L-threonine dehydrogenase from Escherichia coli. Identification of an active site cysteine residue and metal ion studies.
Pure L-threonine dehydrogenase from Escherichia coli is a tetrameric protein (Mr = 148,000) with 6 half-cystine residues/subunit; its catalytic activity as isolated is stimulated 5-10-fold by added Mn2+ or Cd2+. The peptide containing the 1 cysteine/subunit which reacts selectively with iodoacetate, causing complete loss of enzymatic activity, has been isolated and sequenced; this cysteine residue occupies position 38. Neutron activation and atomic absorption analyses of threonine dehydrogenase as isolated in homogeneous form now show that it contains 1 mol of Zn2+/mol of enzyme subunit. Removal of the Zn2+ with 1,10-phenanthroline demonstrates a good correlation between the remaining enzymatic activity and the zinc content. Complete removal of the Zn2+ yields an unstable protein, but the native metal ion can be exchanged by either 65Zn2+, Co2+, or Cd2+ with no change in specific catalytic activity. Mn2+ added to and incubated with the native enzyme, the 65Zn2(+)-, the Co2(+)-, or the Cd2(+)- substituted form of the enzyme stimulates dehydrogenase activity to the same extent. These studies along with previously observed structural homologies further establish threonine dehydrogenase of E. coli as a member of the zinc-containing long chain alcohol/polyol dehydrogenases; it is unique among these enzymes in that its activity is stimulated by Mn2+ or Cd2+.
Epperly
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Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606.
Dekker
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D000429:Alcohol Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002412:Cations; D002614:Chelating Agents; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D012689:Sequence Homology, Nucleic Acid; D013054:Spectrophotometry, Atomic
D000596:Amino Acids; D002412:Cations; D002614:Chelating Agents; D008670:Metals; D000429:Alcohol Oxidoreductases; C023159:L-threonine 3-dehydrogenase; D003545:Cysteine