关键词: 蛋白质酪氨酸磷酸酶；纹状体蛋白质酪氨酸磷酸酶；纹状体；中枢神经系统；蛋白磷酸化

Abstract:

Objective   To explore the essential role of Threonine at position 541 and 330(T541，T330)in the intrinsic phosphatase activity of striatal-enriched protein tyrosine phosphatase(STEP). Methods   STEP wild type(STEP-WT) and its mutants STEP-T330D/T541A were sub-cloned into the PET15b vector. Expression and purification of STEP-WT and its mutants were performed by affinity column and liquid chromatography. The phosphatase activity was measured in vitro with 4nitrophenyl phosphate (pNPP) as substrate. The inhibition by NaVO3 was measured to monitor the effects of mutants on protein folding. The pH-dependence and leaving-group pKa dependence of STEP catalysis were carried out to dissect the underlying molecular mechanism. Results   STEP-WT and STEP-T330D displayed similar catalytic ability toward pNPP at pH 7.0. The kcat of STEP-T541A decreased 3 folds compared to STEP-WT.-STEP-WT and the two mutants had similar Ki for NaVO3. Examination of the kcat versus pH curve revealed that pK2app of STEP-T541A significantly increased and the (kcat)lim dropped by at least 10 folds. In consisitent with these observations, β1g(kcat) of STEP-T541A increased significantly. Conclusion   T541 plays an important role in STEP catalysis, by participating the processes from product formation to phosphate release. Future drugs targeting to STEP for therapeutic usage could be developed through modulating T541 conformations.

Key words: Prtotein tyrosine phosphatase; Striatalenriched protein tyrosine phosphatase; Striatal; Central nervous system; Protein phosphorylation