The catalytic site is the enzyme’s workshop, and it is here that the catalytic reaction occurs. This cleft, which is often buried (sometimes deeply) , houses a relatively small number of amino acids that are involved in binding the substrate (and/or cofactor), and an even smaller subset of these that are vital to the enzyme’s catalytic function.
In order to both study and understand the role and function of the catalytic amino acids in enzymes, it was first necessary to define what is meant by a ‘catalytic residue’. In MACiE, we have taken the definition first proposed by Bartlett et al.  and split that definition into two general categories, such that a catalytic residue is any residue involved in the reaction that: (a) has direct involvement in the reaction mechanism, the so-called reactant residues whose chemical structure is modified during the course of the reaction (for example, the residue is involved in covalent catalysis, electron shuttling or proton shuttling); and (b) has indirect, but essential, involvement in the reaction mechanism, the so-called spectator residues, whose chemical structure does not change during the course of the reaction – these are the residues that polarize or alter the pKa of a residue, a water molecule or part of the substrate directly involved in the reaction, affect the stereospecificity or regiospecificity of the reaction, or stabilize the reactive intermediates (either by stabilizing the transition states or the intermediates themselves, or destabilizing the ground states of the substrates).
All 20 amino acids (Arg, Asp, Cys, Glu, His, Lys, Ser, Thr, Trp, Tyr, Asn, Gln, Ile, Leu, Pro, Gly, Ala, Phe, Met and Val) are seen in MACiE as part of the active site machinery. MACiE does not currently contain any entries that utilize either selenocysteine or pyrrolysine as catalytic residues, so we cannot determine whether this is attributable to a lack of activity or of annotation for these two residues. The nonpolar residues (Ile, Leu, Pro, Gly, Ala, Phe, Met, Val and Trp) very rarely act through their side chains; instead, they act mainly through their main chain portions (usually as either the N-H group or the C=O group). However, only 10 of the catalytic residues annotated in MACiE (Arg, Asp, Cys, Glu, His, Lys, Ser, Thr, Trp and Tyr) are absolutely essential [12,13], in that they perform almost all of the functions associated with catalysis in all classes of enzymes. Nonetheless, their prevalence as catalytic entities in the six different classes of enzyme clearly differs. Catalytic propensity is a measure of how often a residue is catalytic as compared with its background levels in a protein; thus, it is calculated by dividing the percentage of that residue type that is catalytic by the total percentage of that residue in the whole protein dataset. If the propensity is < 1, then the propensity for that residue to be catalytic is less than expected, and if it is > 1, then the residue is more catalytic than might be expected by chance. Figure 1 shows the catalytic propensities of the 10 residues that are most commonly catalytic , along with Asn, Gln and Phe.
Figure 1. Balloon plot showing the propensity of a residue to be catalytic in each of the six classes of enzyme (EC 1, oxidoreductases; EC 2, transferases; EC 3, hydrolases; EC 4, lyases; EC 5, isomerases; EC 6, ligases). The diameter of the circle represents the value of the propensity; thus, the larger the circle, the higher the propensity of the residue to be catalytic. The circle is shown in blue if the propensity is greater than (or equal to) 1, and red if the propensity is < 1 (see Table S1 for exact values).
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What is immediately clear is that whereas some residues (such as His and Cys) are strongly catalytic in all classes, the catalytic propensities of the different residues varies between the six functional classifications of the EC. This difference is not attributable to differences between the background amino acid compositions of these enzymes (a detailed analysis is available from the database analysis and statistics section on the MACiE website). The differences in catalytic propensity are further seen in the functions that the residues are carrying out in the six different enzyme classes (as defined by the EC). It is possible to split the functions that the catalytic residues are performing into seven categories: (a) activation – residues that are responsible for activating other species; (b) steric role – residues that affect the outcome of the reaction through steric considerations; (c) stabilization – residues that (de)stabilize other species; (d) proton shuttling – residues that donate, accept or relay protons; (e) hydrogen radical shuttling – residues that donate, accept or relay hydrogen atoms; (f) electron shuttling – residues that donate, accept or relay electrons, either singly or in pairs; and (g) covalent catalysis – residues that become covalently attached to a reaction intermediate.
We have previously shown  that, with the exception of hydrogen radical shuttling and covalent catalysis (to a lesser degree), all of the residues examined are capable of performing all of the seven categories of residue function to some extent. However, the functional profiles of the residues analysed are different in each of the six enzyme (EC) classes, suggesting that the propensity of the residues to be catalytic in the different EC classes could well be related to the different roles that the residues can play. However, it is still not clear why residues that are capable of performing any one of the seven categories of function annotated have a predilection for performing certain functions in one class and other functions in another. We are currently looking into this phenomenon, including the effect of the local environment and physicochemical properties of these residues, in more detail.