Editing Lysozyme (section)

==== 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]]