Computational Studies of Multi-Active Site Enzymes

Multi-scale computational approaches have been applied to investigate the catalytic mechanisms of (i) yeast mitochondrial threonyl-tRNA synthetase (MST1) pre-transfer editing and, (ii) glutamine deamination by glucosamine-6-phosphate synthase (GlmS). MST1: MD and QM/MM-MD methods were used to examine (i) differences in the binding of its cognate and non-cognate Thr- and Ser-AMP substrates respectively, and (ii) mechanism of hydrolytic pre-transfer editing. In contrast to bound Thr-AMP, bound Ser-AMP is less constrained; i.e., greater positional variability, and as a result more waters are able to permeate the active site. Mechanistically, Thr-AMP hydrolysis occurs in two steps via a tetrahedral oxyanion intermediate. For Ser-AMP, however, formation of the oxyanion proceeds via a metastable intermediate while the second step, cleavage of the Ccarb-OP bond, occurs as for Thr-AMP with similar energy barriers. Umbrella sampling shows that mechanism differences are due to a greater number of active site waters stabilizing the forming oxyanion in Ser-AMP, compared to Thr-AMP. As a result, the relative free energies of the rate-limiting barriers as well as that of the hydrolyzed products for Thr-AMP (14-19 and 4-10 kcal mol-1, respectively) are markedly higher than for Ser-AMP (7-12 and 0-5 kcal mol-1, respectively). That is, MST1 thermodynamically and kinetically preferentially edits against non-cognate substrate Ser-AMP, in agreement with experiment. GlmS: MD and QM/MM studies were performed to examine the (i) protonation state of the mechanistically important amine of its N-terminal cysteinyl (Cys1) and its effect on its glutaminase domain and, (ii) mechanism by which it deaminates its glutamine substrate. Proton affinity studies suggest that at physiological pH, the Cys1-NH2 group prefers to be neutral, and that if protonated, the active site is structurally less consistent. When the Cys1-NH2 group acts as the required mechanistic base the rate limiting step corresponds to nucleophilic attack of a water on the covalently cross-linked thioester intermediate with a free energy barrier of 78.2 kJ mol-1