Using NMR spectroscopy to elucidate the role of molecular motions in enzyme function

Abstract

Conformational motions play an essential role in enzyme function, often facilitating the formation of enzyme-substrate complexes and/or product release. Although considerable debate remains regarding the role of molecular motions in the conversion of enzymatic substrates to products, numerous examples have found motions to be crucial for optimization of enzyme scaffolds, effective substrate binding, and product dissociation. Conformational fluctuations are often rate-limiting to enzyme catalysis, primarily through product release, with the chemical reaction occurring much more quickly. As a result, the direct involvement of motions at various stages along the enzyme reaction coordinate remains largely unknown and untested. In the following review, we describe the use of solution NMR techniques designed to probe various timescales of molecular motions and detail examples in which motions play a role in propagating catalytic effects from the active site and directly participate in essential aspects of enzyme function.

Conformational Rigidity and Protein Dynamics at Distinct Timescales Regulate PTP1B Activity and Allostery

“…A Bracken plot (R 1 * R 2 vs R 2 /R 1 ), which indirectly reports conformational exchange, showed that ~25 residues have significant chemical exchange or motional anisotropy, many of which belong to residues in helices α3 and α7 (Figure S1C) (Kneller et al, 2002). Over the past decades it has become clear that motions in enzymes are often coordinated with function (Boehr et al, 2006; Henzler-Wildman and Kern, 2007; Lisi and Loria, 2016; Mittermaier and Kay, 2009). Thus, these data suggest that helices α3 and α7 play key roles in PTP1B activity.…”

“…Protein dynamics are essential for protein function and regulation (Akimoto et al, 2013; Henzler-Wildman and Kern, 2007; Lisi and Loria, 2016; Masterson et al, 2010; Tzeng and Kalodimos, 2012). Most metabolic enzymes turnover quickly, and thus are highly efficient catalysts that generate large quantities of product.…”

Controlling Active Site Loop Dynamics in the (β/α)8 Barrel Enzyme Indole-3-Glycerol Phosphate Synthase

“…These motions may be important in gating the entry and exit of substrate and products, and creating microenvironments conducive to chemical catalysis. Importantly, many of these loop motions are rate-determining [17,18,21]. Considering their functional importance, understanding and controlling active-site loop motions may be necessary in the design and engineering of next generation enzyme catalysts [26].…”

“…NMR experiments can also provide insight into the structural dynamics of proteins over a wide-range of timescales [17,18,21]. We determined R ex values (Figure 4a), the contribution of conformational exchange on the µs-ms timescale to the R 2 transverse relaxation rates.…”

“…Similar effects are also likely important for IGPS catalysis. The motions of active site loops are often rate-determining [17][18][19][20][21]. For example, in the thermophilic Sulfolobus sulfataricus IGPS (ssIGPS) enzyme, product release is rate-determining at lower [22,23] but [28]), showing the β1α1 loop in purple, catalytic residues Glu51 (blue), Lys53 (red) and Lys110 (orange), and residues modified in this manuscript to change the structure/dynamics of the β1α1 loop, including Arg64, Asp65 (both magenta) and Asn90 (green); (b) schematic highlighting the hydrogen bonding interactions of the β1α1 loop.…”