Enantioselectivity in Candida antarctica lipase B: a molecular dynamics study

Abstract

A major problem in predicting the enantioselectivity of an enzyme toward substrate molecules is that even high selectivity toward one substrate enantiomer over the other corresponds to a very small difference in free energy. However, total free energies in enzyme-substrate systems are very large and fluctuate significantly because of general protein motion. Candida antarctica lipase B (CALB), a serine hydrolase, displays enantioselectivity toward secondary alcohols. Here, we present a modeling study where the aim has been to develop a molecular dynamics-based methodology for the prediction of enantioselectivity in CALB. The substrates modeled (seven in total) were 3-methyl-2-butanol with various aliphatic carboxylic acids and also 2-butanol, as well as 3,3-dimethyl-2-butanol with octanoic acid. The tetrahedral reaction intermediate was used as a model of the transition state. Investigative analyses were performed on ensembles of nonminimized structures and focused on the potential energies of a number of subsets within the modeled systems to determine which specific regions are important for the prediction of enantioselectivity. One category of subset was based on atoms that make up the core structural elements of the transition state. We considered that a more favorable energetic conformation of such a subset should relate to a greater likelihood for catalysis to occur, thus reflecting higher selectivity. The results of this study conveyed that the use of this type of subset was viable for the analysis of structural ensembles and yielded good predictions of enantioselectivity.

Fig. 1.

Reaction mechanism of serine hydrolase catalyzed hydrolysis or esterification.

Fig. 2.

Free energy profile for the conversion of the serine hydrolase acyl enzyme and alcohol to the free enzyme and ester. The figure shows the energy profiles for the fast R enantiomer (dark) and the slow S enantiomer (light) of the substrate. The ground-state energy of the reactants, G^o, is the same for both enantiomers. The activation free energies for R and S enantiomers are given by ΔG^#_R and ΔG^#_S, respectively, and the absolute free energies for the corresponding transition states are given by G^#_S and G^#_R. The difference in free energy between the transition state and its corresponding tetrahedral intermediate is negligible (Hu et al. 1998). Therefore, ΔΔG^# can be approximated to the difference in absolute free energies between the tetrahedral intermediates of the R and S enantiomers, ΔΔG^#_calc.

Fig. 3.

Substrates modeled for present study on Candida antarctica lipase B. (A) Variation in acyl moiety with alcohol moiety remaining constant. (B) Variation in alcohol moiety with acyl moiety remaining constant. The experimentally derived enantiomeric ratios, E, for temperature 300 K with hexane as solvent (Ottosson and Hult, in press; J. Ottosson, unpubl.) and corresponding values of ΔΔG^# are also specified.

Fig. 4.

Comparison of calculated ΔΔG^# values for various subsets (▪, black bars) with experimental values from Candida antarctica lipase B-catalyzed esterification in hexane (□, white bars). Parts A, B, C, E, and F refer to 3-methyl-2-butanol with variation in the acyl chain moiety. '4i' refers to iso-butanoyl; all other acyl moieties are straight chained. Part D refers to variation in the alcohol moiety (2B, 2-butanol; 3M2B, 3-methyl-2-butanol; 33D2B, 3,3-dimethyl-2-butanol); in each case octanoyl is used as the acyl moiety. The graphs denote potential energies for the following cases. (A) Whole of modeled system including waters. (B) and (D) Function-based subset describing the core structural elements of the transition state, as illustrated in Figure 5 ▶, for variation in acyl and alcohol moieties of the substrate, respectively. (C) Smaller function-based subset describing the substrate alcohol oxygen to His 224 hydrogen bond only, and consisting of the alcohol oxygen of the substrate, His 224:N_ε and the corresponding hydrogen atom. (E) Structure-based subset consisting of full substrate and residues lining the active-site cavity, as given in Figure 6 ▶. (F) Energy-based subset consisting of the full substrate and those residues possessing a difference in interaction energy of ≥0.42 kJ mole⁻¹ (0.1 kcal mole⁻¹) between R and S enantiomers of the substrate.

Fig. 5.

Function-based subset (dark) that describes the core structural elements of the modeled transition state in Candida antarctica lipase B.

Fig. 6.

Molecular surface representation of the Candida antarctica lipase B active-site cavity. The inset figure illustrates the size of the active-site (dark patch) as compared with the whole enzyme. The residues selected as lining the active site are as follows: 38–42, 47, 73, 104–109, 132–134, 138, 140, 141, 144, 150, 151, 153, 154, 157, 188–190, 224, 225, 278, 281, 282, 285. The main figure shows the modeled transition state for 3-methyl-2-butyl octanoate. The residues forming the energy-based subset with a difference in interaction energy of ≥0.42 kJ mole⁻¹ (0.1 kcal mole⁻¹) with the whole of this substrate were as follows: 38–40, 73, 104–106, 134, 140, 144, 153, 154, 157, 188–190, 224, 278, 285. The dark patches of the molecular surface in the main figure correspond to residues excluded from the latter subset.