A novel atomistic motional correlation method combined with thermodynamics to delineate the intricate mechanism of substrate specific catalysis: Enzyme engineering perspective

Conference Dates

September 24-28, 2017


Enzymes are powerful and highly specific catalysts, both in the reactions that they catalyze and in their choice of reactants. (1) Enzymes show this partiality towards substrate through a precise mechanism. The precision of this mechanism is governed by a well connected network of residues in and around the catalytic site, in terms of their motions and consequential thermodynamics. In this study we have designed a novel atomistic motional (AM) correlation method which measures the distance and direction of the atoms in motion. Discretizing the variable, 41 different combinations (AM alphabets) of displacement and direction of the motion of a single atom was calculated over a wide range of molecular motions derived from MD simulations. This is the maximum reported number of measurements which quantifies high-frequency harmonic oscillations to slow functional conformational transitions with a higher level of sensitivity. The method was tested to delineate the mechanism of the Michaelis complexes of Penicillin G acylase (PGA) & Penicillin-G (native reaction) and PGA & PGSO (slow reaction). Correlation of AM alphabets of a pair of atoms (i,j) was calculated as a normalized mutual information (MI; ), (2) and this was summed to derive the per residue correlation (PRC;CnMI) . The CnMI was used to generate network models and clustering analysis, post 0.25 μs of simulations of the Michaelis complexes. Results show clear difference between the AM alphabets of the fast and the slow hydrolyzing enzymatic reactions (Fig1.A). Networks formed between the amino acids in the slow reaction are very much different from native reaction (Fig1.B & C), especially cluster 1 and 2 which shows close relation with the substrate of the native reaction is completely decomposed in the slow reaction. Further, CnMI was combined with a novel method that quantitatively weights atomic interactions (qWAI) in conjuncture with high throughput binding free energy calculation deposited over every amino acid (PRB). The three methods CnMI, qWAI and PRB in isolation and in combination showed the precise mechanism of PGA pertaining to substrate specificity. To mention CnMI shows decomposition of cluster 1 and 2 in slow reaction. qWAI shows that the amide bond of PenG was stabilized by βSer1, βThr68, βGln23 and βAla69 and the same in PGSO is stabilized only by βSer1 and βAla69. Finally, the combined score (Fig.1.D) shows that βGln23 and βPro22, forming a part of oxyanion hole are extremely modulated in the slow reaction, resulting in the destabilization of tetrahedral intermediate. The presentation will show the precise mechanism of substrate selectivity of PGA revealed by these three methods in conjunction with insights for enzyme engineering.

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