Abstract
To identify ionizations of the active site metal-bound water in horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase; EC 1.1.1.1), the pH, solvent isotope, temperature, and anion dependences of the steady-state kinetic parameters kcat and kcat/KM have been evaluated under initial velocity conditions for the native and the active site-specific Co(2+)-reconstituted enzyme. In the oxidation of benzyl alcohol, a bell-shaped pattern of four prototropic equilibria was observed under conditions of saturating concentrations of NAD+. It is shown that the ionizations governing kcat (pK1 congruent to 6.7, pK2 congruent to 10.6) belong to the ternary enzyme-NAD(+)-alcohol complex, whereas the ionizations governing kcat/KM (pK1' congruent to 7.5, pK2' congruent to 8.9) belong to the binary enzyme-NAD+ complex. The ionizations pK1 and pK1' are not influenced by metal substitution and are ascribed to His-51 on the basis of experimental estimates of their associated enthalpies of ionization. On the other hand, pK2 and pK2' are significantly decreased (delta pKa congruent to 1.0) in the Co(2+)-enzyme and are attributed to the active site metal-bound water molecule. The shape of the pH profiles requires that the metal ion coordinates a neutral water molecule in the ternary enzyme-NAD(+)-alcohol complex under physiological conditions. The possible catalytic role of the water molecule within a pentacoordinate metal ion complex in the active site is discussed.
Highlights
From the Department of Biochemistry and Molecular Biobgy, The University of Chicago, Cummings Life ScienceCenter, Chicago, Illinois 60637
On the basis of initial velocity data, we confirmed that TFEbehaves competentternary complex is ligatedby a neutral water as a linear, competitive inhibitor for both ZnLADH and CoLADH
11was observed for the oxidation of 2-propanol catalyzed by ZnLADH
Summary
To identify ionizations of tahcetive site metal-bound the chemical step of hydride transfer [7,8,9,10,11]. These molecular waterinhorse liver alcoholdehydrogenase(alcohol:NAD+ oxidoreductaseE;C 1.1.1.1) ,the pH, solvent events arecontrolled by ionizing groups for which assignment has remained conjectural despite numerous structural, chemisotope, temperature, and anion dependences of the ical, and kinetic studies. O n theotherhand,pK2andpK2‘ are significantly of the mechanism of action of LADH, such as assignment of decreased (ApK,, = 1.0) intheCo2+-enzymeandare ionizations of the metal-bound water or the alcohol molecule, attributed to the active site metal-bound water mole- of groups on the enzyme responsible for acid-base catalysis, cule. $Present address: Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, MA 02115
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