The cytosolic malic enzyme from the pigeon liver is sensitive to chemical denaturant urea. When monitored by protein intrinsic fluorescence or circular dichroism spectral changes, an unfolding of the enzyme in urea at 25 degrees C and pH 7.4 revealed a biphasic phenomenon with an intermediate state detected at 4-5 m urea. The enzyme activity was activated by urea up to 1 m but was completely lost before the intermediate state was detected. This suggests that the active site region of the enzyme was more sensitive to chemical denaturant than other structural scaffolds. In the presence of 4 mm Mn(2+), the urea denaturation pattern of malic enzyme changed to monophasic. Mn(2+) helped the enzyme to resist phase I urea denaturation. The [urea](0.5) for the enzyme inactivation shifted from 2.2 to 3.8 m. Molecular weight determined by the analytical ultracentrifuge indicated that the tetrameric enzyme was dissociated to dimers in the early stage of phase I denaturation. In the intermediate state at 4-5 m urea, the enzyme showed polymerization. However, the polymer forms were dissociated to unfolded monomers at a urea concentration greater than 6 m. Mn(2+) retarded the polymerization of the malic enzyme. Three mutants of the enzyme with a defective metal ligand (E234Q, D235N, E234Q/D235N) were cloned and purified to homogeneity. These mutant malic enzymes showed a biphasic urea denaturation pattern in the absence or presence of Mn(2+). These results indicate that the Mn(2+) has dual roles in the malic enzyme. The metal ion not only plays a catalytic role in stabilization of the reaction intermediate, enol-pyruvate, but also stabilizes the overall tetrameric protein architecture. Study holds ProTherm entries: 13012, 13013, 13014, 13015, 13016 Extra Details: biphasic phenomenon; intermediate state; tetrameric enzyme; metal ligand
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:44 p.m.
|Number of data points||5|
|Proteins||NADP-dependent malic enzyme ; NADP-dependent malic enzyme|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: Cm ; Experimental Assay: Tm|
|Libraries||Mutations for sequence KKGYEVLRDPHLNKGMAFTLEERQQLNIHGLLPPCFLGQDAQVYSILKNFERLTSDLDRYILLMSLQDRNEKLFYKVLTSDIERFMPIVYTPTVGLACQHYGLAFRRPRGLFITIHDRGHIATMLQSWPESVIKAIVVTDGERILGLGDLGCYGMGIPVGKLALYTACGGVKPHQCLPVMLDVGTDNETLLKDPLYIGLRHKRIRGQAYDDLLDEFMEAVTSRYGMNCLIQFEDFANANAFRLLHKYRNKYCTFNDDIQGTASVAVAGLLAALRITKNRLSDHTVLFQGAGEAALGIANLIVMAMQKEGVSKEEAIKRIWMVDSKGLIVKGRASLTPEKEHFAHEHCEMKNLEDIVKDIKPTVLIGVAAIGGAFTQQILQDMAAFNKRPIIFALSNPTSKAECTAEQLYKYTEGRGIFASGSPFDPVTLPSGQTLYPGQGNNSYVFPGVALGVISCGLKHIGDDVFLTTAEVIAQEVSEENLQEGRLYPPLVTIQQVSLKIAVRIAKEAYRNNTASTYPQPEDLEAFIRSQVYSTDYNCFVADSYTWPEEAMKVK|