L-threonine Dehydrogenase From Escherichia Coli K-12 Thiol-dependent Activation By Manganese Cation And The Role Of Sulfhydryl Groups In Structure And Function.

Abstract

In previous research efforts, our laboratory succeeded in purifying threonine dehydrogenase (TDH) to homogeneity from a mutant strain of E. coli K-12 (forced to grow on L- threonine as sole carbon source) that has constitutively derepressed levels of the enzyme. By adding 1 mM Mn('2+) to all buffers used in the final fractionation step that provides pure TDH, yield of enzyme was increased (TURNEQ)4-fold 30 to 40 mg of pure TDH are obtained from 100 gm (wet weight) of cells . Threonine dehydrogenase has been determined to be a metal ion-activated enzyme. While TDH is activated (TURNEQ)10-fold by added Mn('2+) with an activation K(,d) of 9.0 (mu)M, the enzyme also has a basal, metal iton-independent level of activity = 20 U/mg. The stoichiometry of binding is 0.86 g-atoms Mn('2+)/TDH subunit with K(,d) = 8.5 (mu)M. A unique role for sulfhydryl groups in Mn('2+)-stimulated TDH activity is suggested by the findings that (a) stimulation of activity by Mn('2+) is dependent on an added thiol; (b) Mn('2+) stimulation is pH-dependent with pK(,a) = 7.95 (a value consistent with that for a thiolate anion); and (c) the carboxymethylation of one equivalent of cysteine/TDH subunit abolishes both the ability of TDH to bind Mn('2+) and the Mn('2+)-stimulatory effect on TDH activity. TDH has been found to contain one disulfide and four thiol groups per subunit. Incubation of TDH with iodoacetic acid (IAA) under non-denaturing conditions results in the carboxymethylation of only 1.3 thiol groups per TDH subunit; the rate of TDH inactivation by IAA is identical with the rate of carboxymethylation of one critical equivalent of cysteine per enzyme subunit. Both L-threonine and NADH provide partial protection against inactivation of TDH by IAA whereas the addition of 10 (mu)M Cd('2+) causes a 4-fold increase in the rate of TDH inactivation by IAA. The results obtained establish a thiol-dependent interaction of 1 g-atom of Mn('2+)/TDH subunit involving a single ionizable group on the protein. The inverse correlation between Mn('2+) binding and the modification of one critical equivalent of cysteine per TDH subunit suggests an interaction between Mn('2+) and a sulfhydryl group on the enzyme.