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

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 mu M 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 mu M 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.