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 an enzyme's active site converts a bimolecular reaction into a unimolecular, intramolecular one. Intramolecular model reactions provide the simplest means of determining the rate acceleration that can be achieved through reactant approximation [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 order [631, 744].

Destabilization of atomic groups transformed in substrates

A susceptible substrate group can be destabilized the moment the substrate binds to the enzyme; the energy required for this destabilization [631] is drawn from the overall binding energy. Destabilization mechanisms may include solvent substitution, charge-charge interactions, and bond-length or bond-angle strain. Such destabilization can be alleviated in the Transition State, which corresponds to a lowering of 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 to such an environment contributes significantly to the observed catalytic rate. The binding energy required to anchor a charged substrate in such an unfavorable environment and to drive the rate increase can be provided by the pyrophosphate group and the pyrimidine ring of the cofactor.

Class="center">

Fig. 11.3. Catalysis of decarboxylation via solvent replacement. 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 here, R₁ = CH₃ and R₂ = H. In organic solvents, the model compound loses CO₂ 50,000 times faster than in water [746]. It is suggested that organic solvents favor the transition state shown in parentheses 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), these cofactors bind to their respective enzymes in extended Conformations, thereby maximizing the number of enzyme-cofactor interactions (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 reactive 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]. Replacing a solvating water molecule with 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 the processes occurring in the D-subsite of Lysozyme. Interaction with the substrate's 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 within a substrate can be distorted upon its binding to the enzyme. An enzyme can act on a chemical group of the substrate by forcing it into a Structure that closely mimics the transition state; The Mechanism of such structural destabilization includes bond-angle deformation, the bringing together of reacting atoms to distances shorter than the sum of their Van der Waals radii, and the stretching of 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 offset 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 the 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 interaction with the trypsin-inhibitor interaction [269, 536]. The complex of trypsin with a pseudosubstrate inhibitor is characterized by an abnormal distance of

2.6 Å between the carbonyl carbon atom of the inhibitor's P1 residue 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, and the ΔG value reaches —18 kcal/mol, despite the strain in the C—O_y 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 standard substrates [749].

Processes within the catalytic center can stabilize the transition state. Up to this point, emphasis has been placed on how long-range interactions supply Free energy to the activated groups within the catalytic center of the enzyme-substrate complex. However, interactions within the catalytic center itself can also stabilize the transition state and thereby contribute 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 backbone nitrogen atoms, along with 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 transformed substrate groups. However, enzymes also possess functional groups that exert more subtle effects on these reactive groups. General Acid-Base Catalysis is quite common in enzymes and can enhance reaction rates by up to a factor of 1,000. In chymotrypsin, this function is carried out by a charge-Relay system that facilitates proton transfer across several reaction steps via hydrogen bonds (Fig. 11.1). In Other Enzymes, such as glutathione reductase, the protein utilizes active groups (FAD and a redox-active Cysteine pair) to transport electrons across the enzyme molecule (Fig. 11.4).

Separate consideration of the effects leading to enhanced enzymatic catalysis

Numerous attempts have been made to quantitatively assess the contributions of various effects (such as proximity, orbital steering, destabilization, general acid-base catalysis, etc.) to the rate acceleration brought about by a given enzyme. However, the example of chymotrypsin demonstrates that these "effects" represent different ways of describing the same events occurring at the active site 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].