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Comparative Study
. 2010 Mar 9;49(9):2008-17.
doi: 10.1021/bi901851y.

Kinetic mechanistic studies of wild-type leucine-rich repeat kinase 2: characterization of the kinase and GTPase activities

Min Liu  1 , Brittany Dobson, Marcie A Glicksman, Zhenyu Yue, Ross L Stein
Affiliations
Comparative Study

Kinetic mechanistic studies of wild-type leucine-rich repeat kinase 2: characterization of the kinase and GTPase activities

Min Liu et al. Biochemistry. .

Abstract

Recent studies have identified mutations in the leucine-rich repeat kinase2 gene (LRRK2) in the most common familial forms and some sporadic forms of Parkinson's disease (PD). LRRK2 is a large and complex protein that possesses kinase and GTPase activities. Some LRRK2 mutants enhance kinase activity and possibly contribute to PD through a toxic gain-of-function mechanism. Given the role of LRRK2 in the pathogenesis of PD, understanding the kinetic mechanism of its two enzymatic properties is critical for the discovery of inhibitors of LRRK2 kinase that would be therapeutically useful in treating PD. In this report, by using LRRK2 protein purified from murine brain, first we characterize kinetic mechanisms for the LRRK2-catalyzed phosphorylation of two peptide substrates: PLK-derived peptide (PLK-peptide) and LRRKtide. We found that LRRK2 follows a rapid equilibrium random mechanism for the phosphorylation of PLK-peptide with either ATP or PLK-peptide being the first substrate binding to the enzyme, as evidenced by initial velocity and inhibition mechanism studies with nucleotide analogues AMP and AMP-PNP, product ADP, and an analogue of the peptide substrate. The binding of the first substrate has no effect on the binding affinity of the second substrate. Identical mechanistic conclusions were drawn when LRRKtide was the phosphoryl acceptor. Next, we characterize the GTPase activity of LRRK2 with a k(cat) of 0.2 +/- 0.02 s(-1) and a K(m) of 210 +/- 29 microM. A SKIE of 0.97 +/- 0.04 was measured on k(cat) for the GTPase activity of LRRK2 in a D(2)O molar fraction of 0.86 and suggested that the product dissociation step is rate-limiting, of the steps governed by k(cat) in the LRRK2-catalyzed GTP hydrolysis. Surprisingly, binding of GTP, GDP, or GMP has no effect on kinase activity, although GMP and GDP inhibit the GTPase activity. Finally, we have identified compound LDN-73794 through screen of LRRK2 kinase inhibitors. Our study revealed that this compound is a competitive inhibitor of the binding of ATP and inhibits the kinase activity without affecting the GTPase activity.

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Figures

Figure 1
Figure 1
Comparison of two kinase assays. The LRRK2-catalyzed PLK-peptide phosphorylation was conducted at the same condition in a TR-FRET assay and a radiometric assay. The initial velocities were measured as a function of [PLK-peptide] at 200 µM ATP, and 24 nM LRRK2. Filled circles correspond to the y-axis of FU/h on the left measured by the TR-FRET assay, while open circles correspond to the y-axis of nM/h on the right determined from the radiometric assay. The data were fit to the simple Michaelis-Menten equation. Best fit parameters are (kcat)app = 31870 FU/h and (KPLK)app = 592 nM for the TR-FRET assay; (kcat)app = 33.6 nM/h and (KPLK)app = 434 nM for the radiometric assay. The inset shows the linear correlation of unit FU/h and unit nM/h.
Figure 2
Figure 2
Steady-state kinetic studies for LRRK2-catalyzed phosphorylation of PLK-peptide. A: dependence of initial velocities on [PLK-peptide] at [ATP] = 200 (●), 100 (○), 50 (▼), 25 (▽), 12.5 (■), and 6.25 µM (□). Each data point is the average of duplicate determinations. The data set was globally fit to the equation reflecting rapid equilibrium random. B & C: ATP concentration dependencies of (kcat)PLK and (kcat/Km)PLK apparent values derived from analysis of the data of panel A.
Figure 3
Figure 3
Inhibition study of the LRRK2-catalyzed phosphorylation of PLK-peptide by AMP. A: Plot of initial velocities vs [ATP] at [AMP] = 25 (●), 10 (▼), 5 (▽), 2.5 (■), and 0 µM (□) all at a fixed PLK-peptide concentration of 100 nM. B & C: AMP concentration dependencies of (kcat)ATP and (kcat/Km)ATP apparent values derived from analysis of the data of panel A. D: Plot of initial velocities vs [PLK-peptide] at [AMP] = 25 (●), 10 (○), 5 (▼), 2.5 (▽), and 0(■) mM all at a fixed ATP concentration of 200 µM. E & F: AMP concentration dependencies of (kcat)PLK and (kcat/Km)PLK apparent values derived from analysis of the data of panel D.
Figure 4
Figure 4
LRRK2-catalyzed GTP hydrolysis. The LRRK2-catalyzed GTP hydrolysis was conducted in buffer containing 20 mM Tris (pH 7.4), 50 mM NaCl, 10 mM MgCl2, 1 mM DTT, BSA 0.5 mg/ml, GTP, and [α-33P]-GTP. The reaction was initiated by the addition of 30 nM LRRK2, and incubated at RT for 20 min. The reaction was stopped by the addition of 20 mM EDTA, and the product [α-33P]-GDP was separated from [α-33P]-GTP by PEI-cellulose TLC developed by 0.5 M KH2PO4 (pH 3.4) developing buffer and analyzed by scintillation counter. Each data point is the average of triplicate determinations, and the error bars represent standard deviation. The data set was fit to the simple Michaelis-Menten equation.
Figure 5
Figure 5
Inhibition of the LRRK2-catalyzed GTP hydrolysis by nucleotide analogues. The GTPase reaction was carried out at 200 µM GTP in the presence of GMP (A), GDP (B), or ADP (C). The dependence of relative rate on I (I = GMP, GDP, ADP) was fit to the simple inhibition expression of general form: νinhib = νcontrol/(1 + [I]/Ki,app), which allowed us to calculate the apparent dissociation constants of 2.9 mM, 97 µM, and 0.6 mM for GMP, GDP, and ADP, respectively. Each data point is the average of triplicate determinations, and the error bars represent standard deviation.
Figure 6
Figure 6
Effects of nucleotides on LRRK2-catalyzed PLK-peptide phosphorylation. The initial velocity of LRRK2-catalyzed phosphorylation of PLK-peptide were measured as a function of concentration of GTP, GDP, or GMP at saturating concentrations of ATP and PLK. Each data point is the average of triplicate determinations, and the error bars represent standard deviation.
Figure 7
Figure 7
Inhibition of the LRRK2-catalyzed GTP hydrolysis. The GTPase reaction was carried out at 200 µM GTP in the presence of 0.5 mM ADP, 0.5 mM ATP, 0.5 mM ATP & 1 µM PLK, or 1 µM PLK, respectively. The control was carried out in the absence of either ADP/ATP or PLK. Each data point is the average of triplicate determinations, and the error bars represent standard deviation.
Figure 8
Figure 8
Inhibition of the LRRK2-catalyzed phosphorylation of PLK-peptide by compound LDN-73794. A: Plot of initial velocities vs [ATP] at [LDN-73794] = 16.7 (●), 5.6 (○), 1.85 (▼), 0.62 (▽), and 0 µM (■) all at a fixed PLK-peptide concentration of 100 nM. B & C: LDN-73794 concentration dependencies of (Vmax)ATP and (Vmax/Km)ATP apparent values derived from analysis of the data of panel A. D: Plot of initial velocities vs [PLK-peptide] at [LDN-73794] = 16.7 (●), 5.6 (○), 1.85 (▼), 0.62 (▽), and 0 µM (■) all at a fixed ATP concentration of 50 µM. E & F: LDN-73794 concentration dependencies of (Vmax)PLK and (Vmax/Km)PLK apparent values derived from analysis of the data of panel D.
Scheme I
Scheme I
The rapid equilibrium random mechanism of LRRK2-catalyzed phosphorylation. A represents ATP and B represents phosphoryl acceptor, either PLK-peptide or LRRKtide.
Figure 10
Figure 10
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