Investigation of a catalytic zinc binding site in Escherichia coli L-threonine dehydrogenase by site-directed mutagenesis of cysteine-38.

L-Threonine dehydrogenase catalyzes the NAD+-dependent oxidation of threonine forming 2-amino-3-ketobutyrate. Chemical modification of Cys-38 of Escherichia coli threonine dehydrogenase, whose residue aligns with the catalytic zinc-binding residue, Cys-46, of related alcohol/polyol dehydrogenases, inactivates the enzyme [B. R. Epperly and E. E. Dekker (1991) J. Biol. Chem. 266, 6086-6092; A. R. Johnson and E. E. Dekker (1996) Protein Sci., 382-390]. To probe its function, Cys-38 was changed to Ser, Asp, and Glu by site-directed mutagenesis. Mutants C38S and C38D were purified to homogeneity and found to be, like the wild-type enzyme, homotetrameric proteins containing one Zn2+ atom per subunit. The circular dichroism spectra of these mutants were essentially identical to that of the wild-type enzyme. Mutant C38S was catalytically inactive but mutant C38D had a specific activity of 0.2 unit/mg, a level approximately 1% that of the wild-type enzyme. After it was incubated with 1 mM Zn2+ and then assayed in the presence of 15 mM Zn2+, mutant C38S showed only a trace of enzymatic activity (i.e., 0.013 unit/mg). Preincubation of mutant C38D with 5 mM Zn2+, Co2+, or Cd2+ increased its activity 57-, 6-, or 3-fold, respectively; 1 mM Mn2+ halved and 0.5 mM Hg2+ abolished activity. Zn2+-stimulated mutant C38D showed these properties: apparent substrate activation at low threonine concentrations, a maximum activity of 27 units/mg with 20 mM threonine, and inhibition by high levels of substrate; an activation Kd = 3 mM Zn2+; and a pH optimum of 8.4 (in contrast to pH 10.3 for the wild-type enzyme). Without added Zn2+, mutant C38D is equally active with threonine and 2-amino-3-hydroxypentanoate, but Zn2+-activated mutant C38D is 10-fold more reactive with threonine than with 2-amino-3-hydroxypentanoate. In the absence of added metal ions, wild-type enzyme similarly uses substrates other than threonine and shows a dramatic increase in activity with only threonine when stimulated by either Cd2+ or Mn2+; added Zn2+ has no effect on activity with threonine. Cys-38 of threonine dehydrogenase, therefore, is located in an activating divalent metal ion-binding site. Having a negatively charged residue like Asp in this position allows the binding of a catalytic Zn2+ ion which enhances activity with threonine and reduces activity with substrate analogs. Whether Cys-38 of wild-type threonine dehydrogenase binds a catalytic metal ion (possibly Zn2+) in vivo remains to be established.