The utility of molecular dynamics simulations for understanding site-directed mutagenesis of glycine residues in biotin carboxylase.

Biotin carboxylase from Escherichia coli catalyzes the ATP-dependent carboxylation of biotin and is one component of the multienzyme complex acetyl-CoA carboxylase, which catalyzes the committed step in long-chain fatty acid synthesis. Comparison of the crystal structures of biotin carboxylase in the absence and presence of ATP showed a central B-domain closure when ATP was bound. Peptidic NH groups from two active site glycine residues (Gly165 and Gly166) that form hydrogen bonds to the phosphate oxygens of ATP were postulated to act as a "trigger" for movement of the B-domain. The function of these two glycine residues in the catalytic mechanism was studied by disruption of the hydrogen bonds using site-directed mutagenesis. Both single (G165V) and (G166V) and double mutants (G165V-G166V) were constructed. The mutations did not affect the maximal velocity of a partial reaction, the bicarbonate-dependent ATPase activity. This suggests that the peptidic NH groups of Gly165 and Gly166 are not triggers for domain movement. However, the K(m) values for ATP for each of the mutants was increased over 40-fold when compared with wild-type indicating the peptidic NH groups of Gly165 and Gly166 play a role in binding ATP. Consistent with ATP binding, the maximal velocity for the biotin-dependent ATPase activity (i.e. the complete reaction) was decreased over 100-fold suggesting the mutations have misaligned the reactants for optimal catalysis. Molecular dynamics studies confirm perturbation of the hydrogen bonds from the mutated residues to ATP, whereas the double mutant exhibits antagonistic effects such that hydrogen bonding from residues 165 and 166 to ATP is similar to that in the wild-type. Consistent with the site-directed mutagenesis results the molecular dynamics studies show that ATP is misaligned in the mutants.