Probing coupled motions in enzymatic hydrogen tunnelling reactions

Abstract

Much work has gone into understanding the physical basis of the enormous catalytic power of enzymes over the last 50 years or so. Nevertheless, the detailed mechanism used by Nature's catalysts to speed chemical transformations remains elusive. DHFR (dihydrofolate reductase) has served as a paradigm to study the relationship between the structure, function and dynamics of enzymatic transformations. A complex reaction cascade, which involves rearrangements and movements of loops and domains of the enzyme, is used to orientate cofactor and substrate in a reactive configuration from which hydride is transferred by quantum mechanical tunnelling. In the present paper, we review results from experiments that probe the influence of protein dynamics on the chemical step of the reaction catalysed by TmDHFR (DHFR from Thermotoga maritima). This enzyme appears to have evolved an optimal structure that can maintain a catalytically competent conformation under extreme conditions.

Microsecond Subdomain Folding in Dihydrofolate Reductase

“…9,10 Moreover, DHFR has served as a paradigm for studying the relationship between the structure, function, and dynamics of enzymatic transformations. 11,12 Relevant to the present study, DHFR has been the target of numerous studies of its folding reaction. Within the first few milliseconds, DHFR accumulates a kinetic folding intermediate (termed I 5 ) in which ∼25% of the far-UV circular dichroism (CD) signal is developed [13][14][15][16] and two subsets of hydrogen-bonding networks, corresponding to two hydrophobic clusters, are formed.…”

Direct Evidence of Catalytic Heterogeneity in Lactate Dehydrogenase by Temperature Jump Infrared Spectroscopy

“…Conformational equilibria that occur on the nanosecond to millisecond time scale are of particular interest because they perturb average values of reactive structures, such as the donor–acceptor distance for hydride transfer, and thereby modulate the catalytic rate. 3−5 Despite the importance of these concepts, the most widely used approach to analyze enzyme kinetics in the literature and textbooks still relies on the Michaelis–Menten model and transition state theory. This approach has served its purpose as an organizing framework for interpreting enzyme kinetics, but it tends to oversimplify enzyme reaction pathways.…”

Increased Dynamic Effects in a Catalytically Compromised Variant ofEscherichia coliDihydrofolate Reductase

“…1 Nevertheless, theoretical frameworks that involve protein “promoting vibrations” or “promoting motions” on femtosecond to millisecond time scales, which are proposed to reduce the height and/or width of the potential energy barrier and thereby enhance enzymatic catalysis, 2−4 have been invoked to interpret experimental data. 5−11 Protein motions occur over a hierarchy of time scales and ranges, 12,13 from femtosecond local bond vibrations to millisecond large scale domain motions. It has been suggested that motions on one time scale may facilitate motions on another.…”

“…5−7,29−33 It catalyzes the NADPH-dependent reduction of 7,8-dihydrofolate (H 2 F) to 5,6,7,8-tetrahydrofolate (H 4 F) by hydride transfer from C4 of NADPH and protonation of N5 of H 2 F (Figure 1). The catalytic cycle of DHFR from E. coli (EcDHFR) has been thoroughly characterized; 33−35 the physical steps of substrate binding and product release involve large scale millisecond time scale conformational motions of the M20 loop (residues 9–24), which forms part of the active site.…”