= Active site
{wiki}
An <active site> is the region of an <enzyme> where a <substrate> binds and undergoes a <chemical reaction>. In a <protein> <enzyme>, it includes the <amino acid residues> that contact the <substrate> and those that directly assist <catalysis>. These roles can overlap: an <amino acid side chain> can both orient a <substrate> and participate in chemistry. The <active site> occupies only part of the folded <protein>; the remaining <amino acid residues> help establish its <protein structure>, stability and movement. An <active site> need not be a consecutive stretch of <protein primary structure>: <protein folding> can bring distant <amino acid residues> together. A <ribozyme> instead supplies an <RNA> <active site>. See https://www.ncbi.nlm.nih.gov/books/NBK26911/[Alberts et al., Molecular Biology of the Cell, Protein Function: binding sites and catalytic sites].
Binding and <catalysis> answer different questions. A <protein binding site> recognizes shape and chemistry through such interactions as <hydrogen bonds>, <electrostatics> and the <hydrophobic effect>. At an <active site>, appropriate positioning also helps a <substrate> reach the <transition state> of a <chemical reaction>. Some <amino acid side chains> transfer a <proton>; others participate transiently in a <covalent bond>. The <enzyme-substrate complex> is the associated <enzyme> and <substrate>, before conversion to a <reaction product>. Merely accumulating this complex does not establish rapid <catalysis>: stabilizing the bound <substrate> without sufficiently stabilizing the <transition state> can leave a large barrier. The important complementarity is chemical as well as geometric. See https://www.ncbi.nlm.nih.gov/books/NBK9921/[Geoffrey M. Cooper, The Cell, The Central Role of Enzymes as Biological Catalysts: substrate binding and transition-state stabilization].
\Image[/active-sites-induced-fit-residue-map.png]
{title=Distant <amino acid residues> can cooperate at an <active site>}
{description=Original twelve-residue <polypeptide chain> cartoon, with the same order and eleven connections in both drawings. Positions 3, 7 and 10 approach a generic <substrate> after an imagined fold. Blue means recognition, orange means catalytic chemistry, and purple means both; these assignments are invented for explanation. Dashed contacts represent cooperation, not specified <hydrogen bonds> or actual atomic coordinates. The other nine positions support the imagined <protein structure>; three highlighted positions are not a universal <active site> size.}
The cartoon makes a useful experimental distinction. Changing a contacting <amino acid residue> might weaken <substrate> binding, change the <reaction rate>, or do both. Changing a distant <amino acid residue> can also disrupt the <active site> by changing <protein folding>. Therefore a loss of activity after a <mutation> alone does not locate a catalytic group: compare binding, <protein structure> and <enzyme kinetics> as well. This is an interpretation guide, not a report of a particular <mutation> experiment.
A concrete example is <hexokinase>: it transfers the terminal <phosphate> group of <ATP> to the $6$-position <hydroxyl group> of a <sugar> such as <glucose>. Specificity need not mean only one acceptable <substrate>: the <hexokinase> reaction class includes several possible acceptor <sugars>. Which are used, and at what <reaction rates>, depends on the particular <enzyme> and conditions. See https://enzyme.expasy.org/EC/2.7.1.1[Swiss Institute of Bioinformatics, ENZYME entry EC 2.7.1.1: hexokinase reaction and acceptor range].
Bennett and Steitz compared <yeast> <hexokinase> A crystallized with <glucose> against <hexokinase> B crystallized without <glucose>. <X-rays> scattered by the crystals supplied <diffraction> measurements from which the authors reconstructed <protein structure>; comparing coordinates located movement, rather than measuring a <reaction rate>. The <glucose>-containing cleft was narrower: one lobe was rotated by about $12^\circ$ relative to the other, with some <protein backbone> displacements reaching $8\,\mathrm{\mathring A}$. This makes the <active site> a moving arrangement of interacting groups, rather than a permanent hole. See https://pmc.ncbi.nlm.nih.gov/articles/PMC336218/[William S. Bennett Jr. and Thomas A. Steitz, Glucose-induced conformational change in yeast hexokinase, PNAS 75 (1978), 4848–4852].
The structural comparison has two explicit limitations: different forms of <hexokinase> and different crystal environments were compared, and static coordinates do not record the order of binding and movement. Closure is consistent with <induced fit>, but those structures alone cannot exclude <conformational selection>. The solution experiment under <induced fit> addresses whether compaction also occurs outside a crystal.
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