title: Zinc ions bound to chimeric His4/lactate dehydrogenase facilitate decarboxylation of oxaloacetate.
abstract: A chemically synthesized DNA linker coding for a peptide fragment that contains four histidines was fused in-frame to the 5'-end of the Bacillus stearothermophilus lactate dehydrogenase gene. The gene product, His4/lactate dehydrogenase, could be purified to homogeneity using either immobilized metal (Zn2+)-affinity chromatography or affinity chromatography on oxamate agarose. The stability against heat and urea for the modified enzymes was decreased as compared to the native lactate dehydrogenase but could be increased if zinc ions were present during the denaturation. In the presence of zinc ions the His4/lactate dehydrogenase could catalyse the sequential reaction from oxaloacetate to L-lactate, hence operating as a semi-synthetic bifunctional enzyme. A small increase in the apparent second-order rate constant (kcat/Km) of the coupled reaction was observed as compared to a corresponding system with native lactate dehydrogenase.
authors.affiliation: Department of Pure and Applied Biochemistry, Chemical Center, Lund, Sweden.
authors.forename: H
authors.initials: H
authors.lastname: Carlsson
authors.forename: V
authors.initials: V
authors.lastname: Prachayasittikul
authors.forename: L
authors.initials: L
authors.lastname: Bülow
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D006639:Histidine; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D007773:Lactates / Q000096:biosynthesis; D019344:Lactic Acid; D008667:Metalloproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D010071:Oxaloacetates / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015032:Zinc
chemical_list: D007773:Lactates; D008667:Metalloproteins; D010071:Oxaloacetates; D010455:Peptides; D011993:Recombinant Fusion Proteins; D009243:NAD; C033223:polyhistidine; D019344:Lactic Acid; D006639:Histidine; D007770:L-Lactate Dehydrogenase; D015032:Zinc