Relay mechanism, charge

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... 

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... 

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. 

The inventory technique is clearly a potentially useful tool for elucidating enzymatic transition states. Deacetylation of acetyl-a-chymotrypsin is consistent with a single proton transfer (Eqn. 45) [25] with a fractionation factor 0.42. It is thought that this result excludes the charge-relay mechanism (Eqn. 46) which would require a quadratic term for the dependence. 

Interpretation of early X-ray and NMR spectroscopic results lead to the hypothesis that the serine hydroxyl is activated via a charge-relay mechanism including proton removal from serine to the buried aspartate anion via the neighbouring histidine (cf. Fig. 4 b). This double proton transfer would yield neutral aspartic acid and an alkoxide anion with enhanced nucleophilicity. Later this was questioned on the basis of more precise NMR and neutron diffraction studies (cf. [241] for references). At variance with earlier quantum chemical calculations [246, 247] predicting the aspartate as the ultimate proton acceptor, we stressed the importance of the electrostatic effect of the environment including the protein dipoles, surrounding water molecules and a counter ion, and concluded that, while the Asp-His couple exists in a neutral form in vacuo, the ion-pair form is stabilized by the environment [239, 241]. These results have been confirmed by recent sophisticated calculations [218]. 

On the basis of well-designed experiments and theoretical calculations it seems at present quite cer in that the charge-relay mechanism is not operative in serine proteases. What is then the source of the rate acceleration by 6-8 orders of magnitude as compared to the uncatalyzed reaction This qustion has been addressed by several authors [233, 235-237, 240, 248]. The problem can be best treated by theoretical calculations since these allow the consideration of hypothetical reaction intermediates, transition-state complexes and reaction routes, as well [237]. Based on simple calculations combining the CNDO/2 and BI methods we stressed the importance of the electrostatic stabilization effect of the buried aspartate on the transition state leading to a rate acceleration [240]. The same conclusion was reached independently using the ab initio method by Umeyama et al. [249]. Later on, Warshel and Russell... 

Streptomyces griseus trypsin [8] and a-lytic protease [9] are serine proteases with a solitary histidine residue. This histidine residue is believed to be involved in the catalytic mechanism via a charge-relay mechanism analogous to that in a-chymotrypsin. Figures 2 and 3 show the reactivity data for the histidine residue in a-lytic protease and S.G. Trypsin. The data for a-lytic protease approximately follow a titration curve... 

The specificity of compound 1 for AChE was demonstrated by treating other proteases of the His-Ser-Asp charge relay mechanism with the potential inhibitor. Trypsin, elastase, and subtilisin were not inactivated by 1. Chymotrypsin and kallikrein showed barely detectable inactivation at 100 IM and 40-45% inactivation at 1 mM. Conpounds 2, 3, and 5 also inactivated the eel AChE. Compound 2 showed 95% inactivation at 100 IM in 15 min. Conpounds 3 and 5 showed IC50 values of 102 XM and 108 1M, respectively. 

W. W. Bachovchin and J. D. Roberts (1978), Nitrogen-15 nuclear magnetic resonance spectroscopy. The state of histidine in the catalytic triad of a-lytic protease. Implications for the charge-relay mechanism of peptide bond cleavage by serine proteases. J. Amer. Chem. Soc. 100, 8041-8047. 

In 1969 it was suggested that the proton from the catalytic histidine may be transferred to the buried aspartate during catalysis and this charge-relay mechanism vastly increases the nucleophilicity of the active serine through its ionization. This process, outlined in many textbooks, is called the double proton-transfer or charge-relay mechanism. Later on, the hypothesis was questioned on the basis of experimental studies, - molecular orbital - and semimacroscopic calculations. All the above studies supported the early proposal by Polgar and Bender who stated that the buried aspartate remains ionized throughout the catalytic process and its role is mainly to increase the stability of the ion pair formed by the tetrahedral intermediate and protonated histidine. 

The movement of the proton from histidine to aspartate (as suggested by the charge relay mechanism in serine proteases, cf. Section 4.1) was excluded on the basis of the higher activation energy obtained for this process. Such a proton transfer may be hindered by the electrostatic effect of the environment favoring the formation of the ion pair. 

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