Conformational Entropy in PEPCK Catalysis: Dynamic Motions Critical to Function

Abstract

Enzymes increase the rate at which chemical reactions occur. How they achieve this rate increase has been an area of intense research for many years. Multiple studies have shown that dynamic motions of individual protein segments can play a role in enzyme function. Our enzyme of interest, phosphoenolpyruvate carboxykinase (PEPCK), has multiple dynamic motions that work in concert during the catalytic cycle. One of these mobile elements, termed the omega-loop due to its architecture, acts like a lid that closes over the active site and has been hypothesized to serve multiple functions during the catalytic cycle. Furthermore, the act of opening and closing the lid domain is thought to be a delicate free energy balance, meaning the lid pays an energetic penalty to close, which is offset by the energetic contribution from substrate binding. To investigate the role of the loop, specifically the open/closed transition, and to probe for unappreciated roles, multiple mutant enzymes were created. In the first set of mutations the lid region was removed to investigate other potential roles for the omega-loop. In the second set two different amino acid residues (Ala-467 and Glu-89) were mutated to either increase the energetic penalty of lid closure or decrease the energetic contribution from substrate binding. The resultant mutants were characterized via structure/function experiments. The data revealed two new roles for the omega-loop lid, confirmed the delicate free energy balance, and shed light on the energetic pathway between the active site and the lid. Taking into account the data from the WT and all seven mutant PEPCK enzymes, a revised model for the role of the omega-loop lid during catalysis is proposed: The role of the lid during the catalytic cycle is to correctly position the substrates in the active site, stabilize the R-loop and N/C-terminal lobe closure, and sequester and protect the reaction intermediate.