1.5 Proteases
1.5.1 Protease classification and catalytic mechanisms
Table 1-1. The four currently recognised classes of proteases. (After Barrett, 1994).
Class Representative Active site residues Diagnostic inhibitor"
protease
Serine I (mammalian) Chymotrypsin Asp102, Ser195, His5? DFP Serine II (bacterial) Subtilisin Asp32, Ser221, His64 DFP Cysteine Papain Cys,25 Hi 159 As 158s , P E-64
Aspartic PenicilIopepsin Asp33, Asp213 Pepstatin
Metallo I (mammalian) CarboxypeptidaseA Zn, Glu270,Trp248 EDTA;
Metallo II (bacterial) Thermolysin Zn, GIUI43,HiS231 1,10-phenanthroline
aE-64,L-trans-epoxysuccinyl-Ieucylamido(4-guanidino)butane;
EDTA, ethylenediaminetetra-acetic acid;
DFP, di-isopropyl fluorophosphate
Serine and cysteine pro teases form covalent enzyme-substrate complexes whereas the aspartic and metallo-proteases do not. However, the overall effect of protease activity, namely, scissile bond cleavage, is identical for all classes and the differences between catalytic mechanisms are fairly subtle. Nucleophilic attack on the carbonyl group requires a nucleophile, in the form of oxygen or sulfur, to approach the slightly electrophilic carbonyl carbon. This process is facilitated by general base catalysis removing a proton from the attacking nucleophile and some electrophilic influence on the carbonyl oxygen to increase the polarisation of the C=O bond. The precise functional groups performing these functions (nucleophilic attack, general base catalysis and electrophilic assistance) differ between the four protease classes. The tetrahedral intermediate formed after the initial nucleophilic attack requires breakdown by general acid catalysis to assist in the release of the amine. Again, the groups playing this role differ for each protease class (Neurath, 1989).
a). Serine proteases.
Michaelis complex formation between enzyme and substrate is facilitated by non-specific binding of S and S / residues to P and P/ residues respectively. The close proximity of crucial active site residues allows Ser195
(chymotrypsin numbering) to transfer a proton to the imidazole ring of His5
? , a charge transfer which is facilitated and stabilised by the negative charge of Asp102. This system boosts the electronegativity of the oxygen of the active site Ser195,
and results in nucleophilic attack on the carbonyl carbon of the scissile bond. At this point, enzyme and substrate are in an unstable tetrahedral transition state, but the oxygen
aruon IS stabilised by hydrogen bonding by -NH- groups of amino acids in the so-called oxyanion hole (01/93, Ser195). In this activated state, cleavage of the peptide bond occurs .
. 57 £ . al
The C-terminal portion of the substrate extracts the proton from HIS to orm a new termm amino group and subsequently dissociates, while the N-terminal portion remains covalently attached to the enzyme via an acyl linkage. Deacylation of the acyl-enzyme intermediate is essentially a reversal of the above process, with a water molecule taking the place of the released substrate polypeptide. The water molecule transfers a proton to His57
and the hydroxyl group makes a nucleophilic attack on the carbonyl carbon of the ester. This results in the formation of a second tetrahedral transition state and subsequent cleavage of the acyl bond. The substrate portion with newly formed carboxy terminus dissociates, and the proton is transferred from His57
back to Ser195
and the enzyme returns to ground state. Thus the catalysis proceeds by an intermediate acyl transfer from substrate, to enzyme via a covalent bond, to water. This is the common feature between serine pro teases and other transferases in biology (Dunn, RM., 1989).
b). Cysteine proteases.
This family bears great mechanistic similarity to the serine pro teases, also proceeding by intermediate acyl transfer to water. In this case the attacking nucleophile is the sulfur atom of the active site Cys25 (papain numbering). A histidine side chain (His159) is again involved in a hydrogen acceptor/shuttle role, stabilised by Asp158. Maintaining the analogy with serine proteases, the -NH- groups of 01n19
and Cys25 provide the stabilising hydrogen bonds of the oxyanion hole.
c). Aspartic proteases.
These enzymes do not rely on nucleophilic attack for catalysis and do not form covalent intermediates with substrates. Catalysis is mediated by two aspartic acid side chains (ASp33 and Asp213 , penicillopepsin numbering) which are in close geometric proximity. In the enzymatically active pH range (pH 2-3), one of these is ionised and the other is not. They share a hydrogen bond between two of their oxygens and, in the native form, are hydrogen bonded to a water molecule. After formation of the Michaelis complex, general acid-general base catalysis occurs with the attack of the hydrogen-bonded water molecule on the substrate carbonyl group. A tetrahedral intermediate is formed and the scissile bond is cleaved. The low pH optimum of aspartic pro teases and the numerous hydrogen bonds within the active site cleft facilitate this catalytic mechanism. Breakdown of the tetrahedral intermediate yields
a non-covalent product complex containing both halves of the substrate. Dissociation of either half can follow to give an acyl product complex or an amino product complex, the final dissociation of which returns the enzyme to ground state.
d). Metallo-proteases.
Like aspartic pro teases, these enzymes do not form covalent intermediates. This class of enzyme has no oxyanion hole component to effect catalysis on the carbonyl group of the scissile bond, but utilises co-ordination to a metal ion instead (Dunn, B.M., 1989). This metal is usually zinc, although other transition metals can substitute. In the native enzyme, the metal atom is tetrahedrally co-ordinated to a water molecule and three amino acid side chains (two histidines and a glutamic acid in carboxypeptidase A and thermolysin). The water molecule may be displaced by co-ordination to the substrate carbonyl, perhaps via a transition state, but is thought to remain in the active site. This water molecule is also hydrogen-bonded to a glutamic acid side chain (Glu27o, carboxypeptidase numbering). The carboxyl group of Glu270 acts a general base, removing a proton from the water molecule and assisting its attack on the peptide carbonyl. The attack is further facilitated by the co-ordination of the substrate to the metal ion, which exerts a strong e1ectrophilic attraction on the carbonyl oxygen. A proton must be transferred to the leaving nitrogen atom and could be derived from Glu270.
Hence the glutamic acid acts as a 'shuttle', analogous to one of the catalytic groups in the aspartic pro teases and the histidine in serine and cysteine pro teases.
e). Inhibition of proteases.
As detailed above, serine and cysteine pro teases have strongly nucleophilic amino acids within their catalytic sites. These are usually aligned with hydrogen bond acceptors to promote dissociation of the nucleophile in the approach to the transition state, thereby increasing the fraction in the hyper-reactive state. Effective inhibitors of these proteases will thus comprise molecules able to introduce electrophilic groups to chemically modify the nucleophile or general base, thereby rendering the catalytic apparatus inactive (Dunn, B.M., 1989). Aspartic and metallo-proteases do not rely on nucleophilic attack but rather upon general acid-general base catalysis of the attack of a water molecule. As the catalytic residues lack the aggressive nucleophilicityof serine and cysteine pro teases, effective inhibition of these pro teases relies more on secondary binding interactions along the active site cleft and on transition state analogues. In the case of the metallo-proteases, the metal ion can be exploited for inhibition by the introduction of functional groups, which lead to nearly irreversible chelation (Dunn, B.M., 1989). Specific inhibitors have proved very useful in the
determination of protease classes (Barrett, 1994) and are often referred to as diagnostic inhibitors, as detailed in Table 1-1.