title: Efficient Calculation of Enzyme Reaction Free Energy Profiles Using a Hybrid Differential Relaxation Algorithm: Application to Mycobacterial Zinc Hydrolases.
abstract: Determination of the free energy profile for an enzyme reaction mechanism is of primordial relevance, paving the way for our understanding of the enzyme's catalytic power at the molecular level. Although hybrid, mostly DFT-based, QM/MM methods have been extensively applied to this type of studies, achieving accurate and statistically converged results at a moderate computational cost is still an open challenge. Recently, we have shown that accurate results can be achieved in less computational time, combining Jarzynski's relationship with a hybrid differential relaxation algorithm (HyDRA), which allows partial relaxation of the solvent during the nonequilibrium steering of the reaction. In this work, we have applied this strategy to study two mycobacterial zinc hydrolases. Mycobacterium tuberculosis infections are still a worldwide problem and thus characterization and validation of new drug targets is an intense field of research. Among possible drug targets, recently two essential zinc hydrolases, MshB (Rv1170) and MA-amidase (Rv3717), have been proposed and structurally characterized. Although possible mechanisms have been proposed by analogy to the widely studied human Zn hydrolases, several key issues, particularly those related to Zn coordination sphere and its role in catalysis, remained unanswered. Our results show that mycobacterial Zn hydrolases share a basic two-step mechanism. First, the attacking water becomes deprotonated by the conserved base and establishes the new C-O bond leading to a tetrahedral intermediate. The intermediate requires moderate reorganization to allow for proton transfer to the amide N and C-N bond breaking to occur in the second step. Zn ion plays a key role in stabilizing the tetrahedral intermediate and balancing the negative charge of the substrate during hydroxide ion attack. Finally, comparative analysis of other Zn hydrolases points to a convergent mechanistic evolution.
authors.affiliation: Instituto de Química Física de los Materiales Medio Ambiente y Energía (INQUIMAE), UBA-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Buenos Aires, Argentina.
authors.forename: Juan Manuel
authors.initials: JM
authors.lastname: Romero
authors.affiliation: Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Buenos Aires, Argentina.
authors.forename: Mariano
authors.initials: M
authors.lastname: Martin
authors.affiliation: Instituto de Química Física de los Materiales Medio Ambiente y Energía (INQUIMAE), UBA-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Buenos Aires, Argentina.
authors.forename: Claudia Lilián
authors.initials: CL
authors.lastname: Ramirez
authors.affiliation: Instituto de Química Física de los Materiales Medio Ambiente y Energía (INQUIMAE), UBA-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Buenos Aires, Argentina; Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Buenos Aires, Argentina.
authors.forename: Victoria Gisel
authors.initials: VG
authors.lastname: Dumas
authors.affiliation: Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Buenos Aires, Argentina; Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN) CONICET, Ciudad Universitaria, Buenos Aires, Argentina. Electronic address: marcelo@qi.fcen.uba.ar.
authors.forename: Marcelo Adrián
authors.initials: MA
authors.lastname: Marti
mesh_terms: D000465:Algorithms; D000581:Amidohydrolases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D055162:Biocatalysis; D002413:Cations, Divalent; D006801:Humans; D056004:Molecular Dynamics Simulation; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000201:enzymology; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000737:chemistry*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011522:Protons; D011789:Quantum Theory; D013045:Species Specificity; D013816:Thermodynamics; D014867:Water / Q000737:chemistry; D015032:Zinc / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D002413:Cations, Divalent; D011522:Protons; D014867:Water; D000581:Amidohydrolases; C417275:N-acetyl-1-D-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside deacetylase; D009238:N-Acetylmuramoyl-L-alanine Amidase; D015032:Zinc
keywords: Free energy; HyDRA; Jarzynski's relationship; M. tuberculosis; MshB; QM/MM; Rv1170; Rv3717; Zn hydrolases