzyme's chemical step is still intensely debated. This chapter examines the possible role of protein motions that display a hierarchy of timescales in enzyme catalysis. The linkage between protein motions and catalysis is investigated in the context of a model enzyme, E. coli dihydrofolate reductase (DHFR), that catalyzes the hydride transfer reaction in the conversion of dihydrofolate to tetrahydrofolate. The results of extensive computer simulations probing the protein motions that are manifest during different steps along the turnover cycle of DHFR are summarized. Evidence is presented that the protein motions modulate the catalytic efficacy of DHFR by generating a conformational ensemble conducive to the hydride transfer. The alteration of the equilibrium conformational ensemble rather than any protein dynamical effects is found to be sufficient to explain the rate-diminishing effects of mutation on the kinetics of the enzyme. These data support the view that the protein motions facilitate catalysis by establishing reaction competent conformations of the enzyme, but they do not directly couple to the chemical reaction itself. These findings have broad implications for our understanding of enzyme mechanisms and the design of novel protein catalysts.