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Metal‐Induced reversible structural interconversion of human mitochondrial NAD(P) + ‐Dependent malic enzyme
Author(s) -
Kuo ChuWei,
Hung HuiChih,
Tong Liang,
Chang GuGang
Publication year - 2004
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.10635
Subject(s) - malic enzyme , isomerization , chemistry , quenching (fluorescence) , enzyme , nad+ kinase , metal , stereochemistry , conformational change , reaction rate constant , crystallography , fluorescence , kinetics , biochemistry , dehydrogenase , organic chemistry , catalysis , physics , quantum mechanics
Human mitochondrial NAD(P) + ‐dependent malic enzyme was strongly inhibited by Lu 3+ . The X‐ray crystal structures indicated a structural change between the metal‐free and Lu 3+ ‐containing enzymes (Yang Z, Batra R, Floyd DL, Hung HC, Chang GG, Tong L. Biochem Biophys Res Commun 2000;274:440–444). We characterized the reversible slow‐binding mechanism and the structural interconversion between Mn 2+ ‐ and Lu 3+ ‐containing human mitochondrial malic enzymes. When Lu 3+ was added, the activity of the human enzyme showed a downward curve over time, similar to that of the pigeon enzyme. The rate of the transformation ( k obs ) from the initial rate to the steady‐state rate increased hyperbolically with the concentration of Lu 3+ , suggesting the involvement of an isomerization step. Lu 3+ had a much higher affinity for the isomerized form ( K * i,Lu (app) = 4.8 μ M ) than that of the native form ( K i,Lu (app) = 148 μ M ). When an excess of Mn 2+ was added to the Lu 3+ ‐inhibited enzyme, assays of the kinetic activity showed an upward trend, indicating reactivation. This result also indicated that the reactivation was a slow process. Fluorescence quenching experiments confirmed that the Lu 3+ ‐induced isomerization was completely reversible. The dynamic quenching constants for the metal‐free, Mn 2+ ‐containing, and Lu 3+ ‐containing enzyme were 3.08, 3.07, and 3.8 M −1 , respectively. When the Lu 3+ ‐containing enzyme was treated with excess Mn 2+ , the dynamic quenching constant returned to the original value (3.09 M −1 ). These results indicated that binding of Mn 2+ did not induce any conformational change in the enzyme. The open form transformed to the closed form only after substrate binding. Lu 3+ , on the other hand, transformed the open form into a catalytically inactive form. Excess Mn 2+ could replace Lu 3+ in the metal binding site and convert the inactive form back into the open form. This reversible process was slow in both directions because of the same but opposite structural change involved. Proteins 2004;54:000–000. © 2004 Wiley‐Liss, Inc.