These conformational changes also bring catalytic residues in the
active site close to the chemical bonds in the substrate that will be altered in the reaction. After binding takes place, one or more mechanisms of catalysis lowers the energy of the reaction's
transition state, by providing an alternative chemical pathway for the reaction. There are six possible mechanisms of "over the barrier" catalysis as well as a "through the barrier" mechanism:
Proximity and orientation Enzyme-substrate interactions align the reactive chemical groups and hold them close together in an optimal geometry, which increases the rate of the reaction. This reduces the
entropy of the reactants and thus makes addition or transfer reactions less unfavorable, since a reduction in the overall entropy when two reactants become a single product. However this is a general effect and is seen in non-addition or transfer reactions where it occurs due to an increase in the "effective concentration" of the reagents. This is understood when considering how increases in concentration leads to increases in reaction rate: essentially when the reactants are more concentrated, they collide more often and so react more often. In enzyme catalysis, the binding of the reagents to the enzyme restricts the conformational space of the reactants, holding them in the 'proper orientation' and close to each other, so that they collide more frequently, of an with the correct geometry, to facilitate the desired reaction. The "effective concentration" is the concentration the reactant would have to be, free in solution, to experiences the same collisional frequency. Often such theoretical effective concentrations are unphysical and impossible to realize in reality – which is a testament to the great catalytic power of many enzymes, with massive rate increases over the uncatalyzed state. However, the situation might be more complex, since modern computational studies have established that traditional examples of proximity effects cannot be related directly to enzyme entropic effects. Also, the original entropic proposal has been found to largely overestimate the contribution of orientation entropy to catalysis.
Proton donors or acceptors Proton donors and acceptors, i.e.
acids and
base may donate and accept protons in order to stabilize developing charges in the transition state. This is related to the overall principle of catalysis, that of reducing energy barriers, since in general transition states are high energy states, and by stabilizing them this high energy is reduced, lowering the barrier. A key feature of enzyme catalysis over many non-biological catalysis, is that both acid and base catalysis can be combined in the same reaction. In many abiotic systems, acids (large [H+]) or bases ( large concentration H+ sinks, or species with electron pairs) can increase the rate of the reaction; but of course the environment can only have one overall pH (measure of acidity or basicity (alkalinity)). However, since enzymes are large molecules, they can position both acid groups and basic groups in their active site to interact with their substrates, and employ both modes independent of the bulk pH. Often general acid or base catalysis is employed to activate
nucleophile and/or
electrophile groups, or to stabilize leaving groups. Many amino acids with acidic or basic groups are this employed in the active site, such as the glutamic and aspartic acid, histidine, cystine, tyrosine, lysine and arginine, as well as serine and threonine. In addition, the peptide backbone, with carbonyl and amide N groups is often employed.
Cystine and
Histidine are very commonly involved, since they both have a
pKa close to neutral
pH and can therefore both accept and donate protons. Many reaction mechanisms involving acid/base catalysis assume a substantially altered pKa. This alteration of pKa is possible through the local environment of the residue. pKa can also be influenced significantly by the surrounding environment, to the extent that residues which are basic in solution may act as proton donors, and vice versa. The modification of the pKa's is a pure part of the electrostatic mechanism. Note that in the example shown, the histidine conjugate acid acts as a general acid catalyst for the subsequent loss of the amine from a tetrahedral intermediate. Evidence supporting this proposed mechanism (Figure 4 in Ref. 13) has, however, been controverted.
Electrostatic catalysis Stabilization of charged transition states can also be by residues in the active site forming
ionic bonds (or partial ionic charge interactions) with the intermediate. These bonds can either come from
acidic or
basic side chains found on
amino acids such as
lysine,
arginine,
aspartic acid or
glutamic acid or come from metal
cofactors such as
zinc. Metal ions are particularly effective and can reduce the pKa of water enough to make it an effective nucleophile. Systematic computer simulation studies have established that electrostatic effects give, by far, the largest contribution to catalysis, and can increase the rate of reaction by a factor of up to 107. In particular, it has been found that enzymes provide an environment which is more polar than water, and that ionic transition states are stabilized by fixed dipoles. This is very different from transition state stabilization in water, where the water molecules must pay with "reorganization energy" in order to stabilize ionic and charged states. Thus, catalysis is associated with the fact that the enzyme polar groups are preorganized. The magnitude of the electrostatic field exerted by an enzyme's active site has been shown to be highly correlated with the enzyme's catalytic rate enhancement. Binding of substrate usually excludes water from the active site, thereby lowering the local dielectric constant to that of an organic solvent. This strengthens the electrostatic interactions between the charged/polar substrates and the active sites. In addition, studies have shown that the charge distributions about the active sites are arranged so as to stabilize the transition states of the catalyzed reactions. In several enzymes, these charge distributions apparently serve to guide polar substrates toward their binding sites so that the rates of these enzymatic reactions are greater than their apparent diffusion-controlled limits. Describing the dielectric constant in the enzyme–substrate complex as a single, low macroscopic value represents a significant oversimplification. In practice, the effective macroscopic dielectric constant of proteins can be relatively high (see, for example,). What is most relevant is that enzyme active sites are highly polar environments, in which polar groups are preorganized to stabilize the transition state.
Covalent catalysis Covalent catalysis involves the substrate forming a transient
covalent bond with residues in the enzyme active site or with a cofactor. This adds an additional covalent intermediate to the reaction, and helps to reduce the energy of later transition states of the reaction. The covalent bond must, at a later stage in the reaction, be broken to regenerate the enzyme. This mechanism is utilised by the
catalytic triad of enzymes such as
proteases like
chymotrypsin and
trypsin, where an acyl-enzyme intermediate is formed. An alternative mechanism is
schiff base formation using the free
amine from a
lysine residue, as seen in the enzyme
aldolase during
glycolysis. Some enzymes utilize non-amino acid
cofactors such as
pyridoxal phosphate (PLP) or
thiamine pyrophosphate (TPP) to form covalent intermediates with reactant molecules. Such covalent intermediates function to reduce the energy of later transition states, similar to how covalent intermediates formed with active site amino acid residues allow stabilization, but the capabilities of cofactors allow enzymes to carryout reactions that amino acid side residues alone could not. Enzymes utilizing such cofactors include the PLP-dependent enzyme
aspartate transaminase and the TPP-dependent enzyme
pyruvate dehydrogenase. Rather than lowering the activation energy for a reaction pathway, covalent catalysis provides an alternative
pathway for the reaction (via to the covalent intermediate) and so is distinct from true catalysis. However, metal ions are advantageous in biological catalysis because they are not affected by changes in pH. Metal ions can also act to ionize water by acting as a
Lewis acid. Metal ions may also be agents of oxidation and reduction.
Bond strain This is the principal effect of induced fit binding, where the affinity of the enzyme to the transition state is greater than to the substrate itself. This induces structural rearrangements which strain substrate bonds into a position closer to the conformation of the transition state, so lowering the energy difference between the substrate and transition state and helping catalyze the reaction. However, the strain effect is, in fact, a ground state destabilization effect, rather than transition state stabilization effect. Furthermore, enzymes are very flexible and they cannot apply large strain effect. In addition to bond strain in the substrate, bond strain may also be induced within the enzyme itself to activate residues in the active site.
Quantum tunneling These traditional "over the barrier" mechanisms have been challenged in some cases by models and observations of "through the barrier" mechanisms (
quantum tunneling). Some enzymes operate with kinetics which are faster than what would be predicted by the classical ΔG‡. In "through the barrier" models, a proton or an electron can tunnel through activation barriers. Quantum tunneling for protons has been observed in
tryptamine oxidation by
aromatic amine dehydrogenase. Quantum tunneling does not appear to provide a major catalytic advantage, since the tunneling contributions are similar in the catalyzed and the uncatalyzed reactions in solution. However, the tunneling contribution (typically enhancing rate constants by a factor of ~1000 However, this study did not include any representation of the enzyme environment, nor did it provide a relevant potential energy surface. Consequently, it cannot be considered a true quantum mechanical study of enzyme catalysis. Such investigations became feasible only with the development of combined quantum mechanical/molecular mechanical (QM/MM) methods.
Active enzyme The binding energy of the enzyme-substrate complex cannot be considered as an external energy which is necessary for the substrate activation. The enzyme of high energy content may firstly transfer some specific energetic group X1 from catalytic site of the enzyme to the final place of the first bound reactant, then another group X2 from the second bound reactant (or from the second group of the single reactant) must be transferred to active site to finish substrate conversion to product and enzyme regeneration. We can present the whole enzymatic reaction as a two coupling reactions: It may be seen from reaction () that the group X1 of the active enzyme appears in the product due to possibility of the exchange reaction inside enzyme to avoid both electrostatic inhibition and repulsion of atoms. So we represent the active enzyme as a powerful reactant of the enzymatic reaction. The reaction () shows incomplete conversion of the substrate because its group X2 remains inside enzyme. This approach as idea had formerly proposed relying on the hypothetical extremely high enzymatic conversions (catalytically perfect enzyme). The crucial point for the verification of the present approach is that the catalyst must be a complex of the enzyme with the transfer group of the reaction. This chemical aspect is supported by the well-studied mechanisms of the several enzymatic reactions. Consider the reaction of peptide bond hydrolysis catalyzed by a pure protein α-chymotrypsin (an enzyme acting without a cofactor), which is a well-studied member of the serine proteases family, see. We present the experimental results for this reaction as two chemical steps: where S1 is a polypeptide, P1 and P2 are products. The first chemical step () includes the formation of a covalent acyl-enzyme intermediate. The second step () is the deacylation step. The group H+, initially found on the enzyme, but not in water, appears in the product before the step of hydrolysis, therefore it may be considered as an additional group of the enzymatic reaction. Thus, the reaction () shows that the enzyme acts as a powerful reactant of the reaction. According to the proposed concept, the H transport from the enzyme promotes the first reactant conversion, breakdown of the first initial chemical bond (between groups P1 and P2). The step of hydrolysis leads to a breakdown of the second chemical bond and regeneration of the enzyme. The proposed chemical mechanism does not depend on the concentration of the substrates or products in the medium. However, a shift in their concentration mainly causes free energy changes in the first and final steps of the reactions () and () due to the changes in the free energy content of every molecule, whether S or P, in water solution. This approach is in accordance with the following mechanism of muscle contraction. The final step of ATP hydrolysis in skeletal muscle is the product release caused by the association of myosin heads with actin. The closing of the actin-binding cleft during the association reaction is structurally coupled with the opening of the nucleotide-binding pocket on the myosin active site. Notably, the final steps of ATP hydrolysis include the fast release of phosphate and the slow release of ADP. The release of a phosphate anion from bound ADP anion into water solution may be considered as an exergonic reaction because the phosphate anion has low molecular mass. Thus, we arrive at the conclusion that the primary release of the inorganic phosphate H2PO4− leads to transformation of a significant part of the free energy of ATP hydrolysis into the kinetic energy of the solvated phosphate, producing active streaming. This assumption of a local mechano-chemical transduction is in accord with Tirosh's mechanism of muscle contraction, where the muscle force derives from an integrated action of active streaming created by ATP hydrolysis. ==Examples of catalytic mechanisms==