Substrate-Based Allosteric Regulation of a Homodimeric Enzyme.

Many enzymes operate through half-of-the sites reactivity wherein a single protomer is catalytically engaged at one time. In the case of the homodimeric enzyme, fluoroacetate dehalogenase, substrate binding triggers closing of a regulatory cap domain in the empty protomer, preventing substrate access to the remaining active site. However, the empty protomer serves a critical role by acquiring more disorder upon substrate binding, thereby entropically favoring the forward reaction. Empty protomer dynamics are also allosterically coupled to the bound protomer, driving conformational exchange at the active site and progress along the reaction coordinate. Here, we show that at high concentrations, a second substrate binds along the substrate-access channel of the occupied protomer, thereby dampening interprotomer dynamics and inhibiting catalysis. While a mutation (K152I) abrogates second site binding and removes inhibitory effects, it also precipitously lowers the maximum catalytic rate, implying a role for the allosteric pocket at low substrate concentrations, where only a single substrate engages the enzyme at one time. We show that this outer pocket first desolvates the substrate, whereupon it is deposited in the active site. Substrate binding to the active site then triggers the empty outer pocket to serve as an interprotomer allosteric conduit, enabling enhanced dynamics and sampling of activation states needed for catalysis. These allosteric networks and the ensuing changes resulting from second substrate binding are delineated using rigidity-based allosteric transmission theory and validated by nuclear magnetic resonance and functional studies. The results illustrate the role of dynamics along allosteric networks in facilitating function.

Understanding Protein Function Through an Ensemble Description: Characterization of Functional States by 19F NMR.

Protein function is a consequence of a complex and dynamic equilibrium between allosterically coupled functional states. However, it is often difficult to distinguish the representative members of an ensemble by spectroscopic means. 19F NMR is particularly useful in this regard owing to the sensitivity of its chemical shift to subtle differences in environment. Here, we address aspects of 19F NMR relevant to the study of ensembles. In particular, we discuss current trends toward: (1) 19F-reporters that can be biosynthetically incorporated into proteins, (2) Approaches to chemical tagging of proteins by 19F reporters, (3) Improving delineation of states by 19F NMR, (4) Distinguishing states by (19F NMR-based) topology measurements that focus on solvent exposure and hydrophobicity, (5) Relaxation experiments and simple approaches to delineating states in fast and slow exchange, (6) Extending resolution of states by 19F NMR, and (7) Validating 19F NMR spectroscopy by computational methods. Many of these advances are demonstrated through recent 19F NMR studies of a homodimeric enzyme, fluoroacetate dehalogenase.