Biochemical and kinetic properties of the complex Roco G-protein cycle

Roco proteins have come into focus after mutations in the gene coding for the human Roco protein Leucine-rich repeat kinase 2 (LRRK2) were discovered to be one of the most common genetic causes of late onset Parkinson's disease. Roco proteins are characterized by a Roc domain responsible for GTP binding and hydrolysis, followed by a COR dimerization device. The regulation and function of this RocCOR domain tandem is still not completely understood. To fully biochemically characterize Roco proteins, we performed a systematic survey of the kinetic properties of several Roco protein family members, including LRRK2. Together, our results show that Roco proteins have a unique G-protein cycle. Our results confirm that Roco proteins have a low nucleotide affinity in the micromolar range and thus do not strictly depend on G-nucleotide exchange factors. Measurement of multiple and single turnover reactions shows that neither Pi nor GDP release are rate-limiting, while this is the case for the GAP-mediated GTPase reaction of some small G-proteins like Ras and for most other high affinity Ras-like proteins, respectively. The KM values of the reactions are in the range of the physiological GTP concentration, suggesting that LRRK2 functioning might be regulated by the cellular GTP level.