Investigation of active site residues and sequence homology in Escherichia coli L-threonine dehydrogenase.

Abstract

This thesis extends our previous knowledge of the structure and function of E. coli L-threonine dehydrogenase by identifying active-site residues, confirming the amino acid sequence derived from the gene sequence, establishing the existence of homology between this enzyme and alcohol dehydrogenases such as LADH, and investigating the occurrence of a 2 disulfide/subunit form of the enzyme. Manual sequencing of two peptides of threonine dehydrogenase confirmed that the nucleotide sequence-derived amino acid sequence of the molecule was accurate. Further, the presence of an Asp-Pro bond, predicted by the amino acid sequence, was demonstrated. Computer searches of amino acid sequence data-banks establish threonine as a member of the long-chain zinc-containing alcohol/polyol dehydrogenase family of enzymes. Selective chemical modification with 2,3-butanedione was used in order to investigate the possible role of the arginine residues in the enzyme. One arginine/subunit was found to be important in catalytic activity; its modification yielded inactive protein. Substrate protection studies suggest that this residue is involved in the binding of the cofactor, NAD$\sp+$, by the enzyme. The one active-site cysteine residue, previously described to be sensitive to selective modification by iodoacetic acid, has been located within the amino acid sequence of the molecule. Manual sequencing of the $\sp{14}$C-carboxymethylated peptide identified Cys-38 as the active-site cysteine residue. The presence of tightly bound zinc (1 mol Zn$\sp{2+}$/mol subunit) in the native enzyme is reported for the first time. The enzyme is inactivated when this Zn$\sp{2+}$ is removed, suggesting it is essential for enzyme catalytic activity. Homology of E. coli threonine dehydrogenase to LADH suggests that Cys-38 is likely one of the ligands to this zinc ion. When stored in Tris$\cdot$HCl buffer containing 5 mM 2-mercaptoethanol, threonine dehydrogenase slowly oxidizes to form a 2 disulfide/subunit form of the enzyme; this form may also be made by incubation with the disulfide compound, 2-hydroxyethyldisulfide. Both the 1 and 2 disulfide forms of threonine dehydrogenase are equally active. An unsuccessful attempt was made to identify the disulfide-forming residues within the sequence of the molecule, after selectively labeling them with $\sp{14}$C-iodoacetic acid.