title: Kinetic, mechanistic, and structural modeling studies of truncated wild-type leucine-rich repeat kinase 2 and the G2019S mutant.
abstract: Leucine-rich repeat kinase 2 (LRRK2), a large and complex protein that possesses two enzymatic properties, kinase and GTPase, is one of the major genetic factors in Parkinson's disease (PD). Here, we characterize the kinetic and catalytic mechanisms of truncated wild-type (t-wt) LRRK2 and its most common mutant, G2019S (t-G2019S), with a structural interpretation of the kinase domain. First, the substitution of threonine with serine in the LRRKtide peptide results in a much less efficient substrate as demonstrated by a 26-fold decrease in k(cat) and a 6-fold decrease in binding affinity. The significant decrease in k(cat) is attributed to a slow chemical transfer step as evidenced by the inverse solvent kinetic isotope effect in the proton inventory and pL (pH or pD)-dependent studies. The shape of the proton inventory and pL profile clearly signals the involvement of a general base (pK(a) = 7.5) in the catalysis with a low fractionation factor in the ground state. We report for the first time that the increased kinase activity of the G2019S mutant is substrate-dependent. Homology modeling of the kinase domain (open and closed forms) and structural analysis of the docked peptide substrates suggest that electrostatic interactions play an important role in substrate recognition, which is affected by G2019S and may directly influence the kinetic properties of the enzyme. Finally, the GTPase activity of the t-G2019S mutant was characterized, and the mutation modestly decreases GTPase activity without significantly affecting GTP binding affinity.
authors.affiliation: Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, Brigham and Women's Hospital, Harvard Medical School, Cambridge, Massachusetts 02139, United States. mliu@rics.bwh.harvard.edu
authors.forename: Min
authors.initials: M
authors.lastname: Liu
authors.forename: Stephanie
authors.initials: S
authors.lastname: Kang
authors.forename: Soumya
authors.initials: S
authors.lastname: Ray
authors.forename: Justin
authors.initials: J
authors.lastname: Jackson
authors.forename: Alexandra D
authors.initials: AD
authors.lastname: Zaitsev
authors.forename: Scott A
authors.initials: SA
authors.lastname: Gerber
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Cuny
authors.forename: Marcie A
authors.initials: MA
authors.lastname: Glicksman
mesh_terms: D000595:Amino Acid Sequence; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010300:Parkinson Disease / Q000201:enzymology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary
chemical_list: D006160:Guanosine Triphosphate; C495280:LRRK2 protein, human; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D017346:Protein Serine-Threonine Kinases