Regulation and Plasticity of Catalysis in Enzymes: Insights from Analysis of Mechanochemical Coupling in Myosin

Abstract

The mechanism of ATP hydrolysis in the myosin motor domain is analyzed using a combination of DFTB3/CHARMM simulations and enhanced sampling techniques. The motor domain is modeled in the pre-powerstroke state, in the post-rigor state, and a hybrid model based on the post-rigor state with a closed nucleotide-binding pocket. The ATP hydrolysis activity is found to depend on the positioning of nearby water molecules, and a network of polar residues facilitates proton transfer and charge redistribution during hydrolysis. Comparison of the observed hydrolysis pathways and the corresponding free energy profiles leads to detailed models for the mechanism of ATP hydrolysis in the pre-powerstroke state and proposes factors that regulate the hydrolysis activity in different conformational states. In the pre-powerstroke state, the scissile Pγ-O3β bond breaks early in the reaction. Proton transfer from the lytic water to the γ-phosphate through active site residues is an important part of the kinetic bottleneck; several hydrolysis pathways that feature distinct proton transfer routes are found to have similar free energy barriers, suggesting a significant degree of plasticity in the hydrolysis mechanism. Comparison of hydrolysis in the pre-powerstroke state and the closed post-rigor model suggests that optimization of residues beyond the active site for electrostatic stabilization and pre-organization is likely important to enzyme design.

Analysis of Phosphoryl-Transfer Enzymes with QM/MM Free Energy Simulations

“…Nevertheless, it is important to emphasize that there are multiple variants of the string method, which treat thermal fluctuations in different ways with different underlying approximations (Maragliano, Fischer, Vanden-Eijnden, & Ciccotti, 2006). For example, in the variant of the string method applied to the myosin example (Lu, Ovchinnikov, Roston, Demapan, & Cui, 2017) in Section 3.2, the entropic contribution associated with thermal fluctuations of the collective variables is treated approximately compared to the finite temperature string approach (Maragliano et al, 2006; Ovchinnikov, Karplus, & Vanden-Eijnden, 2011); therefore, the number of collective variables used to parameterize the string should be carefully selected. Moreover, in both metadynamics and string calculations, there are a number of key parameters that influence the convergence and accuracy of these calculations; an example in the string calculations is the force constant used to control the degree of sampling orthogonal to the path, which determines the accuracy of the mean force (Lu et al, 2016); another issue is the overlap of the configurations sampled along the string, and conducting Hamiltonian replica exchange is useful in this regard (Rosta et al, 2011).…”

“…The mechanism of ATP hydrolysis in myosin has been analyzed by numerous experimental (Málnási-Csizmadia et al, 2001; Malnasi-Csizmadia et al, 2007; Onishi, Mochizuki, & Morales, 2004; Onishi, Ohki, Mochizuki, & Morales, 2002) and computational (Grigorenko et al, 2007; Kiani & Fischer, 2014; Li & Cui, 2004; Lu et al, 2017; Schwarzl, Smith, & Fischer, 2006; Yang & Cui, 2009; Yang, Yu, & Cui, 2008) investigations. For the purpose of this chapter, it is worth noting that debates remain regarding the identity of a catalytic base: one mechanism involves the γ phosphate of ATP as the ultimate base, although the proton transfer from the lytic water is likely mediated by active-site water molecules and/or polar side chains, while another mechanism involves a highly conserved glutamate (Glu459 in Dictyostelium discoideum myosin).…”

“…While glutamate serving as the base for ATP/GTP hydrolysis has been supported in several other molecular motors such as F 1 -ATPase (Hayashi et al, 2012) and kinesin (McGrath et al, 2013), this role in myosin is not expected as Glu459 is engaged in a signature salt-bridge with Arg238; this interaction has been deemed essential to the closure of the active site and also expected to lower the pK a of Glu459, making it less likely to act as a base. Different computations, however, led to different conclusions (Grigorenko et al, 2007; Kiani & Fischer, 2014; Lu et al, 2017), and mutational studies involving this salt-bridge (Onishi et al, 2002) also led to results that were not straightforward to interpret (see below).…”

“…6 for an example). Therefore, the strategy found effective in Lu et al (2017) was to use metadynamics simulations to explore the reaction mechanism with delocalized collective variables; once a complete reaction pathway is sampled (it does not need to feature a converged free energy profile), the structures are fed into string calculations, which are able to use many more structural variables to uniquely define the pathway for meaningful free energy calculations.…”

“…7, the hydrolysis reaction in the prepowerstroke state is exothermic by almost 10 kcal/mol; the experimental data suggested that the hydrolysis is nearly thermoneutral (Geeves & Holmes, 1999). Studies of simple model reactions indicated that the error in exothermicity can be qualitatively understood (Lu et al, 2017), yet the issue of more concern is whether the DFTB3/MM approach captures the proper sequence of events between proton transfer and nucleophilic attack (Mlynsky et al, 2014); this affects the nature of the rate limiting transition state and therefore any factors that stabilize that transition state. Although gas phase models support the use of DFTB3 (Lu et al, 2017), more systematic calibration using solution phase model reactions will be valuable.…”