Metal binding effect in structure stability of pigeon liver malic enzyme
Department of Biochemistry, National Defense Medical Center
Pigeon liver malic enzyme ((S)-malate:NADP+ oxidoreductase (oxaloactate-decarboxylating), EC 1. 1. 1. 40) is a homotetrameric enzyme whose catalytic process is divalent metal ion-dependent. Unfolding of malic enzyme by urea monitored by both circular dichroism and fluorescence revealed biphasic conformational changes at 25 0C and pH 7.4. Enzyme activity was activated by low urea concentration then completely lost before intermediate was formed, suggesting that the active site region was more sensitive to chemical denaturant than other structure domains. After malic enzyme was treated with excess amount Mn2+, the urea denaturation pattern changed to monophasic. The resistance to urea denaturation was elevated in phase I with the same result in enzyme activity. Molecular weight determinated by analytical ultracentrifuge confirmed that the tetramer had dissociated to dimer in the pre-state of phase I, the intermediate state showed polymer and monomer in high concentrations of urea. However malic enzyme was preincubated with Mn2+ the polymer was never found. Three metal ligand mutants (E234Q, D235N, E234QD235N) were preincubated with or without Mn2+ showed biphasic urea denaturation pattern. Secondary structure percentages caculated from CD spectra further indicated that wild-type malic enzyme was more resistant to changes in b-sheet when preincubated with Mn2+, but this effect was not observed in the mutants. Therefore, Mn2+ binding to the active site can prevent phase I urea denaturation and this may be by b-sheet stabilization. The above results indicate that the Mn2+ is not only a catalytic cofactor but also stabilizes protein structure in chemical denaturation.