Michael Shokhen

Is there a weak H-bond LBHB transition on tetrahedral complex formation in serine proteases?†

The transformation of a weak hydrogen bond in the free enzyme into a low‐barrier hydrogen bond (LBHB) in the tetrahedral intermediate has been suggested as an important factor facilitating catalysis in serine proteases. In this work, we examine the structure of the H‐bond in the Asp102–His57 diad of serine proteases in the free enzyme and in a covalent tetrahedral complex (TC) with a trifluoromethylketone inhibitor. We apply ab initio quantum mechanical calculations to models consisting of a large molecular fragment of the enzyme active site, and the combined effect of the rest of the protein body and the solvation by surrounding bulk water was simulated by a self‐consistent reaction field method in our novel QM/SCRF(VS) approach. Potential profiles of adiabatic proton transfer in the Asp102–His57 diad in these model systems were calculated. We conclude that the hydrogen bond in both the free enzyme and in the enzyme‐inhibitor TC is a strong ionic asymmetric one‐well hydrogen bond, in contrast to a previous suggestion that it is a weak H‐bond in the former and a double‐well LBHB in the latter. Proteins 2004;54:000–000.

The mechanism of papain inhibition by peptidyl aldehydes

Various mechanisms for the reversible formation of a covalent tetrahedral complex (TC) between papain and peptidyl aldehyde inhibitors were simulated by DFT calculations, applying the quantum mechanical/self consistent reaction field (virtual solvent) [QM/SCRF(VS)] approach. Only one mechanism correlates with the experimental kinetic data. The His–Cys catalytic diad is in an N/SH protonation state in the noncovalent papain–aldehyde Michaelis complex. His159 functions as a general base catalyst, abstracting a proton from the Cys25, whereas the activated thiolate synchronously attacks the inhibitors carbonyl group. The final product of papain inhibition is the protonated neutral form of the hemithioacetal TC(OH), in agreement with experimental data. The predicted activation barrier g  enz≠ = 5.2 kcal mol−1 is close to the experimental value of 6.9 kcal mol−1. An interpretation of the experimentally observed slow binding effect for peptidyl aldehyde inhibitors is presented. The calculated g  cat≠ is much lower than the rate determining activation barrier of hemithioacetal formation in water, g  w≠ , in agreement with the concept that the preorganized electrostatic environment in the enzyme active site is the driving force of enzyme catalysis. We have rationalized the origin of the acidic and basic pKas on the k2/KS versus pH bell‐shaped profile of papain inhibition by peptidyl aldehydes. Proteins 2011.

Factors determining the relative stability of anionic tetrahedral complexes in serine protease catalysis and inhibition

Quantum mechanical ab initio (RHF/6‐31+G*//RHF/3‐21G) calculations were used to simulate the formation of the tetrahedral complex intermediate (TC) in serine protease active site by substrates and transition‐state analog inhibitors. The enzyme active site was simulated by an assembly of the amino acids participating in catalysis, whereas the substrates and inhibitors were simulated by small ligands, acetamide (1) and trifluoroacetone (2), respectively. For the first time, the principal factors determining the relative stability of the TC in serine proteases are arranged according to their energy contributions. These include (a) formation of the new covalent bond between Ser195 Oγ and the electrophilic center of a ligand; (b) stabilization of the oxyanion in the oxyanion hole; (c) basic catalysis by His57; and (d) hydrogen bond between Asp102 carboxylate and Nδ of the protonated His57. We have directly calculated the gas‐phase relative free energy of formation of TCAS(2) and TCAS(1), the value of ΔΔGg[TCAS(2,1)]. It is ΔEcov, the relative energy of the new covalent bond between the enzyme and the ligand formed in a TC that determines the experimentally observed large difference in the stability of TCs formed by substrates and TS‐analog inhibitors of serine proteases. We demonstrated that the relative stability of TCs formed by a series of mono‐ and dipeptide amides and TFKs, derived from experimental kinetic data, can be rather well approximated by the sum of the theoretically calculated value of ΔΔGg[TCAS(2,1)] and the difference in hydration free energies of isolated ligands. Proteins 2000;40:154–167.

Challenging a paradigm: theoretical calculations of the protonation state of the Cys25-His159 catalytic diad in free papain.

A central mechanistic paradigm of cysteine proteases is that the His–Cys catalytic diad forms an ion‐pair NH(+)/S(−) already in the catalytically active free enzyme. Most molecular modeling studies of cysteine proteases refer to this paradigm as their starting point. Nevertheless, several recent kinetics and X‐ray crystallography studies of viral and bacterial cysteine proteases depart from the ion‐pair mechanism, suggesting general base catalysis. We challenge the postulate of the ion‐pair formation in free papain. Applying our QM/SCRF(VS) molecular modeling approach, we analyzed all protonation states of the catalytic diad in free papain and its SMe derivative, comparing the predicted and experimental pKa data. We conclude that the His–Cys catalytic diad in free papain is fully protonated, NH(+)/SH. The experimental pKa = 8.62 of His159 imidazole in free papain, obtained by NMR‐controlled titration and originally interpreted as the NH(+)/S(−) ⇌ N/S(−)

Screening of the active site from water by the incoming ligand triggers catalysis and inhibition in serine proteases

{\rm NH}(+)/{\rm S}(-)\rightleftharpoons {\rm N/S}(-)

Differentiating serine and cysteine protease mechanisms by new covalent QSAR descriptors.

equilibrium, is now assigned to the NH(+)/SH ⇌ N/SH

The cooperative effect between active site ionized groups and water desolvation controls the alteration of acid/base catalysis in serine proteases.

{\rm NH}(+)/{\rm SH}\rightleftharpoons {\rm N/SH}

Enzyme isoselective inhibitors: application to drug design.

equilibrium. Proteins 2009.

Stepwise Versus Concerted Mechanisms in General-Base Catalysis by Serine Proteases.

The pKa of the catalytic His57 NεH in the tetrahedral complex (TC) of chymotrypsin with trifluoromethyl ketone inhibitors is 4–5 units higher relative to the free enzyme (FE). Such stable TCs, formed with transition state (TS) analog inhibitors, are topologically similar to the catalytic TS. Thus, analysis of this pKa shift may shed light on the role of water solvation in the general base catalysis by histidine. We applied our QM/SCRF(VS) approach to study this shift. The method enables explicit quantum mechanical DFT calculations of large molecular clusters that simulate chemical reactions at the active site (AS) of water solvated enzymes. We derived an analytical expression for the pKa dependence on the degree of water exposure of the ionizable group, and on the total charge in the enzyme AS, Q(A) and Q(B), when the target ionizable functional group (His57 in this study) is in the acidic (A) and basic (B) forms, respectively. Q2(B) > Q2(A) both in the FE and in the TC of chymotrypsin. Therefore, water solvation decreases the relative stability of the protonated histidine in both. Ligand binding reduces the degree of water solvation of the imidazole ring, and consequently elevates the histidine pKa. Thus, the binding of the ligand plays a triggering role that switches on the cascade of catalytic reactions in serine proteases. Proteins 2008.

Enzyme isoselective inhibitors: a tool for binding-trend analysis.

In the first catalytic step in serine proteases, the attacking Ser nucleophile forms an unstable covalent anionic tetrahedral complex, TC(O-), with the carbonyl group of the substrate (Fig. 1a).[1] In the case of reaction coordinate analog inhibitors (RCA)[2] the energy released in the formation of the new O—C covalent bond is sufficient for the thermodynamic stabilization of TC(O-) as the end products,[3] as for instance with α-ketoheterocycle inhibitors.[4] The catalytic mechanism of Cys proteases is still under debate.[5] The thiolate anion of Cys is much weaker nucleophile than the hydroxyl anion of Ser.[6] Therefore, the binding of RCA inhibitors in cysteine proteases may require further stabilization of TC, which could be realized by its protonation and the formation of a neutral TC(OH) (Fig. 1b).[5c, 7] Figure 1 The first catalytic step in proteases. a) Formation of an anionic TC(O-) with serine proteases, and b) formation of a neutral TC(OH) with cysteine proteases. X is the varied substituent modifying the electrophilicity of the carbonyl group. A substrate or a RCA inhibitor is formally comprised of two parts: the chemical site, CS, responsible for the covalent binding, and the recognition site, RS, dominating selectivity of the ligand (substrate or inhibitor) towards the target enzyme. CS of RCA inhibitors can be rationally designed on special sets of isoselective inhibitors with constant RS and varied CS fragments.[8] In developing this approach we introduce now two types of QSAR descriptors W1 and W2. They quantitatively account for the energetic contribution from the enzyme nucleophile (Nuc)-inhibitor (Inh) covalent binding and reorganization of the covalent bonds of the CS fragments of inhibitors during formation of TC(O-) or TC(OH), respectively: W1=Hf[TC(O-)]−Hf(Inh)−Hf(Nuc) (1) W2=Hf[TC(OH)]−Hf(Inh)−Hf(Nuc) (2) W1 and W2 are calculated as heats of formation, Hf, at 25 °C by PM6 semi-empirical QM Hamiltonian applying MOPAC2009[9] on small molecular clusters (see Supporting information, SI). According to Eqs 1 and 2, the difference W2 – W1 is a measure of the proton affinity, PA, or pKa of CS: W2−W1=Hf[TC(OH)]−Hf[TC(O-)] (3) Here we analyze implication of W1 and W2 indices to the mechanism of serine and cysteine proteases, with direct consequence on different binding trends of RCA inhibitors to these enzyme families. The reaction core of CS of the considered RCA inhibitors is the carbonyl group C=O (Fig. 2). The varied substituent X in CS modulates with opposite effects the electrophilicity of the carbon atom, and PA/pKa of the carbonyl oxygen. This is clearly illustrated by the negative slope of the correlation trend of W2 – W1 (PA/pKa) vs. W1 (electrophilicity) for thrombin and cathepsin K inhibitors (Fig. 3). Figure 2 The RCA inhibitors with varied substituent X at the carbonyl group – the reaction core of the CS fragment. Serine protease thrombin inhibitors: a) 21 compounds in the training set (Table 5 in ref. 12), and b) 9 compounds 2b-f,h-k in the test set ... Figure 3 Variation of substituent X causes opposite trends in W2 – W1 (PA/pKa) vs. W1 (electrophilicity). a) Thrombin, b) Cathepsin K. Our method accounts for both covalent interactions of CS in the enzyme active site by W1 and W2, and non-covalent CS interactions by conventional 2D non-covalent and topological descriptors implemented in most drug design software. The QSAR models were generated and optimized by Genetic Function Approximation (GFA),[10] implemented in Accelrys Discovery Studio (DS)[11] (See details in SI). GFA selects the most relevant indices dominating the inhibitors binding trend. Covalent indices W1 or W2 were identified by GFA as an obligatory part of the optimal QSAR model since the varied X substituent considerably modifies the electron distribution on the CS reactivity center. The best QSAR model of the serine protease thrombin, identified by GFA on 21 inhibitors in the training set (Fig. 2a)[12] and 9 inhibitors in the test set (Fig. 2b),[13] contains W1 and W2 in combination with conventional 2D descriptors (Fig. 4a and Table S1 in SI). Ser hydroxyl forms a thermodynamically stable TC(O-) with RCAs. W1 accounts for the modulation of electrophilicity of the carbonyl group by the varied X. A linear combination of W1 and W2 indices, (1-λ)W1 + λW2, where 0 ≤ λ ≤ 1, corresponds to the stabilization of TC(O-) in the active site of serine proteases by hydrogen bonds in the oxyanion hole. Sequential exclusion of W1 and W2 indices from the QSAR model (Fig. 4 and Table S1) demonstrates that for a strong nucleophile – Ser hydroxyl anion, W1 accounting for the modulation of electrophilicity dominates the inhibitors binding trend. Contribution of H-bonds in the oxyanion hole to the TC(O-) stabilization is much smaller than the energy released in the enzyme-inhibitor covalent bond formation. Thus, in serine proteases W2 plays a minor role in comparison with W1, so W2 can be considered as a non-covalent descriptor slightly improving the prediction (Fig. 4a vs. 4c and Table S1). Figure 4 Correlations of experimental and calculated pKis in QSAR models for thrombin, generated on the training set (empty circles and solid line) and examined on the test set (filled circles and dashed line) of varied CSs. a) W1, W2 and 2D descriptors (generated ... In sharp contrast, the W1 index was not identified by the GFA in the cysteine protease. W2 plays an exclusive role in the QSAR model of the cathepsin K series of inhibitors (Fig. 5 and Table S4),[14] divided into 23 molecules for a training set and 7 for a test set (Fig. 2c). We previously observed a similar effect for the human rhinoviral 3C cysteine protease.[8b] Why is the W1 index irrelevant for cysteine proteases or, in other words, why a cysteine nucleophile cannot stabilize the anionic TC(O-) in contrast to a serine nucleophile? Figure 5 Correlations of experimental and calculated pIC50s in QSAR models for cathepsin K, generated on the training set (empty circles and solid line) and examined on the test set (filled cirles and dashed line) of varied CSs. a) W2 and 2D descriptors (generated ... By definition, the W2 index accounts for two energetic effects – formation of the enzyme-inhibitor covalent bond and PA/pKa of TC(OH). We demonstrated previously that the reduced ability of a thiolate anion to stabilize anionic TC(O-) in comparison with hydroxide is due to the larger extent of electron back-donation directed from the electrophiles (carbonyl group) HOMOA to the nucleophiles LUMOD.[6a] Therefore, the energy of the enzyme-inhibitor covalent bond plays a minor role in the inhibitors binding trend and the W1 descriptor is absent in QSAR models for a cysteine protease. Another important observation differentiating serine and cysteine proteases is the opposite signs of the linear regression coefficients for W1 in serine, and W2 in cysteine proteases in the QSAR models (Table S4). Comparing this observation with the graphs in Figure 3, it is obvious that in cysteine protease the effect of X variation on the pKa of TC(OH) dominates the RCA inhibitors binding trend. This stems from redistribution of electron density between atomic orbitals of the reactivity centers in the formed tetrahedral complex.[6a,15] The absolute atomic electronegativity of sulfur is slightly lower than that of carbon (6.22 vs. 6.27, respectively), in contrast to the highly electronegative oxygen (7.54).[16] Therefore, the electron density redistribution between the S and C reactivity centers in the formed TC(OH) should be either negligible or directed from sulfur to carbon, depending on the valent surrounding (X substituent) of the electrophilic center. In the O and C pair in TC(O-), the electronic flow is directed towards the oxygen atom. We previously demonstrated by ab initio QM calculations that indeed the summarized charge transfer to the carbonyl fragment is considerably larger for HS- than for HO- nucleophile.[6a] We have applied QSAR analysis as a mechanistic tool to support the suggestion that the covalent tetrahedral complex in cysteine proteases has a neutral form TC(OH),[5c, 7] in contrast to the anionic TC(O-) in serine proteases. We explained why the varied substituent X at the carbonyl group in CS has different influence on the binding trend of RCA inhibitors of serine and cysteine proteases. Detailed mechanistic analysis of the physical nature of the W1 and W2 indices validates their use for rational design of chemical sites of RCA inhibitors for proteases. Validation of the relevance of the generated QSAR models is presented in the Supporting Information.