Catalytic Fields as a Tool to Analyze Enzyme Reaction Mechanism Variants and Reaction Steps

Abstract

Catalytic fields representing the topology of the optimal molecular environment charge distribution that reduces the activation barrier have been used to examine alternative reaction variants and to determine the role of conserved catalytic residues for two consecutive reactions catalyzed by the same enzyme. Until now, most experimental and conventional top-down theoretical studies employing QM/MM or ONIOM methods have focused on the role of enzyme electric fields acting on broken bonds of reactants. In contrast, our bottom-up approach dealing with a small reactant and transition-state model allows the analysis of the opposite effects: how the catalytic field resulting from the charge redistribution during the enzyme reaction acts on conserved amino acid residues and contributes to the reduction of the activation barrier. This approach has been applied to the family of histidyl tRNA synthetases involved in the translation of the genetic code into the protein amino acid sequence. Activation energy changes related to conserved charged amino acid residues for 12 histidyl tRNA synthetases from different biological species allowed to compare on equal footing the catalytic residues involved in ATP aminoacylation and tRNA charging reactions and to analyze different reaction mechanisms proposed in the literature. A scan of the library of atomic multipoles for amino acid side-chain rotamers within the catalytic field pointed out the change in the Glu83 conformation as the critical catalytic effect, providing, at low computational cost, insight into the electrostatic preorganization of the enzyme catalytic site at a level of detail that has not yet been accessible in conventional experimental or theoretical methods. This opens the way for rational reverse biocatalyst design at a very limited computational cost without resorting to empirical methods.

Figure 1

Partial sequence comparison based on the structure alignment of HisRSs originating from 12 different biological organisms. Only the neighborhoods of conservative charged residues and known class II aaRS motifs are presented for brevity. Hidden parts are marked with vertical blue lines.

Figure 2

Conserved charged residues for 12 members of the HisRS family superimposed on the 1KMM chain C using the corresponding C_α carbon atoms of 9 conserved charged residues, shown as black balls. Blue or red balls indicate terminal carbon atoms C_t of negatively or positively charged side chains, respectively. Golden spheres indicate the positions of phosphorus atoms in ligands, if present.

Scheme 1. Schematic Representation of Both Consecutive Reactions Considered in This Study

PP_i denotes pyrophosphate, and A76 denotes the adenine of the tRNA terminal nucleotide.

Figure 3

Transition state TS0 for the ATP aminoacylation reaction mechanism (Scheme 1a), as proposed by Banik and Nandi, where the thicker representation corresponds to the smaller model used to obtain the corresponding catalytic field Δ_S.

Figure 4

Transition states TS1, TS2, TS3, and TS4 for the His-AMP tRNA charging reaction mechanisms (Scheme 1b), as proposed by Liu and Gauld, where the thicker lines correspond to the smaller model used here to obtain the corresponding catalytic field Δ_S.

Figure 5

Catalytic fields for all considered reaction mechanisms with conserved residues exerting the most pronounced catalytic activity. 3D view of the surface colored with the Δ_S value. The blue color indicates the region where a positively charged catalyst will be beneficial. Red denotes locations beneficial for negatively charged residues. Numerical values in kilocalories per mole denote the lowering of activation energy (DTSS) obtained from the MULTISCAN procedure (conformation denoted by solid color), and the values in parentheses represent the DTSS changes resulting from the use of crystallographic side-chain conformations (faded) and neglecting interactions with remaining charged side chains.

Figure 6

Changes in activation barrier DTSS yielded by the MULTISCAN procedure for conserved charged residues for both reactions and mechanisms catalyzed by HisRS involving transition states TS0, TS1, TS2, TS3, and TS4.