Protein Flexibility and Stiffness Enable Efficient Enzymatic Catalysis

Abstract

The enormous rate accelerations observed for many enzyme catalysts are due to strong stabilizing interactions between the protein and reaction transition state. The defining property of these catalysts is their specificity for binding the transition state with a much higher affinity than substrate. Experimental results are presented which show that the phosphodianion-binding energy of phosphate monoester substrates is used to drive conversion of their protein catalysts from flexible and entropically rich ground states to stiff and catalytically active Michaelis complexes. These results are generalized to other enzyme-catalyzed reactions. The existence of many enzymes in flexible, entropically rich, and inactive ground states provides a mechanism for utilization of ligand-binding energy to mold these catalysts into stiff and active forms. This reduces the substrate-binding energy expressed at the Michaelis complex, while enabling the full and specific expression of large transition-state binding energies. Evidence is presented that the complexity of enzyme conformational changes increases with increases in the enzymatic rate acceleration. The requirement that a large fraction of the total substrate-binding energy be utilized to drive conformational changes of floppy enzymes is proposed to favor the selection and evolution of protein folds with multiple flexible unstructured loops, such as the TIM-barrel fold. The effect of protein motions on the kinetic parameters for enzymes that undergo ligand-driven conformational changes is considered. The results of computational studies to model the complex ligand-driven conformational change in catalysis by triosephosphate isomerase are presented.

Maximum Entropy Production Theorem for Transitions between Enzyme Functional States and Its Applications

“…Triosephosphate isomerase is often described as a fully evolved enzyme with near-maximal reaction rate [ 39 ], but our results suggest that there is still room for additional improvement of the TIM enzyme because a reaction rate increase and the increase of the enzyme catalytic efficiency is still possible. In addition, MTEP optimizations have the potential to focus our attention at critical transitions coupled to directed movements of elementary particles, atoms, and amino acid residues, a helpful procedure to get a deeper insight into balancing flexibility and stiffness during enzyme catalysis [ 40 ].…”

Unexpected NADPH Hydratase Activity in the Nitrile Reductase QueF from Escherichia coli

“…The hydration rates were not accelerated in the presence of enzyme (Figure S4). In an effort to facilitate binding of the 1,4‐dihydronicotinamides to the enzyme, we considered an approach of “substrate in pieces” and additionally added ADP (1.11 m m ). However, the hydration rate in the presence of D197A variant was not affected by ADP (Figure S4).…”

Cryo-EM Structures of the SARS-CoV-2 Endoribonuclease Nsp15

“…While more work is needed to establish the functional significance of the endoU wobble, there are several possibilities as to why this could be critical for Nsp15 function. Flexible active sites that become stiff upon engaging substrates have been shown to promote catalysis by reducing the substrate-binding energy (25). Another possibility is that the wobbling of the endoU domain is important for Nsp15 to accommodate different RNA substrates.…”