Structural Reorganization and Preorganization in Enzyme Active Sites: Comparisons of Experimental and Theoretically Ideal Active Site Geometries in the Multistep Serine Esterase Reaction Cycle

Abstract

Many enzymes catalyze reactions with multiple chemical steps, requiring the stabilization of multiple transition states during catalysis. Such enzymes must strike a balance between the conformational reorganization required to stabilize multiple transition states of a reaction and the confines of a preorganized active site polypeptide tertiary structure. Here we investigate the compromise between structural reorganization during the catalytic process, and preorganization of the active site for a multi-step enzyme-catalyzed reaction, the hydrolysis of esters by the Ser-His-Asp/Glu catalytic triad. Quantum mechanical transition states were used to generate ensembles of geometries that can catalyze each individual step in the mechanism. These geometries are compared to each other by superpositions of catalytic atoms to find "consensus" geometries that can catalyze all steps with minimal rearrangement. These consensus geometries are found to be excellent matches for the natural active site. Preorganization is therefore found to be the major defining characteristic of the active site, and reorganizational motions often proposed to promote catalysis have been minimized. The variability of enzyme active sites observed by X-ray crystallography was also investigated empirically. A catalog of geometrical parameters relating active site residues to each other and to bound inhibitors was collected from a set of crystal structures. The crystal-structure-derived values were then compared to the ranges found in quantum mechanically optimized structures along the entire reaction coordinate. The empirical ranges are found to encompass the theoretical ranges when thermal fluctuations are taken into account. Therefore, the active sites are preorganized to a geometry that can be objectively and quantitatively defined as minimizing conformational reorganization while maintaining optimal transition state stabilization for every step during catalysis. The results provide a useful guiding principle for de novo design of enzymes with multi-step mechanisms.

Installing hydrolytic activity into a completely de novo protein framework

“…37 In these, the Cys or Ser side chain acts as the nucleophile; the imidazole group of a proximal histidine (His, H) deprotonates the thiol or hydroxyl moiety; and an aspartic acid (Asp, D) or glutamic acid (Glu, E) residue polarizes the imidazole to activate the system further.…”

Origins of stereoselectivity in evolved ketoreductases

“…Following Pauling's postulate, it is now generally accepted that enzymes are complementary in structure to the TS of the reaction that they catalyze (17). In accordance with this idea, we used MD simulations to provide a detailed description of the active site near the reduction TSs for selected DE variants (16,18). The activated complexes were built into the protein structure from the optimal QM geometries and atomic partial charges of the diastereomeric theozymes described above for substrates 1 and 2 (Fig.…”

Promotion of Enzyme Flexibility by Dephosphorylation and Coupling to the Catalytic Mechanism of a Phosphohexomutase

“…The binding of structurally variable ligands may need to be accommodated, and multiple transition states may need to be stabilized (1,2). Often different steps of the reaction are accompanied by conformational or dynamic rearrangements of the enzyme (3).…”