Hydrolase catalytic groups

Fig. 3.2. Common catalytic groups of hydrolases involved in ester and amide bond hydrolysis (Z+ = electrophilic component polarizing the carbonyl group Y = nucleophilic group attacking the carbonyl C-atom H-B = proton donor transforming the -OR or -NR R" moiety into...

Threonine peptidases (and some cysteine and serine peptidases) have only one active site residue, which is the N-terminus of the mature protein. Such a peptidase is known as an N-terminal nucleophile hydrolase or Ntn-hydrolase. The amino group of the N-terminal residue performs the role of the general base. The catalytic subunits of the proteasome are examples of Ntn-hydrolases. 

The mechanism for the lipase-catalyzed reaction of an acid derivative with a nucleophile (alcohol, amine, or thiol) is known as a serine hydrolase mechanism (Scheme 7.2). The active site of the enzyme is constituted by a catalytic triad (serine, aspartic, and histidine residues). The serine residue accepts the acyl group of the ester, leading to an acyl-enzyme activated intermediate. This acyl-enzyme intermediate reacts with the nucleophile, an amine or ammonia in this case, to yield the final amide product and leading to the free biocatalyst, which can enter again into the catalytic cycle. A histidine residue, activated by an aspartate side chain, is responsible for the proton transference necessary for the catalysis. Another important factor is that the oxyanion hole, formed by different residues, is able to stabilize the negatively charged oxygen present in both the transition state and the tetrahedral intermediate. 

In this article are discussed the results of those studies which have become available over the past 15 years and which permit some generalizations on the catalytic mechanism of glycoside hydrolases from widely differing sources and with different sugar and aglycon specificities. It will be seen that, with few exceptions, the data support a mechanism almost identical to that proposed by Phillips and his group for lysozyme. ... 

The role of the serine residue in hydrolysis was further examined using pseudo-substrates, e.g. p-nitrophenylacetate—substrates which were only very slowly utilized by the enzyme. The p-nitrophenyl group was slowly released and the acyl group became attached to the same serine in hydrolases which had been detected by DIPF (Kilby and Youatt, 1954). Mechanisms for peptide and ester hydrolysis were therefore proposed in which the acyl group became transiently and covalently bound to serines in catalytic sites (see Hartley et al. 1969). 

These three catalytic functionalities are similar in practically all hydrolytic enzymes, but the actual functional groups performing the reactions differ among hydrolases. Based on the structures of their catalytic sites, hydrolases can be divided into five classes, namely serine hydrolases, threonine hydrolases, cysteine hydrolases, aspartic hydrolases, and metallohydrolases, to which the similarly acting calcium-dependent hydrolases can be added. Hydrolases of yet unknown catalytic mechanism also exist. 

The serine hydrolases, threonine hydrolases, and cysteine hydrolases, the attacking nucleophile of which is a serine or threonine OH group or a cysteine thiolate group, respectively, and which form an intermediate covalent complex (i. e., the acylated enzyme). Here, an activated H20 molecule enters the catalytic cycle in the second step, i.e., hydrolysis of the covalent intermediate to regenerate the enzyme. 

Fig. 3.3. Major steps in the hydrolase-catalyzed hydrolysis of peptide bonds, taking chymo-trypsin, a serine hydrolase, as the example. Asp102, His57, and Ser195 represent the catalytic triad the NH groups of Ser195 and Gly193 form the oxyanion hole . Steps a-c acylation Steps d-f deacylation. A possible mechanism for peptide bond synthesis by peptidases is represented by the reverse sequence Steps f-a.

The mechanism of hydrolysis of cysteine peptidases, in particular cysteine endopeptidases (EC 3.4.22), shows similarities and differences with that of serine peptidases [2] [3a] [55 - 59]. Cysteine peptidases also form a covalent, ac-ylated intermediate, but here the attacking nucleophile is the SH group of a cysteine residue, or, rather, the deprotonated thiolate group. Like in serine hydrolases, the imidazole ring of a histidine residue activates the nucleophile, but there is a major difference, since here proton abstraction does not appear to be concerted with nucleophilic substitution but with formation of the stable thiolate-imidazolium ion pair. Presumably as a result of this specific activation of the nucleophile, a H-bond acceptor group like Glu or Asp as found in serine hydrolases is seldom present to complete a catalytic triad. For this reason, cysteine endopeptidases are considered to possess a catalytic dyad (i.e., Cys-S plus H-His+). The active site also contains an oxyanion hole where the terminal NH2 group of a glutamine residue plays a major role. 

The transesterification of cocaine to cocaethylene is an enzymatic reaction catalyzed by microsomal carboxylesterases and blocked by inhibitors of serine hydrolases [124][125], In Chapt. 3, we have discussed the mechanism of serine hydrolases, showing how a H20 molecule enters the catalytic cycle to hydrolyze the acylated serine residue in the active site of the enzyme. In the case of cocaine, the acyl group is the benzoylecgoninyl moiety (Fig. 7.9,d ), which undergoes esterification with ethanol according to Steps e and/ (Fig. 7.9). 

The overall reaction catalyzed by epoxide hydrolases is the addition of a H20 molecule to an epoxide. Alkene oxides, thus, yield diols (Fig. 10.5), whereas arene oxides yield dihydrodiols (cf. Fig. 10.8). In earlier studies, it had been postulated that epoxide hydrolases act by enhancing the nucleo-philicity of a H20 molecule and directing it to attack an epoxide, as pictured in Fig. 10.5, a [59] [60], Further evidence such as the lack of incorporation of 180 from H2180 into the substrate, the isolation of an ester intermediate, and the effects of group-selective reagents and carefully designed inhibitors led to a more-elaborate model [59][61 - 67]. As pictured in Fig. 10.5,b, nucleophilic attack of the substrate is mediated by a carboxylate group in the catalytic site to form an ester intermediate. In a second step, an activated H20... 

Fig. 10.5. Catalytic models of epoxide hydrolase (modified from [59]). a) In an earlier model, a basic group in the enzyme activates a H20 molecule during nucleophilic attack on the epoxide, b) A more-elaborate model showing a carboxylate group in the catalytic site that carries out the nucleophilic attack on the substrate to form an ester intermediate. Only in the second step is the intermediate hydrolyzed by an activated H20 molecule, leading to enzyme reactivation and product liberation. 

The most recently discovered group of proteases are the N-terminal threonine hydrolases of the multi-catalytic protease complex (MPC) of proteasomes. The enzymes are arranged in a regular array inside proteasomal compartments as shown in Box 7-A. The active site is a catalytic dyad formed from the amino group at the N terminus of the P subunits.345-3463... 

All presently known nonredox enzymes with a cubane as catalytic center are hydrolases (see later discussion). In these enzymes Fea is formally equivalent to the tetrahedral Zn(II) site in hydrolytic zinc enzymes. Thus, when the enzyme is ready to act but not yet in turnover (the substrate-free enzyme), its cluster is probably best described as structure ii in Figure 1 with X a hydroxyl group. In shorthand notation this could be written as [FeaOH—3Feb—4S]1+ (Cys)3 or as [Fea3Feb-4S]2+OH(Cys)3 where it has been assumed that, on average, each iron is formally Fe2 5+. 

---