Peptide Hydrolysis in Thermolysin:  Ab Initio QM/MM Investigation of the Glu143-Assisted Water Addition Mechanism

Abstract

Thermolysin (TLN) is one of the best-studied zinc metalloproteases. Yet the mechanism of action is still under debate. In order to investigate the energetic feasibility of the currently most favored mechanism, we have docked a tripeptide to the active site of TLN and computed the free energy profile at the quantum mechanics/molecular mechanics level of theory. The mechanism consists of three distinct steps: (i) a Zn-bound water molecule is deprotonated by Glu143 and attacks the carbonyl bond of the substrate; (ii) Glu143 transfers the proton to the amide nitrogen atom; (iii) the nitrogen atom is protonated and the peptide bond is irreversibly broken. The free energy barriers for steps i and iii have almost equal heights, 14.8 and 14.7 kcal/mol, respectively, and are in good agreement with the effective experimental activation barrier obtained for similar substrates, 12.1-13.6 kcal/mol. Transition state stabilization for nucleophilic attack is achieved by formation of a weak coordination bond between the substrate carbonyl oxygen atom and the Zn ion and of three strong hydrogen bonds between the substrate and protonated His231 and two solvent molecules. The transition state for the nucleophilic attack (step i) is more tightly bonded than the enzyme-substrate complex, implying that TLN complies with Pauling's hypothesis regarding transition-state stabilization. Glu143, at first unfavorably oriented for protonation of the amide nitrogen atom, displayed large structural fluctuations that facilitated reorganization of the local hydrogen-bond network and transport of the proton to the leaving group on the nanosecond time scale. The present simulations give further evidence that Glu143 is a highly effective proton shuttle which should be assigned a key role in any reaction mechanism proposed for TLN.

Efficient Calculation of Enzyme Reaction Free Energy Profiles Using a Hybrid Differential Relaxation Algorithm

“…Interestingly, the substrate carbonyl is not coordinated in the reactive structure and becomes coordinated during the reaction, strongly interacting in the tetrahedral intermediate. More interesting is that the same mechanism was also reported for termolysin (Blumberger, Lamoureux, & Klein, 2007), ACE (Zhang, Wu, & Xu, 2013), and the anthrax lethal factor peptidase (Smith, Smith, Yang, Xu, & Guo, 2010), all displaying barriers for the nucleophilic attack in the 13-18 kcal/mol range, and for the C-N bond breaking in the 5-14.7 kcal/mol range. In summary, the different studies of Zn hydrolases reactions point toward a conserved (or converged) mechanism among them.…”

Theoretical Aspects of Hydrolysis of Peptide Bonds by Zinc Metalloenzymes

“…[83] Model systems: Four model systems were used for the assessment of the accuracy of the quantum chemical methods. The set consisted of the models of metal-free 1) formamide and 2) Ala-Ala hydrolysis, 3) single metal ion (Zn 2 + )-assisted peptide hydrolysis using the thermolysin active-site structure, [52,84] and 4) the dizinc metallopeptidase active site based on the glutamate carboxypeptidase II structure and the QM/MM transition states described earlier. [7] The reactant (Michaelis) complexes (MC X ) are depicted in Figure 7.…”

Modeling the charge distribution at metal sites in proteins for molecular dynamics simulations

“…The substrate is modeled by a Gly-Leu-Ala tripeptide with neutral termini, and is docked into the enzyme (Fig. 1A, Blumberger et al, 2006).…”