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 Cy s-38 of Escherichia coil threonine dehydrogenase, whose residue aligns with the catalytic zinc-binding residue, Cys-46, of related alcohol/p olyol dehydrogenases, inactivates the enzyme [B. R. Epperly and E. E. Dekker (1991) J. Biol. Chem. 266, 6086-6092; A. R. Johnson and E. E. D ekker (1996) Protein Sci. 5, 382-390]. To probe its function, Cys-38 w as changed to Ser, Asp, and Glu by site-directed mutagenesis. Mutants C38S and C38D were purified to homogeneity and found to be, like the w ild-type enzyme, homotetrameric proteins containing one Zn2+ atom per subunit. The circular dichroism spectra of these mutants were essentia lly identical to that of the wild-type enzyme. Mutant C38S was catalyt ically inactive but mutant C38D had a specific activity of 0.2 unit/mg , a level similar to 1% that of the wild-type enzyme. After it was inc ubated 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 uni t/mg). Preincubation of mutant C38D with 5 mM Zn2+, Co2+, Or Cd2+ incr eased 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 conc entrations, a maximum activity of 27 units/mg with 20 mM threonine, an d inhibition by high levels of substrate; an activation K-d = 3 mM Zn2 +; and a pH optimum of 8.4 (in contrast to pH 10.3 for the wild-type e nzyme). Without added Zn2+, mutant C38D is equally active with threoni ne and 2-amino-3-hydroxypentanoate, but Zn2+-activated mutant C38D is 10-fold more reactive with threonine than with 8-amino3-hydroxypentano ate, In the absence of added metal ions, wild-type enzyme similarly us es substrates other than threonine and shows a dramatic increase in ac tivity with only threonine when stimulated by either Cd2+ or Mn2+; add ed Zn2+ has no effect on activity with threonine. Cys-38 of threonine dehydrogenase, therefore, is located in an activating divalent metal i on-binding site. Having a negatively charged residue like Asp in this position allows the binding of a catalytic Zn2+ ion which enhances act ivity with threonine and reduces activity with substrate analogs. Whet her Cys-38 of wild-type threonine dehydrogenase binds a catalytic meta l ion (possibly Zn2+) in vivo remains to be established. (C) 1998 Acad emic Press.