Transition state stabilization by general acid catalysis, water expulsion, and enzyme reorganization in Medicago savita chalcone isomerase

Abstract

In aqueous solution, Medicago savita chalcone isomerase (CHI) enhances the reaction rate for the unimolecular rearrangement of chalcone (CHN) into flavanone by seven orders of magnitude. Conformations of CHN and their relative free energies in water and CHI were investigated by the thermodynamic perturbation method. In water, CHN adopts two conformations (I and II) with conformation I being higher in energy than conformation II by 3 kcal͞mol. Only I can give rise to a near attack conformer (NAC) where the nucleophile O2 and the electrophile C9 are placed in proximity. In CHI, I binds less tightly than II by Ϸ2 kcal͞mol, resulting in the free energy for NAC formation being Ϸ2 kcal͞mol higher in the enzyme than in water. This unfavorable feature in the ground state of the CHI reaction requires the predominant catalytic advantage to be taken in the step of NAC 3 transition state (TS). From the molecular dynamics simulations of apo-CHI, CHI complexed with CHN (CHI⅐CHN) and CHI⅐TS, we found: (i) Lys-97-general-acid catalysis of the O2 ؊ nucleophilic addition; (ii) expulsion of three water molecules in the process of TS formation; (iii) release of enzyme structural distortion on TS formation. In the conclusion, CHI's remarkable efficiency of stabilizing the TS and its relatively poor ability in organizing the ground state is compared with chorismate mutase whose catalytic prowess, when compared with water, originates predominantly from the enhanced NAC population at the active site.T he intramolecular ring-closure reaction of chalcone 3 flavanone (see Scheme 1) has been studied extensively structurally and mechanistically (1-3). The rate constant k cat is 38 s Ϫ1 for the enzymatic reaction, and k non is 10 Ϫ6 s Ϫ1 for the water reaction (1). The pH dependences of the noncatalyzed and catalyzed reactions are much the same, suggesting that ionization of the substrate phenolic O2Ј occurs in water for both enzymatic and nonenzymatic reactions (1). The crystal structures of chalcone isomerase (CHI) complexed with flavanones reveal an absence of an enzyme-based nucleophilic species near the substrate, showing that CHI is not directly involved in the enzymatic reaction through covalent interaction (3). Drawing on all experimental data, Noel and colleagues (2, 3) explain the catalytic mechanism of CHI as a combination of two effects. One is active site molding of the substrate into the reactive conformation and the other is stabilization of the enolate-like transition state (TS) by forming a hydrogen-bonding network with Thr-48 and Tyr-106 at the ketone O7. However, quantitative comparison of these two contributions has not been reached because of the experimental difficulty of perturbing each component selectively. Here we report our computational studies to assess the relative importance of ground state and TS effects in the CHI catalysis.We have used two methods to achieve this goal. First, we measure the free energies (⌬G N°v alues) for forming reactive ground-state conformers [near attack conformers (NACs), ref. 4] in ...

Mechanisms of Soybean Roots' Tolerances to Salinity Revealed by Proteomic and Phosphoproteomic Comparisons Between Two Cultivars

“…Subsequently, malonyl-CoA is added to p-coumaroyl-CoA and yields tetrahydroxychalcone by the enzyme chalcone synthase. Finally, chalcone isomerase converts the C15 compound tetrahydroxychalcone into (2S)-flavanones (85)(86)(87). These flavonoids, including a diverse family of polyphenols, have been proven with health-promoting effects especially in preventing various human pathological risks (88,89).…”

Partial Reconstruction of Flavonoid and Isoflavonoid Biosynthesis in Yeast Using Soybean Type I and Type II Chalcone Isomerases

“…In contrast, the type II CHIs appear to be legume specific and possess additional catalytic activity, which allows them to convert 4, 2#, 4#-trihydroxychalcone (isoliquiritigenin) into (2S)-7, 4#-dihydroxyflavanone (liquiritigenin). A type II CHI isolated from alfalfa (Medicago sativa) has been extensively studied structurally and mechanistically (Jez et al, 2000Hur et al, 2004). These structure-function analyses suggest that the formation of a hydrogen bond network between the active site of CHI and its substrates is crucial for the enzyme's catalytic activity Hur et al, 2004).…”

“…A type II CHI isolated from alfalfa (Medicago sativa) has been extensively studied structurally and mechanistically (Jez et al, 2000Hur et al, 2004). These structure-function analyses suggest that the formation of a hydrogen bond network between the active site of CHI and its substrates is crucial for the enzyme's catalytic activity Hur et al, 2004). Naringenin and liquiritigenin are the precursors of many flavonoid and isoflavonoid compounds.…”

cDNA cloning and characterization of chalcone isomerase-fold proteins from snapdragon (Antirrhinum majus L.) flowers

“…The deduced amino acid sequence of AmCHI1 displayed a 56% identity to that of MsCHI, a type-II CHI, which has been subjected to intense structural and mechanistic studies (Hur and Bruice 2003;Hur et al 2004;Jez et al 2000;Ruiz-Pernia et al 2008). Those studies revealed the importance of hydrogen bonds in the active site of MsCHI for catalysis, which involved Thr48, Tyr106, Asn113 and Thr190 as key residues (Figure 7).…”