Enzyme catalysis

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:

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, and 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.{{cite journal | doi = 10.1021/ja972723x | vauthors = Stanton RV, Peräkylä M, Bakowies D, Kollman PA | year = 1998 | title = Combined ab initio and Free Energy Calculations To Study Reactions in Enzymes and Solution: Amide Hydrolysis in Trypsin and Aqueous Solution | journal = J. Am. Chem. Soc. | volume = 120 | issue = 14| pages = 3448–3457 | bibcode = 1998JAChS.120.3448S }}{{cite journal | doi = 10.1021/ja992218v | vauthors = Kuhn B, Kollman PA | year = 2000 | title = QM-FE and Molecular Dynamics Calculations on Catechol O-Methyltransferase: Free Energy of Activation in the Enzyme and in Aqueous Solution and Regioselectivity of the Enzyme-Catalyzed Reaction | journal = J. Am. Chem. Soc. | volume = 122 | issue = 11| pages = 2586–2596 }}{{cite journal | doi = 10.1021/ar960131y | vauthors = Bruice TC, Lightstone FC | year = 1999 | title = Ground State and Transition State Contributions to the Rates of Intramolecular and Enzymatic Reactions | journal = Acc. Chem. Res. | volume = 32 | issue = 2| pages = 127–136 }} Also, the original entropic proposal{{cite journal | vauthors = Page MI, Jencks WP | title = Entropic contributions to rate accelerations in enzymic and intramolecular reactions and the chelate effect | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 68 | issue = 8 | pages = 1678–1683 | date = August 1971 | pmid = 5288752 | pmc = 389269 | doi = 10.1073/pnas.68.8.1678 | doi-access = free | bibcode = 1971PNAS...68.1678P }} has been found to largely overestimate the contribution of orientation entropy to catalysis.{{cite journal | vauthors = Warshel A, Parson WW | title = Dynamics of biochemical and biophysical reactions: insight from computer simulations | journal = Quarterly Reviews of Biophysics | volume = 34 | issue = 4 | pages = 563–679 | date = November 2001 | pmid = 11852595 | doi = 10.1017/s0033583501003730 | s2cid = 28961992 }}

{{See also|Protein pKa calculations}}

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.{{cn|date=January 2025}}

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.{{Citation needed|date=November 2015}}

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. The catalytic effect of the above example is mainly associated with the reduction of the pKa of the oxyanion and the increase in the pKa of the histidine, while the proton transfer from the serine to the histidine is not catalyzed significantly since it is not the rate determining barrier.{{cite journal | vauthors = Warshel A, Naray-Szabo G, Sussman F, Hwang JK | title = How do serine proteases really work? | journal = Biochemistry | volume = 28 | issue = 9 | pages = 3629–3637 | date = May 1989 | pmid = 2665806 | doi = 10.1021/bi00435a001 }} 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){{cite journal | vauthors = Fersht AR, Requena Y | title = Mechanism of the -chymotrypsin-catalyzed hydrolysis of amides. pH dependence of k c and K m . Kinetic detection of an intermediate | journal = Journal of the American Chemical Society | volume = 93 | issue = 25 | pages = 7079–7087 | date = December 1971 | pmid = 5133099 | doi = 10.1021/ja00754a066 }} has, however, been controverted.{{cite journal | vauthors = Zeeberg B, Caswell M, Caplow M | title = Concerning a reported change in rate-determining step in chymotrypsin catalysis | journal = Journal of the American Chemical Society | volume = 95 | issue = 8 | pages = 2734–2735 | date = April 1973 | pmid = 4694533 | doi = 10.1021/ja00789a081 | bibcode = 1973JAChS..95.2734Z }}

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,{{cite journal | vauthors = Warshel A, Sharma PK, Kato M, Xiang Y, Liu H, Olsson MH | title = Electrostatic basis for enzyme catalysis | journal = Chemical Reviews | volume = 106 | issue = 8 | pages = 3210–3235 | date = August 2006 | pmid = 16895325 | doi = 10.1021/cr0503106 }} and can increase the rate of reaction by a factor of up to 10⁷.{{cite book| vauthors = Voet D, Voet JG |title=Biochemistry |date= 2011 |publisher=John Wiley & Sons|oclc=808679090}} 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"{{cite journal | vauthors = Marcus RA | year = 1965 | title = On the Theory of Electron-Transfer Reactions. VI. Unified Treatment for Homogeneous and Electrode Reactions | url = http://authors.library.caltech.edu/33657/1/MARjcp65b.pdf| journal = J. Chem. Phys. | volume = 43 | issue = 2| pages = 679–701 | doi=10.1063/1.1696792|bibcode = 1965JChPh..43..679M }} in order to stabilize ionic and charged states. Thus, catalysis is associated with the fact that the enzyme polar groups are preorganized.{{cite journal | vauthors = Warshel A | title = Energetics of enzyme catalysis | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 75 | issue = 11 | pages = 5250–5254 | date = November 1978 | pmid = 281676 | pmc = 392938 | doi = 10.1073/pnas.75.11.5250 | doi-access = free | bibcode = 1978PNAS...75.5250W }}

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.{{cite journal | vauthors = Fried SD, Bagchi S, Boxer SG | title = Extreme electric fields power catalysis in the active site of ketosteroid isomerase | journal = Science | location = New York, N.Y. | volume = 346 | issue = 6216 | pages = 1510–4 | date = December 2014 | pmid = 25525245 | pmc = 4668018 | doi = 10.1126/science.1259802 | bibcode = 2014Sci...346.1510F }}

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{{citation needed|date=November 2015}}.

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.Toney, M. D. "Reaction specificity in pyridoxal enzymes." Archives of biochemistry and biophysics (2005) 433: 279-287{{Cite web |url=http://lpi.oregonstate.edu/infocenter/vitamins/thiamin/ |title=Micronutrient Information Center, Oregon State University |access-date=30 September 2009 |archive-date=21 March 2015 |archive-url=https://web.archive.org/web/20150321165821/http://lpi.oregonstate.edu/infocenter/vitamins/thiamin/ |url-status=dead }} 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.{{cite book |title= Biochemistry | vauthors = Voet D, Voet JG |year= 2004 |publisher= John Wiley & Sons Inc. |isbn= 978-0-471-25090-6 |pages= [https://archive.org/details/biochemistry00voet_1/page/986 986–989] |url= https://archive.org/details/biochemistry00voet_1/page/986 }}{{cite book |title= Biochemistry | vauthors = Voet D, Voet JG |year= 2004 |publisher= John Wiley & Sons Inc. |isbn= 978-0-471-25090-6 |pages= [https://archive.org/details/biochemistry00voet_1/page/604 604–606] |url= https://archive.org/details/biochemistry00voet_1/page/604 }}

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. For example, the energetics of the covalent bond to the serine molecule in chymotrypsin should be compared to the well-understood covalent bond to the nucleophile in the uncatalyzed solution reaction. A true proposal of a covalent catalysis (where the barrier is lower than the corresponding barrier in solution) would require, for example, a partial covalent bond to the transition state by an enzyme group (e.g., a very strong hydrogen bond), and such effects do not contribute significantly to catalysis.

A metal ion in the active site participates in catalysis by coordinating charge stabilization and shielding. Because of a metal's positive charge, only negative charges can be stabilized through metal ions.{{cite journal | vauthors = Piccirilli JA, Vyle JS, Caruthers MH, Cech TR | title = Metal ion catalysis in the Tetrahymena ribozyme reaction | journal = Nature | volume = 361 | issue = 6407 | pages = 85–88 | date = January 1993 | pmid = 8421499 | doi = 10.1038/361085a0 | s2cid = 4326584 | bibcode = 1993Natur.361...85P }} However, metal ions are advantageous in biological catalysis because they are not affected by changes in pH.{{cite book|title=Reactions of Coordinated Ligands| vauthors = Bender ML |date=1962-01-01|publisher= American Chemical Society |isbn=978-0-8412-0038-8 |series=Advances in Chemistry|volume=37|pages=19–36|doi=10.1021/ba-1963-0037.ch002|chapter = Metal Ion Catalysis of Nucleophilic Organic Reactions in Solution}} Metal ions can also act to ionize water by acting as a Lewis acid.{{cite journal| vauthors = Fife TH, Przystas TJ |date=1985-02-01|title=Divalent metal ion catalysis in the hydrolysis of esters of picolinic acid. Metal ion promoted hydroxide ion and water catalyzed reactions|journal=Journal of the American Chemical Society|volume=107|issue=4|pages=1041–1047|doi=10.1021/ja00290a048|bibcode=1985JAChS.107.1041F |issn=0002-7863}} Metal ions may also be agents of oxidation and reduction.{{cite journal | vauthors = Stadtman ER | title = Metal ion-catalyzed oxidation of proteins: biochemical mechanism and biological consequences | journal = Free Radical Biology & Medicine | volume = 9 | issue = 4 | pages = 315–325 | date = 1990-01-01 | pmid = 2283087 | doi = 10.1016/0891-5849(90)90006-5 | url = https://zenodo.org/record/1258619 }}

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.{{cite book | vauthors = Jencks WP | author-link=William Jencks | title=Catalysis in Chemistry and Enzymology | year=1987 | orig-year=1969 | location=New York | publisher=Dover Publications | series=McGraw-Hill series in advanced chemistry | edition=reprint | isbn=978-0-486-65460-7 }}{{page needed|date=January 2015}} Furthermore, enzymes are very flexible and they cannot apply large strain effect.{{cite journal | vauthors = Warshel A, Levitt M | title = Theoretical studies of enzymic reactions: dielectric, electrostatic and steric stabilization of the carbonium ion in the reaction of lysozyme | journal = Journal of Molecular Biology | volume = 103 | issue = 2 | pages = 227–249 | date = May 1976 | pmid = 985660 | doi = 10.1016/0022-2836(76)90311-9 }}{{open access}}

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.

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.{{cite journal | vauthors = Garcia-Viloca M, Gao J, Karplus M, Truhlar DG | title = How enzymes work: analysis by modern rate theory and computer simulations | journal = Science | volume = 303 | issue = 5655 | pages = 186–195 | date = January 2004 | pmid = 14716003 | doi = 10.1126/science.1088172 | s2cid = 17498715 | bibcode = 2004Sci...303..186G }}{{cite journal | vauthors = Olsson MH, Siegbahn PE, Warshel A | title = Simulations of the large kinetic isotope effect and the temperature dependence of the hydrogen atom transfer in lipoxygenase | journal = Journal of the American Chemical Society | volume = 126 | issue = 9 | pages = 2820–2828 | date = March 2004 | pmid = 14995199 | doi = 10.1021/ja037233l | bibcode = 2004JAChS.126.2820O }} Quantum tunneling for protons has been observed in tryptamine oxidation by aromatic amine dehydrogenase.{{cite journal | vauthors = Masgrau L, Roujeinikova A, Johannissen LO, Hothi P, Basran J, Ranaghan KE, Mulholland AJ, Sutcliffe MJ, Scrutton NS, Leys D | display-authors = 6 | title = Atomic description of an enzyme reaction dominated by proton tunneling | journal = Science | volume = 312 | issue = 5771 | pages = 237–241 | date = April 2006 | pmid = 16614214 | doi = 10.1126/science.1126002 | s2cid = 27201250 | bibcode = 2006Sci...312..237M }}

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.{{cite journal | vauthors = Hwang JK, Warshel A | title = How important are quantum mechanical nuclear motions in enzyme catalysis | doi = 10.1021/ja962007f | journal = J. Am. Chem. Soc. | year = 1996 | volume = 118| issue = 47 | pages = 11745–11751 | bibcode = 1996JAChS.11811745H }}{{cite journal | vauthors = Ball P | title = Enzymes: by chance, or by design? | journal = Nature | volume = 431 | issue = 7007 | pages = 396–397 | date = September 2004 | pmid = 15385982 | doi = 10.1038/431396a | s2cid = 228263 | doi-access = free | bibcode = 2004Natur.431..396B }}{{cite journal | vauthors = Olsson MH, Parson WW, Warshel A | title = Dynamical contributions to enzyme catalysis: critical tests of a popular hypothesis | journal = Chemical Reviews | volume = 106 | issue = 5 | pages = 1737–1756 | date = May 2006 | pmid = 16683752 | doi = 10.1021/cr040427e }} However, the tunneling contribution (typically enhancing rate constants by a factor of ~1000 compared to the rate of reaction for the classical 'over the barrier' route) is likely crucial to the viability of biological organisms. This emphasizes the general importance of tunneling reactions in biology.

In 1971-1972 the first quantum-mechanical model of enzyme catalysis was formulated.{{cite journal | vauthors = Vol'kenshtein MV, Dogonadze RR, Madumarov AK, Urushadze ZD, Kharkats YI | title = The theory of enzyme catalysis | journal = Molecular Biology | location = Moscow | volume = 6 | issue = 3 | pages = 347–353 | date = 1972 | pmid = 4645409 }}{{cite book | vauthors = Volkenshtein MV, Dogonadze RR, Madumarov AK, Urushadze ZD, Kharkats Yu I | chapter = Electronic and Conformational Interactions in Enzyme Catalysis. | title = Konformatsionnie Izmenenia Biopolimerov v Rastvorakh | location = Moscow | publisher = Nauka Publishing House | date = 1973 | pages = 153–157 }}{{third-party inline|reason=The original papers do not in themselves prove that this was the first such model.|date=April 2013}}

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.{{cite journal | vauthors = Foigel AG | title = Is the enzyme a powerful reactant of the biochemical reaction? | journal = Molecular and Cellular Biochemistry | volume = 352 | issue = 1–2 | pages = 87–89 | date = June 2011 | pmid = 21318350 | doi = 10.1007/s11010-011-0742-4 | s2cid = 11133081 }}

We can present the whole enzymatic reaction as a two coupling reactions:

{{NumBlk|:|{{chem2|S1 + EX1 -> S1EX1 -> P1 + EP2}}|{{EquationRef|1}}}}

{{NumBlk|:|{{chem2|S2 + EP2 -> S2EP2 -> P2 + EX2}}|{{EquationRef|2}}}}

It may be seen from reaction ({{EquationNote|1}}) 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 ({{EquationNote|2}}) 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).{{cite journal | vauthors = Fogel AG | title = Cooperativity of enzymatic reactions and molecular aspects of energy transduction | journal = Molecular and Cellular Biochemistry | volume = 47 | issue = 1 | pages = 59–64 | date = August 1982 | pmid = 7132966 | doi = 10.1007/bf00241567 | s2cid = 21790380 }}

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.{{cite journal | vauthors = Hengge AC, Stein RL | title = Role of protein conformational mobility in enzyme catalysis: acylation of alpha-chymotrypsin by specific peptide substrates | journal = Biochemistry | volume = 43 | issue = 3 | pages = 742–747 | date = January 2004 | pmid = 14730979 | doi = 10.1021/bi030222k }}

We present the experimental results for this reaction as two chemical steps:

{{NumBlk|:|{{chem2|S1 + EH -> P1 + EP2}}|{{EquationRef|3}}}}

{{NumBlk|:|{{chem2|EP2 + H\sO\sH -> EH + P2}}|{{EquationRef|4}}}}

where S₁ is a polypeptide, P₁ and P₂ are products. The first chemical step ({{EquationNote|3}}) includes the formation of a covalent acyl-enzyme intermediate. The second step ({{EquationNote|4}}) is the deacylation step. It is important to note that 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 ({{EquationNote|3}}) 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 ({{EquationNote|1}}) and ({{EquationNote|2}}) 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.{{cite journal | vauthors = Lymn RW, Taylor EW | title = Mechanism of adenosine triphosphate hydrolysis by actomyosin | journal = Biochemistry | volume = 10 | issue = 25 | pages = 4617–4624 | date = December 1971 | pmid = 4258719 | doi = 10.1021/bi00801a004 }} 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.{{cite journal | vauthors = Holmes KC, Angert I, Kull FJ, Jahn W, Schröder RR | title = Electron cryo-microscopy shows how strong binding of myosin to actin releases nucleotide | journal = Nature | volume = 425 | issue = 6956 | pages = 423–427 | date = September 2003 | pmid = 14508495 | doi = 10.1038/nature02005 | s2cid = 2686184 | bibcode = 2003Natur.425..423H }}

Notably, the final steps of ATP hydrolysis include the fast release of phosphate and the slow release of ADP.{{cite journal | vauthors = Siemankowski RF, Wiseman MO, White HD | title = ADP dissociation from actomyosin subfragment 1 is sufficiently slow to limit the unloaded shortening velocity in vertebrate muscle | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 82 | issue = 3 | pages = 658–662 | date = February 1985 | pmid = 3871943 | pmc = 397104 | doi = 10.1073/pnas.82.3.658 | doi-access = free | bibcode = 1985PNAS...82..658S }}{{cite journal | vauthors = White HD, Belknap B, Webb MR | title = Kinetics of nucleoside triphosphate cleavage and phosphate release steps by associated rabbit skeletal actomyosin, measured using a novel fluorescent probe for phosphate | journal = Biochemistry | volume = 36 | issue = 39 | pages = 11828–11836 | date = September 1997 | pmid = 9305974 | doi = 10.1021/bi970540h }}

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.{{cite journal | vauthors = Tirosh R, Low WZ, Oplatka A | title = Translational motion of actin filaments in the presence of heavy meromyosin and MgATP as measured by Doppler broadening of laser light scattering | journal = Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology | volume = 1037 | issue = 3 | pages = 274–280 | date = March 1990 | pmid = 2178685 | doi = 10.1016/0167-4838(90)90025-b }}{{cite journal | vauthors = Tirosh R | year = 2006 | title = Ballistic protons and microwave-induced water solutions (solitons) in bioenergetic transformations | journal = Int. J. Mol. Sci. | volume = 7 | issue = 9| pages = 320–345 | doi=10.3390/i7090320| doi-access = free }}

In reality, most enzyme mechanisms involve a combination of several different types of catalysis.

Triose phosphate isomerase ({{EC number|5.3.1.1}}) catalyses the reversible interconversion of the two triose phosphates isomers dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate.

Trypsin ({{EC number|3.4.21.4}}) is a serine protease that cleaves protein substrates after lysine or arginine residues using a catalytic triad to perform covalent catalysis, and an oxyanion hole to stabilise charge-buildup on the transition states.

Aldolase ({{EC number|4.1.2.13}}) catalyses the breakdown of fructose 1,6-bisphosphate (F-1,6-BP) into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP).

The advent of single-molecule studies in the 2010s led to the observation that the movement of untethered enzymes increases with increasing substrate concentration and increasing reaction enthalpy.{{cite journal | vauthors = Muddana HS, Sengupta S, Mallouk TE, Sen A, Butler PJ | title = Substrate catalysis enhances single-enzyme diffusion | journal = Journal of the American Chemical Society | volume = 132 | issue = 7 | pages = 2110–2111 | date = February 2010 | pmid = 20108965 | pmc = 2832858 | doi = 10.1021/ja908773a | bibcode = 2010JAChS.132.2110M }}{{closed access}} Subsequent observations suggest that this increase in diffusivity is driven by transient displacement of the enzyme's center of mass, resulting in a "recoil effect that propels the enzyme".{{cite journal | vauthors = Riedel C, Gabizon R, Wilson CA, Hamadani K, Tsekouras K, Marqusee S, Pressé S, Bustamante C | display-authors = 6 | title = The heat released during catalytic turnover enhances the diffusion of an enzyme | journal = Nature | volume = 517 | issue = 7533 | pages = 227–230 | date = January 2015 | pmid = 25487146 | pmc = 4363105 | doi = 10.1038/nature14043 | bibcode = 2015Natur.517..227R }}{{closed access}}

Similarity between enzymatic reactions ([http://www.chem.qmul.ac.uk/iubmb/enzyme/ EC]) can be calculated by using bond changes, reaction centres or substructure metrics ([http://www.ebi.ac.uk/thornton-srv/software/rbl/ EC-BLAST] {{Webarchive|url=https://web.archive.org/web/20190530132101/http://www.ebi.ac.uk/thornton-srv/software/rbl/ |date=30 May 2019 }}).{{cite journal | vauthors = Rahman SA, Cuesta SM, Furnham N, Holliday GL, Thornton JM | title = EC-BLAST: a tool to automatically search and compare enzyme reactions | journal = Nature Methods | volume = 11 | issue = 2 | pages = 171–174 | date = February 2014 | pmid = 24412978 | pmc = 4122987 | doi = 10.1038/nmeth.2803 }}

• Catalytic triad
• Enzyme assay
• Enzyme inhibitor
• Enzyme kinetics
• Enzyme promiscuity
• Protein dynamics
• Pseudoenzymes, whose ubiquity despite their catalytic inactivity suggests omic implications
• Quantum tunnelling
• The Proteolysis Map
• Time resolved crystallography

{{Reflist}}

{{refbegin}}

• {{cite book | vauthors = Fersht A |title=Structure and Mechanism in Protein Science : A Guide to Enzyme Catalysis and Protein Folding |date=1998 |publisher=W.H. Freeman |location=New York |isbn=978-0-7167-3268-6}}
• {{cite journal | vauthors = Sutcliffe M, Munro A | title = Quantum catalysis in enzymes—beyond the transition state theory. | journal = Philosophical Transactions B | date = August 2006 | volume = 361 | issue = 1472 | pages = 1291–1455 | doi = 10.1098/rstb.2006.1879 | doi-access = free | pmc = 1647302 }}

{{refend}}

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