Updated on 27 August 2012
Apart from this, transformations of desolvation parameters and intra hydrogen bond energies are analyzed to compute the stability of the proteins. The filtered modifications enter the next level in the framework called 'the transition state analysis', which generates reaction coordinates of different stages of transition states like E-S transition state, E-SE-P transition state and the E+P transition state. This is done using techniques like quantum mechanics/molecular mechanics (QM/MM) simulations, steered molecular dynamics and transition path sampling. Finally, the transition state reaction coordinates are used to predict the kinetic properties, activation energy and rate limiting steps of the mutant enzymes.
For example, we successfully identified the pressure points (as indicated in the graph below), which are the local energy barriers across the enzyme that lead to an un-catalyzed reaction. This information narrows down the hotpots for mutagenesis, a key step towards engineering the enzyme to produce a synthetic beta-lactam based antibiotic.
Moreover, E-S reaction coordinates during the transition state analysis reveal the crucial active site residues (as shown in the figure below) that are involved in substrate product conversion and product dissociation.
There is a consensus across industries that many biologically available enzymes have a great potential to be exploited if they can be productive at reasonable physical conditions.
However, their inherent thermodynamics calls for higher investment in the production process leading to the final high cost to the end-consumer and the constant quest to optimize the enzyme by engineering it for suitable is driving the R&D of many well known successful biotechnology and biopharmaceutical companies. We hope that our in silico framework can significantly contribute to the both the industry as well as the end-consumer.
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2. DeSouza VR, SilvaACG, Pinotti LM, Ara´ujo HSS, Giordano DLMC; Characterization of the penicillin G acylase from Bacillus megaterium ATCC 14945; Braz Arch Biol Technol 2005; 48:105-11
3. Arroyo M, De la Mata I, Acebal C, Castillon PM; Biotechnological applications of penicillin acylases: state of the art; Appl Microbiol Biotechnol 2003; 60:507-14
4. Ignasi Buch, Toni Giorgino, and Gianni De Fabritiis; Complete reconstruction of an enzyme-inhibitor binding process by molecular dynamics simulations; PNAS 2011; Doi:10.1073/pnas.1103547108