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We present a semi-empirical (PM6-based) computational method forsystematically estimating the effect of all possible single mutants, within acertain radius of the active site, on the barrier height of an enzymaticreaction. The intent of this method is not a quantitative prediction of thebarrier heights, but rather to identify promising mutants for furthercomputational or experimental study. The method is applied to identifypromising single and double mutants of Bacillus circulans xylanase (BCX) withincreased hydrolytic activity for the artificial substrate ortho-nitrophenyl\beta-xylobioside (ONPX$_2$). The estimated reaction barrier for wild-type (WT)BCX is 18.5 kcal/mol, which is in good agreement with the experimentalactivation free energy value of 17.0 kcal/mol extracted from the observedk$_\text{cat}$ using transition state theory (Joshi et al., Biochemistry 2001,40, 10115). The PM6 reaction profiles for eight single point mutations arerecomputed using FMO-MP2/PCM/6-31G(d) single points. PM6 predicts an increasein barrier height for all eight mutants while FMO predicts an increase for sixof the eight mutants. Both methods predict that the largest change in barrieroccurs for N35F, where PM6 and FMO predict a 9.0 and 15.8 kcal/mol increase,respectively. We thus conclude that PM6 is sufficiently accurate to identifypromising mutants for further study. We prepared a set of all theoreticallypossible (342) single mutants in which every amino acid of the active site(except for the catalytically active residues E78 and E172) was mutated toevery other amino acid. Based on results from the single mutants we construct aset of 111 double mutants consisting of all possible pairs of single mutantswith the lowest barrier for a particular position and compute their reactionprofile. None of the mutants have, to our knowledge, been preparedexperimentally[...].

peerj

A computational method for the systematic screening of reaction barriers  in enzymes: Searching for Bacillus circulans xylanase mutants with greater  activity towards a synthetic substrate