Interaction energies between tetrahydrobiopterin analogues and aromatic residues in tyrosine hydroxylase and phenylalanine hydroxylase

The phenylalanine residues 300 and 309 in the enzyme tyrosine hydroxylase are known to aid in the positioning and binding of tetrahydrobiopterin (BH4) to the enzyme active site. The residues phenylalanine 254 and tyrosine 325 similarly aid in binding BH4 in phenylalanine hydroxylase. BH4 is a cofactor necessary for enzyme function, and mutations in these residues have been shown to cause a decrease in enzyme function. We examine the pairwise interactions between each aromatic residue and BH4 using second-order Moller Plesset theory and density functional theory to determine the amount of binding due to these aromatic residues. Further, we perform in silico point mutations of these residues to determine if several likely mutations can cause a decrease in protein function. Our results show that dispersion dominates these interactions, and electrostatics alone is not enough to bind the BH4.     

Metal ligand aromatic cation-pi interactions in metalloproteins: ligands coordinated to metal interact with aromatic residues

Cation-pi interactions between aromatic residues and cationic amino groups in side chains and have been recognized as noncovalent bonding interactions relevant for molecular recognition and for stabilization and definition of the native structure of proteins. We propose a novel type of cation-pi interaction in metalloproteins; namely interaction between ligands coordinated to a metal cation--which gain positive charge from the metal--and aromatic groups in amino acid side chains. Investigation of crystal structures of metalloproteins in the Protein Data Bank (PDB) has revealed that there exist quite a number of metalloproteins in which aromatic rings of phenylalanine, tyrosine, and tryptophan are situated close to a metal center interacting with coordinated ligands. Among these ligands are amino acids such as asparagine, aspartate, glutamate, histidine, and threonine, but also water and substrates like ethanol. These interactions play a role in the stability and conformation of metalloproteins, and in some cases may also be directly involved in the mechanism of enzymatic reactions, which occur at the metal center. For the enzyme superoxide dismutase, we used quantum chemical computation to calculate that Trp163 has an interaction energy of 10.09 kcal mol(-1) with the ligands coordinated to iron.     

Characterization of nucleobase-amino acid stacking interactions utilized by a DNA repair enzyme

The present work characterizes the gas-phase stacking interactions between four aromatic amino acid residues (histidine, phenylalanine, tyrosine, and tryptophan) and adenine or 3-methyladenine due to the proposed utilization of these interactions by enzymes that repair DNA alkylation damage. The MP2 potential energy surfaces of the stacked dimers are considered as a function of four variables (vertical displacement, angle of rotation, horizontal displacement, and tilt angle) using a variety of basis sets. It is found that the maximum stacking interaction energy decreases with the amino acid according to TRP > TYR approximately HIS > PHE for both nucleobases. However, the magnitude of the stacking interaction significantly increases upon alkylation (by 50-115%). Comparison of the stacking energies calculated using our surface scans to those estimated from experimental crystal structures indicates that the stacking interactions within the active site of 3-methyladenine DNA glycosylase can account for 65-75% of the maximum possible stacking interaction between the relevant molecules. The decrease in stacking in the crystal structure arises due to significant differences in the relative orientations of the nucleobase and amino acid. Nevertheless, alkylation is found to significantly increase the stacking energy when the crystal structure geometries are considered. Our calculations provide computational support for suggestions that alkylation enhances the stacking interactions within the active site of DNA repair enzymes, and they give a measure of the magnitude of this enhancement. Our results suggest that alkylation likely plays a more important role in substrate identification and removal than the nature of the aromatic amino acid that interacts with the substrate via stacking interactions.     

Evaluation of density functional theory methods for the electronic interactions between indole and substituted benzene: Applications to horseradish peroxidase

In an effort to evaluate and design fast, accurate density functional theory (DFT) methods for calculating electrostatic and dispersion interactions between proteins and ligands, we have set up a model system examining interactions between mono‐substituted benzene and indole in seven different stable conformations. We first optimized the geometries of the monomers at the B3LYP/6‐31G level, and then scanned the potential curves with MP2, HF, B3LYP, SVWN, and HCTH407 [all at the 6‐311++G(d,p) level] to find the optimum separation. We used the approximate counterpoise method to calculate the basis set superposition error‐corrected interaction energies at the optimum geometries. We then applied these methods to the interactions between aromatic active site residues of horseradish peroxidase C with indole‐3‐propionic acid at two different binding sites. © 2007 Wiley Periodicals, Inc. Int J Quantum Chem, 2007     

Interactions of cyclodextrins with aromatic amino acids: a basis for protein interactions

Cyclodextrins (CyD) have proven effects on the stability of proteins and can be used in the formulation of aggregation prone therapeutic proteins. This effect stems from specific interactions between the CyD (preferably β-CyD) and solvent exposed amino acid residues. Here the interaction with hydrophobic aromatic amino acid residues stands out and the interaction between CyDs and these amino acid residues holds the key to understanding the observed effects, which CyDs exerts on proteins and peptides. Here we present a comparative study of the interactions between free and peptide bound aromatic amino acids and their derivatives with α, β and γ-CyDs using NMR spectroscopy. We propose a novel, quantitative means of assessing the penetration depth of guest molecules in CyD cavities, the penetration gauge Π, and apply it to the observed interaction patterns from ROESY NMR spectra. We demonstrate that the penetration depths of the aromatic rings within the CyDs rely highly on the nature of the remainder of the guest molecule. Thus the presence of charges, neighboring amino acids and the specific positioning on the surface of a protein highly influences the penetration depth and geometry of guest–CyD interactions.     

A QM/MM study of fluoroaromatic interactions at the binding site of carbonic anhydrase II, using a DFT method corrected for dispersive interactions

The interaction of the fluorinated benzyl ring of a series of inhibitors of carbonic anhydrase II (CAII), fluorine-substituted N-(4-sulfamylbenzoyl)benzylamines (SBB), with nearby residues in the active site has been studied using a hybrid QM/MM model. To account for the important dispersive interactions between the fluorinated benzenes and these residues, a density functional method with an empirical dispersive term, (DFT-D), is used as the QM part of the model. The major interactions are found to be between the substituted benzenes and the aromatic ring of a nearby phenylalanine residue. However, the intermolecular separations between these two groups span a greater range than that found for comparable interactions between isolated molecules, showing the importance of interactions with other residues, which have been quantified. A decomposition of the interaction energy between the fluorobenzenes and each residue has been carried out which shows the dispersive interactions to be dominant. This work has shown that a QM(DFT-D)/MM model is a computationally feasible and accurate way of studying substrate-protein interactions.     

Modelling of carbohydrate–aromatic interactions: <Emphasis Type="Italic">ab initio </Emphasis>energetics and force field performance

Summary Aromatic amino acid residues are often present in carbohydrate-binding sites of proteins. These binding sites are characterized by a placement of a carbohydrate moiety in a stacking orientation to an aromatic ring. This arrangement is an example of CH/π interactions. Ab initio interaction energies for 20 carbohydrate–aromatic complexes taken from 6 selected ultra-high resolution X-ray structures of glycosidases and carbohydrate-binding proteins were calculated. All interaction energies of a pyranose moiety with a side chain of an aromatic residue were calculated as attractive with interaction energy ranging from −2.8 to −12.3 kcal/mol as calculated at the MP2/6-311+G(d) level. Strong attractive interactions were observed for a wide range of orientations of carbohydrate and aromatic ring as present in selected X-ray structures. The most attractive interaction was associated with apparent combination of CH/π interactions and classical H-bonds. The failure of Hartree–Fock method (interaction energies from +1.0 to −6.9 kcal/mol) can be explained by a dispersion nature of a majority of the studied complexes. We also present a comparison of interaction energies calculated at the MP2 level with those calculated using molecular mechanics force fields (OPLS, GROMOS, CSFF/CHARMM, CHEAT/CHARMM, Glycam/AMBER, MM2 and MM3). For a majority of force fields there was a strong correlation with MP2 values. RMSD between MP2 and force field values were 1.0 for CSFF/CHARMM, 1.2 for Glycam/AMBER, 1.2 for GROMOS, 1.3 for MM3, 1.4 for MM2, 1.5 for OPLS and to 2.3 for CHEAT/CHARMM (in kcal/mol). These results show that molecular mechanics approximates interaction energies very well and support an application of molecular mechanics methods in the area of glycochemistry and glycobiology.     

Stabilizing interactions between aromatic and basic side chains in alpha-helical peptides and proteins. Tyrosine effects on helix circular dichroism

Here we investigate the structures and energetics of interactions between aromatic (Phe or Tyr) and basic (Lys or Arg) amino acids in alpha-helices. Side chain interaction energies are measured using helical peptides, by quantifying their helicities with circular dichroism at 222 nm and interpreting the results with Lifson-Roig-based helix/coil theory. A difficulty in working with Tyr is that the aromatic ring perturbs the CD spectrum, giving an incorrect helicity. We calculated the effect of Tyr on the CD at 222 nm by deriving the intensities of the bands directly from the electronic and magnetic transition dipole moments through the rotational strengths corresponding to each excited state of the polypeptide. This gives an improved value of the helix preference of Tyr (from 0.48 to 0.35) and a correction to the helicity for the peptides containing Tyr. We find that Phe-Lys, Lys-Phe, Phe-Arg, Arg-Phe, and Tyr-Lys are all stabilizing by -0.10 to -0.18 kcal.mol-1 when placed i, i + 4 on the surface of a helix in aqueous solution, despite the great difference in polarity between these residues. Interactions between these side chains have previously been attributed to cation-pi bonds. A survey of protein structures shows that they are in fact predominantly hydrophobic interactions between the CH2 groups of Lys or Arg and the aromatic rings.     

A preference for edgewise interactions between aromatic rings and carboxylate anions: the biological relevance of anion-quadrupole interactions

Noncovalent interactions are quite important in biological structure-function relationships. To study the pairwise interaction of aromatic amino acids (phenylalanine, tyrosine, tryptophan) with anionic amino acids (aspartic and glutamic acids), small molecule mimics (benzene, phenol or indole interacting with formate) were used at the MP2 level of theory. The overall energy associated with an anion-quadrupole interaction is substantial (-9.5 kcal/mol for a benzene-formate planar dimer at van der Waals contact distance), indicating the electropositive ring edge of an aromatic group can interact with an anion. Deconvolution of the long-range coplanar interaction energy into fractional contributions from charge-quadrupole interactions, higher-order electrostatic interactions, and polarization terms was achieved. The charge-quadrupole term contributes between 30 to 45% of the total MP2 benzene-formate interaction; most of the rest of the interaction arises from polarization contributions. Additional studies of the Protein Data Bank (PDB Select) show that nearly planar aromatic-anionic amino acid pairs occur more often than expected from a random angular distribution, while axial aromatic-anionic pairs occur less often than expected; this demonstrates the biological relevance of the anion-quadrupole interaction. While water may mitigate the strength of these interactions, they may be numerous in a typical protein structure, so their cumulative effect could be substantial.     

van der Waals interactions of polycyclic aromatic hydrocarbon dimers

Density functional theory is in principle exact and includes also long-range interactions, such as the van der Waals interactions. These are, however, part of the exchange-correlation energy functional that needs to be approximated, and are absent in the local and semilocal standard implementations. Recently a density functional which includes van der Waals interactions for planar systems has been developed, which we show can be extended to provide a treatment of planar molecules. We use this functional to calculate binding distances and energies for dimers of three of the smallest polycyclic aromatic hydrocarbons (PAHs)--naphthalene, anthracene, and pyrene.     

Substituent effects on edge-to-face aromatic interactions

Chemical double mutant cycles have been used to measure the magnitude of edge-to-face aromatic interactions in hydrogen-bonded zipper complexes as a function of substituents on both aromatic rings. The interaction energies vary depending on the combination of substituents from +1.0 kJ mol-1 (repulsive), to -4.9 kJ mol-1 (attractive). The results correlate with the Hammett substituent constants which indicates that electrostatic interactions are responsible for the observed differences in interaction energy. The experiments can be rationalised based on local electrostatic interactions between the protons on the edge ring and the pi-electron density on the face ring as well as global electrostatic interactions between the overall dipoles on the two aromatic groups.     

牛视紫红质蛋白质中视黄醛的活性位点

视紫红质蛋白是一个跨膜蛋白, 视黄醛(RET)在该蛋白中的活性结合位点涉及到视觉过程机理, 与一些眼科疾病病理有关. 基于牛视紫红质蛋白1U19的蛋白质晶体结构数据, 采用密度泛函理论的B3LYP方法计算RET-Lys296残基与视黄醛分子周围半径为0.6 nm的空间范围30个氨基酸残基相互作用和结合能. 数值显示1U19蛋白中的残基Glu113、Glu181和Glu122是质子化的RET-Lys296残基的活性结合位点, 结合能分别为-333.38、-205.67和-194.56 kJ·mol-1. 这些氨基酸残基带有一个负电荷, 与质子化的RET-Lys296残基发生强烈的离子静电相互作用. 另外几个残基Ala292、Cys187、Phe293、Pro291以及Trp265等与质子化RET-Lys296残基也有相互吸引作用. 当RET-Lys296残基非质子化, 上述相互作用消失, 促使视黄醛分子与视蛋白分离. 研究发现残基Glu113和Glu181周围各自有一个结晶水分子通过双氢键形式起着稳定作用.     

Effects of hydrogen-bonding and stacking interactions with amino acids on the acidity of uracil

The effects of hydrogen-bonding interactions with amino acids on the (N1) acidity of uracil are evaluated using (B3LYP) density functional theory. Many different binding arrangements of each amino acid to three uracil binding sites are considered. The effects on the uracil acidity are found to significantly depend upon the nature of the amino acid and the binding orientation, but weakly depend on the binding site. Our results reveal that in some instances small models for the amino acids can be used, while for other amino acids larger models are required to properly describe the binding to uracil. The gas-phase acidity of uracil is found to increase by up to approximately 60 kJ mol(-1) due to discrete hydrogen-bonding interactions. Although (MP2) stacking interactions with aromatic amino acids decrease the acidity of uracil, unexpected increases in the acidity are found when any of the aromatic amino acids, or the backbone, hydrogen bond to uracil. Consideration of enzymatic and aqueous environments leads to decreases in the effects of the amino acids on the acidity of uracil. However, we find that the magnitude of the decrease varies with the nature of the molecule bound, as well as the (gas-phase) binding orientations and strengths, and therefore solvation effects should be considered on a case-by-case basis in future work. Nevertheless, the effects of amino acid interactions within enzymatic environments are as much as approximately 35 kJ mol(-1). The present study has general implications for understanding the nature of active site amino acids in enzymes, such as DNA repair enzymes, that catalyze reactions involving anionic nucleobase intermediates.     

CH/pi interactions in DNA and proteins. A theoretical study

A systematic study of the CH/pi interactions of methane with the purine and pyrimidine bases of nucleic acids and with the lateral chains of the four natural aromatic amino acids has been carried out for the first time. The MPWB1K/6-31+G(d,p) method has shown to be adequate for the study of these weak interactions in which dispersion forces play a main role. It has been shown that two different kinds of clusters exist, depending on whether one or two CH bonds point to the aromatic system. The latter one, which we have called bifurcated, is usually more stable. With regard to aromatic amino acids, our calculations agree with experimental data in the fact that tryptophan leads to the strongest interaction, while hystidine leads to the weakest one. In the case of nucleic acid bases, the differences in binding energies are not large. This is specially true for thymine and uracil, showing that these two bases have a similar acceptor character in CH/pi interactions.     

Tightening of active site interactions en route to the transition state revealed by single-atom substitution in the guanosine-binding site of the Tetrahymena group I ribozyme

Protein enzymes establish intricate networks of interactions to bind and position substrates and catalytic groups within active sites, enabling stabilization of the chemical transition state. Crystal structures of several RNA enzymes also suggest extensive interaction networks, despite RNA's structural limitations, but there is little information on the functional and the energetic properties of these inferred networks. We used double mutant cycles and presteady-state kinetic analyses to probe the putative interaction between the exocyclic amino group of the guanosine nucleophile and the N7 atom of residue G264 of the Tetrahymena group I ribozyme. As expected, the results supported the presence of this interaction, but remarkably, the energetic penalty for introducing a CH group at the 7-position of residue G264 accumulates as the reaction proceeds toward the chemical transition state to a total of 6.2 kcal/mol. Functional tests of neighboring interactions revealed that the presence of the CH group compromises multiple contacts within the interaction network that encompass the reactive elements, apparently forcing the nucleophile to bind and attack from an altered, suboptimal orientation. The energetic consequences of this indirect disruption of neighboring interactions as the reaction proceeds demonstrate that linkage between binding interactions and catalysis hinges critically on the precise structural integrity of a network of interacting groups.     

Contribution of aromatic interactions to alpha-helix stability

The influence of natural and unnatural i, i + 4 aromatic side chain-side chain interactions on alpha-helix stability was determined in Ala-Lys host peptides by circular dichroism (CD). All interactions investigated provided some stability to the helix; however, phenylalanine-phenylalanine (F-F) and phenylalanine-pentafluorophenylalanine (F-f5F) interactions resulted in the greatest enhancement in helicity, doubling the helical content over i, i + 5 control peptides at internal positions. Quantification of these interactions using AGADIR multistate helix-coil algorithm revealed that the F-F and F-f5F interaction energies are equivalent at internal positions in the sequence (deltaGF-F = deltaGF-f5F = -0.27 kcal/mol), despite the differences in their expected geometries. As the strength of a face-to-face stacked phenyl-pentafluorophenyl interaction should surpass an edge-to-face or offset-stacked phenyl-phenyl interaction, we believe this result reflects the inability of the side chains in F-f5F to attain a fully stacked geometry within the context of an alpha-helix. Positioning the interactions at the C-terminus led to much stronger interactions (deltaGF-F = -0.8 kcal/mol; deltaGF-f5F = -0.55 kcal/mol) likely because of favorable chi(1) rotameric preferences for aromatic residues at C-capping regions of alpha-helices, suggesting that aromatic side chain-side chain interactions are an effective alpha-helix C-capping method.     

Modifying the OPLS-AA force field to improve hydration free energies for several amino acid side chains using new atomic charges and an off-plane charge model for aromatic residues

The hydration free energies of amino acid side chains are an important determinant of processes that involve partitioning between different environments, including protein folding, protein complex formation, and protein-membrane interactions. Several recent papers have shown that calculated hydration free energies for polar and aromatic residues (Trp, His, Tyr, Asn, Gln, Asp, Glu) in several common molecular dynamics force fields differ significantly from experimentally measured values. We have attempted to improve the hydration energies for these residues by modifying the partial charges of the OPLS-AA force field based on natural population analysis of density functional theory calculations. The resulting differences between calculated hydration free energies and experimental results for the seven side chain analogs are less than 0.1 kcal/mol. Simulations of the synthetic Trp-rich peptide Trpzip2 show that the new charges lead to significantly improved geometries for interacting Trp-side chains. We also investigated an off-plane charge model for aromatic rings that more closely mimics their electronic configuration. This model results in an improved free energy of hydration for Trp and a somewhat altered benzene-sodium potential of mean force with a more favorable energy for direct benzene-sodium contact.     

Stacking and T-shape competition in aromatic-aromatic amino acid interactions

The potential of mean force of interacting aromatic amino acids is calculated using molecular dynamics simulations. The free energy surface is determined in order to study stacking and T-shape competition for phenylalanine-phenylalanine (Phe-Phe), phenylalanine-tyrosine (Phe-Tyr), and tyrosine-tyrosine (Tyr-Tyr) complexes in vacuo, water, carbon tetrachloride, and methanol. Stacked structures are favored in all solvents with the exception of the Tyr-Tyr complex in carbon tetrachloride, where T-shaped structures are also important. The effect of anchoring the two alpha-carbons (C(alpha)) at selected distances is investigated. We find that short and large C(alpha)-C(alpha) distances favor stacked and T-shaped structures, respectively. We analyze a set of 2396 protein structures resolved experimentally. Comparison of theoretical free energies for the complexes to the experimental analogue shows that Tyr-Tyr interaction occurs mainly at the protein surface, while Phe-Tyr and Phe-Phe interactions are more frequent in the hydrophobic protein core. This is confirmed by the Voronoi polyhedron analysis on the database protein structures. As found from the free energy calculation, analysis of the protein database has shown that proximal and distal interacting aromatic residues are predominantly stacked and T-shaped, respectively.     

The role of cation-pi interactions in biomolecular association. Design of peptides favoring interactions between cationic and aromatic amino acid side chains.

Cation-pi interactions between amino acid side chains are increasingly being recognized as important structural and functional features of proteins and other biomolecules. Although these interactions have been found in static protein structures, they have not yet been detected in dynamic biomolecular systems. We determined, by (1)H NMR spectroscopic titrations, the energies of cation-pi interactions of the amino acid derivative AcLysOMe (1) with AcPheOEt (2) and with AcTyrOEt (3) in aqueous and three organic solvents. The interaction energy is substantial; it ranges from -2.1 to -3.4 kcal/mol and depends only slightly on the dielectric constant of the solvent. To assess the effects of auxiliary interactions and structural preorganization on formation of cation-pi interactions, we studied these interactions in the association of pentapeptides. Upon binding of the positively-charged peptide AcLysLysLysLysLysNH(2) (5) to the negatively-charged partner AcAspAspXAspAspNH(2) (6), in which X is Leu (6a), Tyr (6b), and Phe (6c), multiple interactions occur. Association of the two pentapeptides is dynamic. Free peptides and their complex are in fast exchange on the NMR time-scale, and 2D (1)H ROESY spectra of the complex of the two pentapeptides do not show intermolecular ROESY peaks. Perturbations of the chemical shifts indicated that the aromatic groups in peptides 6b and 6c were affected by the association with 5. The association constants K(A) for 5 with 6a and with 6b are nearly equal, (4.0 +/- 0.7) x 10(3) and (5.0 +/- 1.0) x 10(3) M(-)(1), respectively, while K(A) for 5 with 6c is larger, (8.3 +/- 1.3) x 10(3) M(-)(1). Molecular-dynamics (MD) simulations of the pentapeptide pairs confirmed that their association is dynamic and showed that cation-pi contacts between the two peptides are stereochemically possible. A transient complex between 5 and 6 with a prominent cation-pi interaction, obtained from MD simulations, was used as a template to design cyclic peptides C(X) featuring persistent cation-pi interactions. The cyclic peptide C(X) had a sequence in which X is Tyr, Phe, and Leu. The first two peptides do, but the third does not, contain the aromatic residue capable of interacting with a cationic Lys residue. This covalent construct offered conformational stability over the noncovalent complexes and allowed thorough studies by 2D NMR spectroscopy. Multiple conformations of the cyclic peptides C(Tyr) and C(Phe) are in slow exchange on the NMR time-scale. In one of these conformations, cation-pi interaction between Lys3 and Tyr9/Phe9 is clearly evident. Multiple NOEs between the side chains of residues 3 and 9 are observed; chemical-shift changes are consistent with the placement of the side chain of Lys3 over the aromatic ring. In contrast, the cyclic peptide C(Leu) showed no evidence for close approach of the side chains of Lys3 and Leu9. The cation-pi interaction persists in both DMSO and aqueous solvents. When the disulfide bond in the cyclic peptide C(Phe) was removed, the cation-pi interaction in the acyclic peptide AC(Phe) remained. To test the reliability of the pK(a) criterion for the existence of cation-pi interactions, we determined residue-specific pK(a) values of all four Lys side chains in all three cyclic peptides C(X). While NOE cross-peaks and perturbations of the chemical shifts clearly show the existence of the cation-pi interaction, pK(a) values of Lys3 in C(Tyr) and in C(Phe) differ only marginally from those values of other lysines in these dynamic peptides. Our experimental results with dynamic peptide systems highlight the role of cation-pi interactions in both intermolecular recognition at the protein-protein interface and intramolecular processes such as protein folding.     

Cation-pi interaction in the polyolefin cyclization cascade uncovered by incorporating unnatural amino acids into the catalytic sites of squalene cyclase

It has been assumed that the pi-electrons of aromatic residues in the catalytic sites of triterpene cyclases stabilize the cationic intermediates formed during the polycyclization cascade of squalene or oxidosqualene, but no definitive experimental evidence has been given. To validate this cation-pi interaction, natural and unnatural aromatic amino acids were site-specifically incorporated into squalene-hopene cyclase (SHC) from Alicyclobacillus acidocaldarius and the kinetic data of the mutants were compared with that of the wild-type SHC. The catalytic sites of Phe365 and Phe605 were substituted with O-methyltyrosine, tyrosine, and tryptophan, which have higher cation-pi binding energies than phenylalanine. These replacements actually increased the SHC activity at low temperature, but decreased the activity at high temperature, as compared with the wild-type SHC. This decreased activity is due to the disorganization of the protein architecture caused by the introduction of the amino acids more bulky than phenylalanine. Then, mono-, di-, and trifluorophenylalanines were incorporated at positions 365 and 605; these amino acids reduce cation-pi binding energies but have van der Waals radii similar to that of phenylalanine. The activities of the SHC variants with fluorophenylalanines were found to be inversely proportional to the number of the fluorine atoms on the aromatic ring and clearly correlated with the cation-pi binding energies of the ring moiety. No serious structural alteration was observed for these variants even at high temperature. These results unambiguously show that the pi-electron density of residues 365 and 605 has a crucial role for the efficient polycyclization reaction by SHC. This is the first report to demonstrate experimentally the involvement of cation-pi interaction in triterpene biosynthesis.