Editing Lysozyme (section)

=== Mechanism ===

==== Phillips ====
The Phillips mechanism proposed that the enzyme's catalytic power came from both steric strain on the bound substrate and electrostatic stabilization of an [[Oxocarbenium|oxo-carbenium]] intermediate. From X-ray crystallographic data, Phillips proposed the active site of the enzyme, where a hexasaccharide binds. The lysozyme distorts the fourth sugar (in the D or -1 subsite) in the hexasaccharide into a half-chair conformation. In this stressed state, the glycosidic bond is more easily broken.<ref>{{cite journal | vauthors = Blake CC, Johnson LN, Mair GA, North AC, Phillips DC, Sarma VR | title = Crystallographic studies of the activity of hen egg-white lysozyme | journal = Proceedings of the Royal Society of London. Series B, Biological Sciences | volume = 167 | issue = 1009 | pages = 378–388 | date = April 1967 | pmid = 4382801 | doi = 10.1098/rspb.1967.0035 | s2cid = 35094695 | bibcode = 1967RSPSB.167..378B }}</ref> An ionic intermediate containing an [[Oxocarbenium|oxo-carbenium]] is created as a result of the glycosidic bond breaking.<ref name="Application of secondary alpha-deut">{{cite journal | vauthors = Dahlquist FW, Rand-Meir T, Raftery MA | title = Application of secondary α-deuterium kinetic isotope effects to studies of enzyme catalysis. Glycoside hydrolysis by lysozyme and β-glucosidase | journal = Biochemistry | volume = 8 | issue = 10 | pages = 4214–4221 | date = October 1969 | pmid = 5388150 | doi = 10.1021/bi00838a045 }}</ref> Thus distortion causing the substrate molecule to adopt a strained conformation similar to that of the [[transition state]] will lower the energy barrier of the reaction.<ref name="McKenzie_1991">{{cite journal | vauthors = McKenzie HA, White FH | title = Lysozyme and α-lactalbumin: structure, function, and interrelationships | journal = Advances in Protein Chemistry | volume = 41 | pages = 173–315 | year = 1991 | pmid = 2069076 | doi = 10.1016/s0065-3233(08)60198-9 | isbn = 978-0-12-034241-9 }}</ref>

The proposed oxo-carbonium intermediate was speculated to be electrostatically stabilized by aspartate and glutamate residues in the active site by [[Arieh Warshel]] in 1978. The electrostatic stabilization argument was based on comparison to bulk water, the reorientation of water dipoles can cancel out the stabilizing energy of charge interaction. In Warshel's model, the enzyme acts as a super-solvent, which fixes the orientation of ion pairs and provides super-[[solvation]] (very good stabilization of ion pairs), and especially lower the energy when two ions are close to each other.<ref>{{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 }}</ref>

The [[rate-determining step]] (RDS) in this mechanism is related to formation of the [[Oxocarbenium|oxo-carbenium]] intermediate. There were some contradictory results to indicate the exact RDS. By tracing the formation of product ([[4-Nitrophenol|p-nitrophenol]]), it was discovered that the RDS can change over different temperatures, which was a reason for those contradictory results. At a higher temperature the RDS is formation of glycosyl enzyme intermediate and at a lower temperature the breakdown of that intermediate.<ref>{{cite journal | vauthors = Weber JP, Fink AL | title = Temperature-dependent change in the rate-limiting step of β-glucosidase catalysis | journal = The Journal of Biological Chemistry | volume = 255 | issue = 19 | pages = 9030–9032 | date = October 1980 | pmid = 6773958 | doi = 10.1016/S0021-9258(19)70521-3 | doi-access = free }}</ref>
[[File:Lysozyme glycosyl covalent intermediate.gif|thumb|Covalent intermediate of lysozyme enzyme, with covalent bond in black and experimental evidence as blue mesh.<ref>{{Cite web|url=http://proteopedia.org/wiki/index.php/Lysozyme#Covalent_intermediate_and_product_complex|title = Hen Egg-White (HEW) Lysozyme - Proteopedia, life in 3D}}</ref>]]

==== Covalent mechanism ====
[[File:LysozymeIntermediates copy.png|thumb|400px|Substrates in Vocadlo's experiment]]
In an early debate in 1969, Dahlquist proposed a covalent mechanism for lysozyme based on [[kinetic isotope effect]],<ref name="Application of secondary alpha-deut"/> but for a long time the ionic mechanism was more accepted. In 2001, a revised mechanism was proposed by Vocadlo via a covalent but not ionic intermediate. Evidence from [[Electrospray ionization|ESI]]-[[Mass spectrometry|MS]] analysis indicated a covalent intermediate. A 2-fluoro substituted substrate was used to lower the reaction rate and accumulate an intermediate for characterization.<ref name="Vocadlo_2001">{{cite journal | vauthors = Vocadlo DJ, Davies GJ, Laine R, Withers SG | title = Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate | journal = Nature | volume = 412 | issue = 6849 | pages = 835–838 | date = August 2001 | pmid = 11518970 | doi = 10.1038/35090602 | s2cid = 205020153 | bibcode = 2001Natur.412..835V | url = https://eprints.whiterose.ac.uk/131/1/daviesgj1.pdf }}</ref> The amino acid side-chains glutamic acid 35 (Glu35) and aspartate 52 (Asp52) have been found to be critical to the activity of this enzyme. Glu35 acts as a proton donor to the glycosidic bond, cleaving the C-O bond in the substrate, whereas Asp52 acts as a [[nucleophile]] to generate a glycosyl enzyme intermediate. The Glu35 reacts with water to form hydroxyl ion, a stronger [[nucleophile]] than water, which then attacks the glycosyl enzyme intermediate, to give the product of hydrolysis and leaving the enzyme unchanged.<ref name="isbn0-495-11912-12">{{cite book | title = Biochemistry | vauthors = Grisham CM, Garrett RH | publisher = Thomson Brooks/Cole | year = 2007 | isbn = 978-0-495-11912-8 | location  =Australia | pages = 467–9 | chapter = Chapter 14: Mechanism of enzyme action | chapter-url = https://books.google.com/books?id=W4o_5YGqfYsC&q=lysozyme%20mechanism%20of%20action%20glu-35%20asp-52&pg=PA468}}</ref> This type of covalent mechanism for enzyme catalysis was first proposed by [[Daniel E. Koshland Jr.|Koshland]].<ref>{{cite journal | vauthors = Koshland DE | date = November 1953 | title = Stereochemistry and the Mechanism of Enzymatic Reactions | journal = Biological Reviews | volume = 28 | issue = 4 | pages = 416–436 | doi = 10.1111/j.1469-185X.1953.tb01386.x | s2cid = 86709302 | url = https://digital.library.unt.edu/ark:/67531/metadc1255185/ }}</ref>

More recently, quantum mechanics/ molecular mechanics (QM/MM) [[molecular dynamics]] simulations have been using the crystal of HEWL and predict the existence of a covalent intermediate.<ref name = "Bowman_2008">{{cite journal | vauthors = Bowman AL, Grant IM, Mulholland AJ | title = QM/MM simulations predict a covalent intermediate in the hen egg white lysozyme reaction with its natural substrate | journal = Chemical Communications | issue = 37 | pages = 4425–4427 | date = October 2008 | pmid = 18802578 | doi = 10.1039/b810099c }}</ref> Evidence for the ESI-MS and X-ray structures indicate the existence of covalent intermediate, but primarily rely on using a less active mutant or non-native substrate. Thus, QM/MM molecular dynamics provides the unique ability to directly investigate the mechanism of wild-type HEWL and native substrate. The calculations revealed that the covalent intermediate from the covalent mechanism is ~30 kcal/mol more stable than the ionic intermediate from the Phillips mechanism.<ref name="Bowman_2008" /> These calculations demonstrate that the ionic intermediate is extremely energetically unfavorable and the covalent intermediates observed from experiments using less active mutant or non-native substrates provide useful insight into the mechanism of wild-type HEWL.{{cn|date=July 2024}}
[[File:JBSlysozymemechanism copy2.jpg|thumb|Two Possible Mechanisms of Lysozyme]]