Zinc ions bound to chimeric His4/lactate dehydrogenase facilitate decarboxylation of oxaloacetate

A chemically synthesized DNA linker coding for a peptide fragmentthat contains four histidines was fused in-frame to the 5'-endof the Bacillus stearothermophilus lactate dehydrogenase gene.The gene product, His₄/lactate dehydrogenase, could be purifiedto homogeneity using either immobilized metal (Zn²⁺)-affinitychromatography or affinity chromatography on oxamate agarose.The stability against heat and urea for the modified enzymeswas decreased as compared to the native lactate dehydrogenasebut could be increased if zinc ions were present during thedenaturation. In the presence of zinc ions the His₄/lactatedehydrogenase could catalyse the sequential reaction from oxaloacetateto L-lactate, hence operating as a semi-synthetic bifunctionalenzyme. A small increase in the apparent secondorder rate constant(k_cat/K_m) of the coupled reaction was observed as compared toa corresponding system with native lactate dehydrogenase.