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.

The effect of type-2 diabetes conditions on neutrophil rolling adhesion.

OBJECTIVE: Type 2 diabetes mellitus (T2D) is the result of a dysregulation of insulin production and signalling, leading to an increase in both glucose concentration and pro-inflammatory cytokines such as interleukin (IL)-6 and tumour necrosis factor (TNF)-α. Previous work showed that T2D patients exhibited immune dysfunction associated with increased adhesion molecule expression on endothelial cell surfaces, accompanied by decreased neutrophil rolling velocity on the endothelial cell surface. Changes in cell rolling adhesion have direct vascular and immune complications such as atherosclerosis and reduced healing time in T2D patients. While previous studies focused primarily on how endothelial cells affect neutrophil rolling under T2D conditions, little is known about changes to neutrophils that affect their rolling. In this study, we aim to show how the rolling behaviour of neutrophils is affected by T2D conditions on a controlled substrate. RESULTS: We found that neutrophils cultured in T2D-serum mimicking media increased cell rolling velocity compared to neutrophils under normal conditions. Specifically, glucose alone is responsible for higher rolling velocity. While cytokines further increase the rolling velocity, they also reduce the cell size. Both glucose and cytokines likely reduce the function of P-selectin Glycoprotein Ligand-1 (PSGL-1) on neutrophils.