Charge relay

FIGURE 7.2. Two alternative mechanisms for the catalytic reaction of serine proteases. Route a corresponds to the electrostatic catalysis mechanism while route b corresponds to the double proton transfer (or the charge relay mechanism), gs ts and ti denote ground state, transition state and tetrahedral intermediate, respectively. 

The considerations presented above were based on the specific assumption that the catalytic reaction of the serine proteases involves mechanism a of Fig. 7.2. However, one can argue that the relevant mechanism is mechanism b (the so-called charge-relay mechanism ). In principle the proper procedure, in case of uncertainty about the actual mechanism, is to perform the calculations for the different alternative mechanisms and to find out which of the calculated activation barriers reproduces the observed one. This procedure, however, can be used with confidence only if the calculations are sufficiently reliable. Fortunately, in many cases one can judge the feasibility of different mechanisms without fully quantitative calculations by a simple conceptual consideration based on the EVB philosophy. To see this point let us consider the feasibility of the charge-relay mechanism (mechanism b) as an alternative to mechanism a. Starting from Fig. 7.2 we note that the energetics of route b can be obtained from the difference between the activation barriers of route b and route a by... 

Catalysis, specific acid, 163 Catalytic triad, 171,173 Cavity radius, of solute, 48-49 Charge-relay mechanism, see Serine proteases, charge-relay mechanism Charging processes, in solutions, 82, 83 Chemical bonding, 1,14 Chemical bonds, see also Valence bond model... 

Double proton transfer mechanism, see Serine proteases, charge-relay mechanism... 

Figure 7-7. Catalysis by chymotrypsin. The charge-relay system removes a proton from Ser 195, making it a stronger nucleophile. Activated Ser 195 attacks the peptide bond, forming a transient tetrahedral intermediate. Release of the amino terminal peptide is facilitated by donation of a proton to the newly formed amino group by His 57 of the charge-relay system, yielding an acyl-Ser 195 intermediate. His 57 and Asp 102 collaborate to activate a water molecule, which attacks the acyl-Ser 195, forming a second tetrahedral intermediate. The charge-relay system donates a proton to Ser 195, facilitating breakdown of tetrahedral intermediate to release the carboxyl terminal peptide .

CHj)3C.CO.OE, by attack of the serine hydroxyl group on the carbonyl carbon atom of 4-nitrophenyl trimethylacetate. This attack is assisted by proton removal from the hydroxyl group by the charge-relay mechanism, Scheme 13. It is also considered that breakdown of trimethylacetylchymo-trypsin may be assisted by the charge-relay mechanism. In this case, a proton... 

A charge relay system (Blow, 1976) exists in a number of hydrolytic enzymes. In such systems, an aspartate carboxylate group buried in a hydrophobic microenvironment activates a seryl hydroxyl group through hydrogen bonding. Thus, it is interesting to study the effect of carboxylate ions on other nucleophiles in aprotic media. 

A similar charge-relay model system has been proposed for the couple of carboxylate ion-thiol (Kobuke and Yoshida, 1977). 

The mechanism schematized above is a summary of the current knowledge. The role of Asp102 has long been controversial [10], Indeed, the catalytic triad has been depicted as a charge-relay system, meaning that the activation of the serine residue involves a concerted transfer of two protons, i.e., from serine to histidine and then to aspartic acid. More recent studies have shown that aspartic acid remains ionized and serves to stabilize the ionic transition state [6] [14-16],... 

R. L. Schowen, Structural and Energetic Aspects of Proteolytic Catalysis by Enzymes Charge-Relay Catalysis in the Function of Serine Proteases , in Mechanistic Principles of Enzyme Activity , Eds. J. F. Liebman, A. Greenberg, VCH, New York, 1988, p. 119 — 165. 

A critical input in unraveling the catalytic mechanism of epoxide hydrolases has come from the identification of essential residues by a variety of techniques such as analysis of amino acid sequence relationships with other hydrolases, functional studies of site-directed mutated enzymes, and X-ray protein crystallography (e.g., [48][53][68 - 74]). As schematized in Fig. 10.6, the reaction mechanism of microsomal EH and cytosolic EH involves a catalytic triad consisting of a nucleophile, a general base, and a charge relay acid, in close analogy to many other hydrolases (see Chapt. 3). 

Fig. 10.6. Simplified representation of the postulated catalytic cycle of microsomal and cytosolic epoxide hydrolases, showing the roles played by the catalytic triad (i.e., nucleophile, general base, and charge relay acid) and some other residues, a) Nucleophilic attack of the substrate to form a /3-hydroxyalkyl ester intermediate, b) Nucleophilic attack of the /Thydroxyal-kyl ester by an activated H20 molecule, c) Tetrahedral transition state in the hydrolysis of the /f-hydroxyalkyl ester, d) Product liberation, with the enzyme poised for a further catalytic... 

Schowen, R.L. (1988). Structural and energetic aspects of protolytic catalysis by enzymes charge-relay catalysis in the function of serine proteases. In Mechanistic Principles of Enzyme Activity, Liebman, J.P. and Greenberg, A. (eds), pp. 119-168. VCH Publishers, New York... 

Crystallographic studies (Blow, 1976) of the structure of the enzyme, enzyme-substrate complexes and enzyme-product complexes have identified a common feature in catalysis by the serine protease enzymes such as a-chymotrypsin. This is the well-known charge-relay system (44), in which... 

Hunkapiller et al. (1973) have recently inferred from NMR data that the pKa-value of ca. 7 in serine proteases is actually that of aspartic acid while histidine has a lower pK (< 4) so that at pH-values from 3-6 7 these residues will be neutral. A mechanism was proposed, utilizing the asp-102-hist-57-ser-195 charge relay system which would avoid charge separation. 

Does the charge relay mechanism play an important role and, if so, what rate enhancement would such a mechanism provide It appears that it will not be necessary to invoke a charge-relay mechanism to account for the rates of a-chymotrypsin reactions in terms of known chemistry. The presence of aspartic acid in the interior of serine proteases could, of course, have structural rather than mechanistic significance. 

CHARGE RELAY SYSTEM CATASTROPHE THEORY Catastrophic depolymerization of microtubules,... 

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