Comparison of the Role of Protein Dynamics in Catalysis by Dihydrofolate Reductase from E. coli and H. sapiens

Dihydrofolate reductase (DHFR) is a well-studied, clinically relevant enzyme known for being highly dynamic over the course of its catalytic cycle. However, the role dynamic motions play in the explicit hydride transfer from the nicotinamide cofactor to the dihydrofolate substrate remains unclear because reaction initiation and direct spectroscopic examination on the appropriate time scale for such femtosecond to picosecond motions is challenging. Here, we employ presteady-state kinetics to observe the hydride transfer as directly as possible in two different species of DHFR: Escherichia coli and Homo sapiens. While the hydride transfer has been well-characterized in DHFR from E. coli, improvements in time resolution now allow for sub-millisecond dead times for stopped-flow spectroscopy, which reveals that the maximum rate is indeed faster than previously recorded. The rate in the human enzyme, previously only estimated, is also able to be directly observed using cutting-edge stopped-flow instrumentation. In addition to the pH dependence of the hydride transfer rates for both enzymes, we examine the primary H/D kinetic isotope effect to reveal a temperature dependence in the human enzyme that is absent from the E. coli counterpart. This dependence, which appears above a temperature of 15 degrees C is a shared feature among other hydride transfer enzymes and is also consistent with computational work suggesting the presence of a fast promoting-vibration that provides donor-acceptor compression on the time scale of catalysis to facilitate the chemistry step.

Automated summary

This study directly observed the hydride transfer in DHFR enzymes from Escherichia coli and Homo sapiens using presteady-state kinetics. The results revealed that the hydride transfer rate in DHFR from E. coli is faster than previously recorded, and the rate in the human enzyme can be directly observed using advanced stopped-flow instrumentation. Additionally, the study found that the hydride transfer rates in both enzymes are pH-dependent, and the human enzyme exhibits a temperature dependence.