Differential Effects of Divalent Manganese and Magnesium on the Kinase Activity of Leucine-Rich Repeat Kinase 2 (LRRK2)

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• 作者：Brian Lovitt ; Erica C. VanderPorten ; Zejuan Sheng ; Haitao Zhu ; Jake Drummond ; Yichin Liu
• 刊名：Biochemistry
• 出版年：2010
• 出版时间：April 13, 2010
• 年：2010
• 卷：49
• 期：14
• 页码：3092-3100
• 全文大小：906K
• 年卷期：v.49,no.14(April 13, 2010)
• ISSN：1520-4995

文摘

Various mutations in leucine-rich repeat kinase 2 (LRRK2) have been linked to susceptibility for both familial and idiopathic late-onset Parkinson’s disease (PD). In this study, we have demonstrated that phosphorylation of MBP and LRRKtide by the LRRK2 G2019S mutant was activated by Mn²⁺ in vitro. This enhanced G2019S kinase activity was due to the combination of an increase in kinase and a decrease in ATPase activity by Mn²⁺. Compared to 10 mM Mg²⁺, 1 mM Mn²⁺ reduced ATP K_m for G2019S from 103 to 1.8 μM and only modestly reduced k_cat (2.5-fold); as a result, the Mn²⁺ increased its k_cat/K_m by 22-fold. This change in ATP K_m was due in large part to an increase in nucleotide affinity. While Mn²⁺ also increased ATP affinity and had similar effects on k_cat/K_m for LRRK2 WT and R1441C enzymes, it reduced their k_cat values significantly by 13−17-fold. Consequently, the difference in the kinase activity between G2019S and other LRRK2 variants was enhanced from about 2-fold in Mg²⁺ to 10-fold in Mn²⁺ at saturating ATP concentrations relative to its K_m. Furthermore, while Mg²⁺ yielded optimal V_max values at Mg²⁺ concentration greater than 5 mM, the optimal Mn²⁺ concentration for activating LRRK2 catalysis was in the micromolar range with increasing Mn²⁺ above 1 mM causing a decrease in enzyme activity. Finally, despite the large but expected differences in IC₅₀ tested at 100 μM ATP, the apparent K_i values of a small set of LRRK2 ATP-competitive inhibitors were within 5-fold between Mg²⁺- and Mn²⁺-mediated reactions except AMP-CPP, an ATP analogue.