Principles of Protein Structure - G. Schultz 1982

Structural Basis of Protein Mechanism, Action, and Function
Enzymatic Catalysis
Effects Contributing to High Reaction Rates

Proximity, orientation, orbital steering, and other Entropy effects

Enzymes can accelerate Chemical Reactions by optimizing the orientation of reactants within the Active Site [741, 742]. Such orientation may also involve orbital steering: the precise mutual alignment of the bonding orbitals of reacting atomic partners [743, 744].

The binding of two separate molecules within the enzyme's active site converts a bimolecular reaction into a monomolecular, intramolecular reaction. Intramolecular model reactions provide the simplest means of determining the rate acceleration that can be achieved through reactant proximity [631, 745]. In other words, the entropy effect of an enzyme manifests as an increase in the effective Substrate Concentration. Since chemical reaction rates are proportional to reactant concentrations, a 10³-fold rate enhancement can be expected in localized regions of high concentration and ordering [631, 744].

Destabilization of atomic groups transformed within substrates

The substrate group undergoing transformation can be destabilized at the moment the substrate binds to the enzyme; the energy required for this destabilization [631] is drawn from the overall binding energy. Mechanisms of destabilization may include solvent substitution, charge-charge interactions, and bond-length or bond-angle strain. Destabilization can be alleviated in the Transition State, which implies a reduction in the activation energy required to reach that transition state.

Placing chemical groups in a nonpolar environment can increase the reaction rate by up to 50,000-fold. A simple mechanism proposed for Pyruvate decarboxylase, a thiamine pyrophosphate-dependent enzyme, is based on two observations [746]. First, the adduct of pyruvate and a cofactor analogue (Fig. 11.3) undergoes decarboxylation in organic Solvents 10⁴ to 10⁵ times faster than in Water; second, the pyruvate-binding site in pyruvate decarboxylase is highly hydrophobic. The rate enhancement expected upon transferring the pyruvate-cofactor adduct into such an environment makes a significant contribution to the observed catalytic rate. The binding energy required to anchor a charged substrate in this unfavorable environment and to secure the rate acceleration can be provided by the pyrophosphate group and the pyrimidine ring of the cofactor.

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Fig. 11.3. Decarboxylation catalysis via solvent substitution. The reactive moiety of thiamine pyrophosphate, a cofactor for decarboxylation enzymes, is the thiazole ring. In thiamine pyrophosphate, R₁ is the pyrimidine moiety and R₂ is the pyrophosphate moiety; in the model compound discussed, R₁ = CH₃ and R₂ = H. In organic solvents, the model compound loses CO₂ 50,000 times faster than in water [746]. It is hypothesized that organic solvents favor the transition state shown in brackets in the figure.

The ADP moieties of Cofactors such as ATP, FAD, NAD(P), or coenzyme A can perform a function analogous to that of the pyrimidine and pyrophosphate groups of thiamine pyrophosphate. In all cases studied (Section 10.4), cofactors bind to their enzymes in extended Conformations, thereby maximizing the number of interactions with the enzyme (Fig. 11.4). The binding energy expended for the specific requirements of other cofactor fragments is invariably provided by the ADP moiety.

Charged groups of the enzyme become more active when solvating water molecules are replaced by the substrate. Desolvation-based destabilization appears to contribute to the catalytic action of the Zn²⁺ ion bound to Carboxypeptidase A [747]. The replacement of a solvating water molecule by the substrate decreases the Dielectric Constant of the metal ion's microenvironment and enhances its ability to polarize the substrate's acyl group, which is necessary for nucleophilic attack. A similar effect contributes to processes in the D-subsite of Lysozyme. Interaction with the substrate of the desolvated carboxyl group of the Asp-42 residue leads to local destabilization of the enzyme-substrate complex; this destabilization is alleviated during The formation of a transition state resembling an oxocarbenium ion [531].

Bond Lengths and Bond Angles in the substrate can become distorted upon its binding to the enzyme. The enzyme can act upon a chemical group of the substrate, imparting a Structure close to that of the transition state; The Mechanism of such structural destabilization includes deformation of bond angles, bringing reacting atoms closer together to interatomic distances less than the sum of their Van der Waals radii, and stretching covalent bonds beyond the sum of the covalent radii of the bonded atoms [631, 739, 740]. The best-studied case of structural destabilization occurs upon the binding of an N-acetylaminosugar residue to the D-subsite of lysozyme [748]. The tetrahedral (sp)³ carbon-1 atom is distorted, and this strain is relieved in the transition state where carbon-1 becomes planar (sp²). Taking into account the desolvation of the Asp-42 residue upon substrate binding, the destabilization energy is at least +8.6 kcal/mol. This energy is compensated by the overall binding energy of the oligosaccharide substrate.

Fig. 11.4. Active Site of Glutathione reductase [124].

The dimeric enzyme contains two identical active sites. Reducing equivalents follow a pathway (from right to left): the nicotinamide moiety of NADPH → the isoalloxazine ring of FAD (shown from the side) → the redox-active disulfide → the glutathione disulfide bond. The glutathione-binding site and the catalytic center are formed by residues from both subunits. Note that NADPH and FAD bind to the enzyme in extended conformations.

Binding interactions distant from the catalytic center stabilize the Trypsin-inhibitor complex. This is evident from a comparison of the trypsin-substrate and trypsin-inhibitor interactions [269, 536]. The complex of trypsin with a pseudosubstrate inhibitor is characterized by an anomalous distance of

2.6 Å between the carbonyl carbon atom of residue-15 of the pseudosubstrate and the Ser-195 residue of the enzyme. Typical substrates of Chymotrypsin and trypsin, which form several favorable contacts, require an activation energy of +5 to +15 kcal/mol to reach the acylation stage. However, upon Formation of the trypsin-inhibitor complex, numerous other interactions are optimized, resulting in a ΔG value of -18 kcal/mol, despite the strain in the C—O^γ adduct (Table 5.6). Thus, the difference in stabilization energies can be explained by the differences in the contact areas within the complexes that trypsin forms with the inhibitor versus ordinary substrates [749].

Processes within the catalytic center can stabilize the transition state. Up to this point, emphasis has been placed on the fact that long-range interactions supply Free energy to the activated groups in the catalytic center of the enzyme-substrate complex. However, interactions within the catalytic center itself can also stabilize the transition state, thereby contributing to the efficiency of enzymatic catalysis. In chymotrypsin, the energy gain provided by the formation of two Hydrogen Bonds between the activated substrate and the main-chain nitrogen atoms, as well as the partial charge compensation of the buried Asp-102 residue (Fig. 11.1), helps offset the energy required to form the strained bond between the enzyme and the substrate in the tetrahedral complex [537].

Chemical groups as parts of enzymes

Destabilizing effects within the enzyme-substrate complex influence the state of the substrate groups undergoing transformation. However, enzymes also possess functional groups that exert more subtle effects on these transforming groups. General Acid-Base Catalysis is quite common in enzymes and can enhance reaction rates by up to 1,000-fold. In chymotrypsin, this function is performed by a charge-Relay system that mediates proton transfer through hydrogen bonds across several reaction steps (Fig. 11.1). In Other Enzymes, such as glutathione reductase, the protein utilizes active groups (FAD and a redox-active Cysteine pair) for electron Transport Across the enzyme molecule (Fig. 11.4).

Separate analysis of effects leading to rate enhancement in enzymatic catalysis

Numerous attempts have been made to quantitatively assess the contributions of various effects (e.g., proximity, orbital steering, destabilization, general acid-base catalysis, etc.) to the rate acceleration produced by a given enzyme. However, the example of chymotrypsin demonstrates that these "effects" represent different ways of describing the same active-site phenomena and cannot truly be separated from one another. On the other hand, the concepts developed through the analysis of enzyme-substrate interactions have broadened our understanding of chemical catalysis and facilitated the design of non-peptide polymer-based enzyme analogues [745, 750].