Editing Trypsin (section)

== Mechanism ==
The enzymatic mechanism is similar to that of other serine proteases. These enzymes contain a [[catalytic triad]] consisting of [[histidine]]-57, [[aspartate]]-102, and [[serine]]-195.<ref name="pmid16003488">{{cite journal | vauthors = Polgár L | title = The catalytic triad of serine peptidases | journal = Cellular and Molecular Life Sciences | volume = 62 | issue = 19–20 | pages = 2161–72 | date = October 2005 | pmid = 16003488 | doi = 10.1007/s00018-005-5160-x | s2cid = 3343824 | pmc = 11139141 }}</ref> This catalytic triad was formerly called a charge relay system, implying the abstraction of protons from serine to histidine and from histidine to aspartate, but owing to evidence provided by NMR that the resultant alkoxide form of serine would have a much stronger pull on the proton than does the imidazole ring of histidine, current thinking holds instead that serine and histidine each have effectively equal share of the proton, forming short [[low-barrier hydrogen bond]]s therewith.<ref>{{cite book | title = Biochemistry | vauthors = Voet D, Voet JG |date=2011 |publisher=John Wiley & Sons |isbn=978-0-470-57095-1 |edition=4th |location=Hoboken, NJ |oclc=690489261}}</ref>{{Page needed|date=April 2020}} By these means, the [[nucleophile|nucleophilicity]] of the [[active site]] serine is increased, facilitating its attack on the amide carbon during proteolysis. The enzymatic reaction that trypsin catalyzes is [[thermodynamics|thermodynamically]] favorable, but requires significant [[activation energy]] (it is "[[enzyme kinetics|kinetically]] unfavorable"). In addition, trypsin contains an "oxyanion hole" formed by the backbone amide hydrogen atoms of Gly-193 and Ser-195, which through hydrogen bonding stabilize the negative charge which accumulates on the amide oxygen after nucleophilic attack on the planar amide carbon by the serine oxygen causes that carbon to assume a tetrahedral geometry. Such stabilization of this tetrahedral intermediate helps to reduce the energy barrier of its formation and is concomitant with a lowering of the free energy of the transition state. Preferential binding of the transition state is a key feature of enzyme chemistry.

The negative aspartate residue (Asp 189) located in the catalytic pocket (S1) of trypsin is responsible for attracting and stabilizing positively charged lysine and/or arginine, and is, thus, responsible for the specificity of the enzyme. This means that trypsin predominantly cleaves [[protein]]s at the [[carboxyl]] side (or "[[C-terminal]] side") of the [[amino acid]]s lysine and arginine except when either is bound to a C-terminal [[proline]],<ref name="urlwww.promega.com">{{cite web | url = http://www.promega.com/tbs/9piv511/9piv511.pdf | title = Sequencing Grade Modified Trypsin | date = 2007-04-01 | publisher = promega.com | access-date = 2009-02-08 | archive-date = 2003-05-19 | archive-url = https://web.archive.org/web/20030519223600/http://www.promega.com/tbs/9piv511/9piv511.pdf }}</ref> although large-scale mass spectrometry data suggest cleavage occurs even with proline.<ref name="pmid18067249">{{cite journal | vauthors = Rodriguez J, Gupta N, Smith RD, Pevzner PA | title = Does trypsin cut before proline? | journal = Journal of Proteome Research | volume = 7 | issue = 1 | pages = 300–5 | date = January 2008 | pmid = 18067249 | doi = 10.1021/pr0705035 | url = http://proteomics.ucsd.edu/pavel/papers/does_trypsin_cut_before_proline.pdf | citeseerx = 10.1.1.163.4052 | access-date = 2017-10-25 | archive-date = 2020-08-13 | archive-url = https://web.archive.org/web/20200813233625/http://proteomics.ucsd.edu/pavel/papers/does_trypsin_cut_before_proline.pdf | url-status = dead }}</ref>   Trypsin is considered an [[endopeptidase]], i.e., the cleavage occurs within the polypeptide chain rather than at the terminal amino acids located at the ends of [[peptide|polypeptides]].