A View at the Millennium: the Efficiency of Enzymatic Catalysis

Understanding how electric fields and their fluctuations in the active site of enzymes affect efficient catalysis represents a critical objective of biochemical research. We have directly measured the dynamics of the electric field in the active site of a highly proficient enzyme, Δ 5 -3-ketosteroid isomerase (KSI), in response to a sudden electrostatic perturbation that simulates the charge displacement that occurs along the KSI catalytic reaction coordinate. Photoexcitation of a fluorescent analog (coumarin 183) of the reaction intermediate mimics the change in charge distribution that occurs between the reactant and intermediate state in the steroid substrate of KSI. We measured the electrostatic response and angular dynamics of four probe dipoles in the enzyme active site by monitoring the time-resolved changes in the vibrational absorbance (IR) spectrum of a spectator thiocyanate moiety (a quantitative sensor of changes in electric field) placed at four different locations in and around the active site, using polarization-dependent transient vibrational Stark spectroscopy. The four different dipoles in the active site remain immobile and do not align to the changes in the substrate electric field. These results indicate that the active site of KSI is preorganized with respect to functionally relevant changes in electric fields.enzyme catalysis | electrostatic preorganization | Stark effect | visible-pump IR probe | time-resolved anisotropy E nzymes catalyze the vast majority of biochemical reactions and accelerate the rate of these reactions by many orders of magnitude compared to the uncatalyzed reactions in solution. The origins of the enormous catalytic power of enzymes are still not well understood despite enormous effort (1-6). In particular, the functional role of fast picosecond protein motions in catalysis and the dynamic nature of the transition state barrier crossing is a subject of ongoing and current debate (7-13). Theoretical studies have suggested that fast vibrations in enzymes might generate transition state conformations conducive to the chemical reaction (7,8,(14)(15)(16)(17)(18)(19)(20)(21), a familiar concept for reactions in ordinary solvents (22). In an alternative viewpoint, preorganization effects have been suggested to be a major contributing factor to enzyme catalysis (23-28). It has been postulated that enzymes have partially oriented dipoles of polar and charged groups in the active site that interact with electrostatic features present in the catalytic transition state more favorably than water can. Such preorganization of active site dipoles and charges has been suggested to result in a reduction in the reorganization energy and provide enormous catalytic advantage over the reaction in solution, where water molecules must rearrange in order to solvate charge rearrangements during the chemical reaction (24,(29)(30)(31). The relative merits of these opposing viewpoints remains unclear largely because of the paucity of direct experimental assessment of the key discrepancies betwee...

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confidence: 99%

Direct measurement of the protein response to an electrostatic perturbation that mimics the catalytic cycle in ketosteroid isomerase

Jha¹,

Gaffney

et al. 2011

“…The first step is the formation of a monometallic amido complex via reaction of the free ligand with 1.0 eq of Zr(NMe 2 ) 4 . This intermediate product is then subjected to reaction with 1.0 eq of Ti(NMe 2 ) 4 to obtain the heterobimetallic amido complex Ti 1 Zr 1 (NMe 2 ) 4 . The sequence of first introducing Zr(NMe 2 ) 2 then Ti(NMe 2 ) 2 is used because the former ligand substitution process is far more rapid and more selective.…”

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confidence: 99%

“…catalysis ͉ polymer ͉ polyolefin E nzymes achieve superior reactivity and selectivity, in part, by their efficacy in creating high local reagent concentrations and special, conformationally advantageous active site-substrate proximities and interactions (1)(2)(3)(4). In this regard, the possibility of unique and more efficient abiotic catalytic transformations based on cooperative effects between adjacent active centers in multinuclear transition metal complexes is currently of great interest.…”

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confidence: 99%

Nuclearity and cooperativity effects in binuclear catalysts and cocatalysts for olefin polymerization

Marks

2006

225

109

A series of bimetallic organo-group 4 ''constrained geometry'' catalysts and binuclear bisborane and bisborate cocatalysts have been synthesized to probe catalyst center-catalyst center cooperativity effects on olefin enchainment in homogenous olefin polymerization and copolymerization processes. Significant nuclearity effects are found versus mononuclear controls, and the effect can be correlated with metal-metal approach distances and ion pairing effects. Novel polymer structures can be obtained by using such binuclear catalyst͞cocatalyst systems.catalysis ͉ polymer ͉ polyolefin E nzymes achieve superior reactivity and selectivity, in part, by their efficacy in creating high local reagent concentrations and special, conformationally advantageous active site-substrate proximities and interactions (1-4). In this regard, the possibility of unique and more efficient abiotic catalytic transformations based on cooperative effects between adjacent active centers in multinuclear transition metal complexes is currently of great interest. Within the context of the rapidly advancing and technologically significant field of homogeneous single-site olefin polymerization catalysis (5-10), the conjecture that cooperative effects involving two or more metal centers in close proximity might achieve more efficient chain propagation and͞or novel polymer architectures motivated the research that is the subject of this contribution. Single-Site Olefin Polymerization ProcessesOne of the most exciting developments in the areas of catalysis, organometallic chemistry, and polymer science in recent years has been the intense development of new polymerization technologies based on well defined single-site metallocene and coordination complex olefin polymerization catalysts (5-10). The active catalytic species is typically generated by combining a transition-metal organometallic precursor with an activator or cocatalyst. The resulting electrophilic͞coordinatively unsaturated, strongly ionpaired species then undergoes rapid olefin enchainment. In optimum cases (e.g., group 4 metal and cyclopentadienyl-type ligand), catalysts with truly exceptional productivities and selectivities for high-molecular-weight polyolefins are produced. Constrained Geometry Catalysts: Testbeds for Multinuclear Cooperativity EffectsGroup 4 ''constrained geometry'' catalysts (CGCs) (A; Fig. 1) are well known single-site polymerization agents (16, 17) that produce unusual branched, hence far more processable, polyethylenes with high productivity and selectivity. One of the key features of these catalysts is the coordinatively open nature of the active site, which allows rapid enchainment of sterically encumbered olefin comonomers into the polyethylene backbone. Under certain conditions, these catalysts yield vinyl-terminated, chain-transferred macromonomers (a macromolecule that acts as a monomer) that diffuse away and then undergo competitive reinsertion into a growing polymer chain to produce macromolecules with long chain branching (Fig. 19, which is published ...

“…T he contributions of ground-state (GS) conformations and transition-state (TS) stabilization to the proficiency of an enzymatic reaction can be more easily understood by studying a simple one-substrate enzyme reaction that involves the intramolecular rearrangement of a substrate to product without formation of a covalent intermediate (1,2). The Claisen rearrangement of chorismate to prephenate is a well studied example (3) as shown in Fig.…”

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confidence: 99%

The proficiency of a thermophilic chorismate mutase enzyme is solely through an entropic advantage in the enzyme reaction

Zhang

Bruice

2005

Self Cite

A study of the Thermus thermophilus chorismate mutase (TtCM) is described by using quantum mechanics (self-consistent-charge density-functional tight binding)͞molecular mechanics, umbrella sampling, and the weighted histogram analysis method. The computed free energies of activation for the reactions in water and TtCM are comparable to the experimental values. The free energies for formation of near attack conformer have been determined to be 8.06 and 0.05 kcal͞mol in water and TtCM, respectively. The near attack conformer stabilization contributes Ϸ90% to the proficiency of the enzymatic reaction compared with the reaction in water. The transition state (TS) structures and partial atom charges are much the same in the enzymatic and water reactions. The difference in the electrostatic interactions of Arg-89 with O13 in the enzymesubstrate complex and enzyme-TS complex provides the latter with but 0.55 kcal͞mol of 1.92 kcal͞mol total TS stabilization. Differences in electrostatic interactions between components at the active site in the enzyme-substrate complex and enzyme-TS complex are barely significant, such that TS stabilization is of minor importance and the enzymatic catalysis is through an entropic advantage.potential of mean force ͉ self-consistent-charge density-functional tight binding ͉ quantum mechanics͞molecular mechanics T he contributions of ground-state (GS) conformations and transition-state (TS) stabilization to the proficiency of an enzymatic reaction can be more easily understood by studying a simple one-substrate enzyme reaction that involves the intramolecular rearrangement of a substrate to product without formation of a covalent intermediate (1, 2). The Claisen rearrangement of chorismate to prephenate is a well studied example (3) as shown in Fig. 1 where the reactive conformation near attack conformer (NAC) is defined.Our choice to research the Thermus thermophilus chorismate mutase (TtCM) relates to our current interest in the chorismate 3 prephenate reaction (4-6) and the questions of the temperature specificity of thermophilic enzymes (7). TtCM belongs to a rare group of the AroH-type monofunctional CMs that are principally found in Gram-positive bacteria and have been discovered as a thermophilic enzyme in the Gram-negative T. thermophilus (8, 9). The experimental free energies of activation ⌬G exp ‡ are 15.2 and 24.2 kcal͞mol for the reactions in TtCM (343 K, the bacterium's optimal growth temperature) and water (298 K) (10), respectively. In this article, we describe a quantum mechanics (QM)͞molecular mechanics (MM) (11) and umbrella sampling (12) study of the reactions at 343 K for TtCM and 298 K for water. MethodsThe initial coordinates of TtCM were modified from the x-ray structure of the F55S mutant of the TtCM trimer (Protein Data Bank ID code 1ODE; resolution, 1.65 Å) (8). The QM͞MM simulations were carried out with CHARMM version 31b1 by the use of the self-consistent-charge density functional tight binding (SCCDFTB) module (13-15). For the simulations in the active site of ...