Netaly Khazanov

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.

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(−)

Plant phenolic volatiles inhibit quorum sensing in pectobacteria and reduce their virulence by potential binding to ExpI and ExpR proteins

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

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

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.

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

Quorum sensing (QS) is a population density-dependent regulatory system in bacteria that couples gene expression to cell density through accumulation of diffusible signaling molecules. Pectobacteria are causal agents of soft rot disease in a range of economically important crops. They rely on QS to coordinate their main virulence factor, production of plant cell wall degrading enzymes (PCWDEs). Plants have evolved an array of antimicrobial compounds to anticipate and cope with pathogens, of which essential oils (EOs) are widely recognized. Here, volatile EOs, carvacrol and eugenol, were shown to specifically interfere with QS, the master regulator of virulence in pectobacteria, resulting in strong inhibition of QS genes, biofilm formation and PCWDEs, thereby leading to impaired infection. Accumulation of the signal molecule N-acylhomoserine lactone declined upon treatment with EOs, suggesting direct interaction of EOs with either homoserine lactone synthase (ExpI) or with the regulatory protein (ExpR). Homology models of both proteins were constructed and docking simulations were performed to test the above hypotheses. The resulting binding modes and docking scores of carvacrol and eugenol support potential binding to ExpI/ExpR, with stronger interactions than previously known inhibitors of both proteins. The results demonstrate the potential involvement of phytochemicals in the control of Pectobacterium.

A novel image-based high-throughput screening assay discovers therapeutic candidates for adult polyglucosan body disease

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.

Specific stabilization of CFTR by phosphatidylserine

What is the driving force that alters the catalytic function of His57 in serine proteases between general base and general acid in each step along the enzymatic reaction? The stable tetrahedral complexes (TC) of chymotrypsin with trifluoromethyl ketone transition state analogue inhibitors are topologically similar to the catalytic transition state. Therefore, they can serve as a good model to study the enzyme catalytic reaction. We used DFT quantum mechanical calculations to analyze the effect of solvation and of polar factors in the active site of chymotrypsin on the pKa of the catalytic histidine in FE (the free enzyme), EI (the noncovalent enzyme inhibitor complex), and TC. We demonstrated that the acid/base alteration is controlled by the charged groups in the active site—the catalytic Asp102 carboxylate and the oxyanion. The effect of these groups on the catalytic His is modulated by water solvation of the active site.

Potentiators exert distinct effects on human, murine, and Xenopus CFTR

Common methodologies of computer‐assisted drug design focus on noncovalent enzyme–ligand interactions. We introduced enzyme isoselective inhibition trend analysis as a tool for the expert analysis of covalent reversible inhibitors. The methodology is applied to predict the binding affinities of a series of transition‐state analogue inhibitors of medicinally important serine and cysteine hydrolases. These inhibitors are isoselective: they have identical noncovalent recognition fragments (RS) and different reactive chemical fragments (CS). Furthermore, it is possible to predict the binding affinities of a series of isoselective inhibitors toward a prototype enzyme and to extrapolate the data to a target medicinally important enzyme of the same family. Rational design of CS fragments followed by conventional RS optimization could be used as a novel approach to drug design.