Metal-Induced reversible structural interconversion of XXXX mitochondrial NAD(P)+-dependent malic enzyme.

@entity1 @entity3281 @entity70989
@entity1 mitochondrial NAD(P)+-dependent malic enzyme was strongly inhibited by @entity70989 . The X-ray crystal structures indicated a structural change between the metal-free and @entity70989 -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 @entity3281 - and @entity70989 -containing @entity1 mitochondrial malic enzymes. When @entity70989 was added, the activity of the @entity1 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 @entity70989 , suggesting the involvement of an isomerization step. @entity70989 had a much higher affinity for the isomerized form (K*(i,Lu (app)) = 4.8 microM) than that of the native form (K(i,Lu (app)) = 148 microM). When an excess of @entity3281 was added to the @entity70989 -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 @entity70989 -induced isomerization was completely reversible. The dynamic quenching constants for the metal-free, @entity3281 -containing, and @entity70989 -containing enzyme were 3.08, 3.07, and 3.8 M(-1), respectively. When the @entity70989 -containing enzyme was treated with excess @entity3281 , the dynamic quenching constant returned to the original value (3.09 M(-1)). These results indicated that binding of @entity3281 did not induce any conformational change in the enzyme. The open form transformed to the closed form only after substrate binding. @entity70989 , on the other hand, transformed the open form into a catalytically inactive form. Excess @entity3281 could replace @entity70989 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.
