Metabolic and mechanistic studies on threonine degradation in Escherichia coli via the L-threonine dehydrogenase-initiated pathway.

Abstract

The major route for the degradation of scL-threonine in both prokaryotes and eukaryotes is initiated by the enzymes, scL-threonine dehydrogenase and 2-amino-3-ketobutyrate (AKB) CoA lyase. Threonine dehydrogenase catalyzes the oxidation of scL-threonine to AKB with concomitant reduction of NAD$\sp+.$ The unstable $\beta$-keto acid formed in this reaction can then either undergo decarboxylation with release of aminoacetone or be utilized as a substrate (along with CoA) by the lyase to form glycine plus acetyl CoA. The first portion of this research focuses on the dynamics of the threonine dehydrogenase/AKB CoA lyase coupled reaction in the reverse direction (i.e. the formation of threonine from glycine (plus acetyl CoA and NADH)). In vitro and in vivo studies showed that AKB CoA lyase and threonine dehydrogenase are able to catalyze formation of threonine. The stability of the AKB intermediate was then examined. Half-life determinations showed a value that ranged from 8.6 minutes at pH 5.9 to 140 minutes at pH 11.1. Furthermore, the pH dependence of the decarboxylation rate constant predicted a pK$\sb{\rm a}$ of 8.15 for the $\alpha$-amino group of AKB. The relation of AKB to 2-amino-3-ketoadipate, which decarboxylates to 5-aminolevulinate, is also discussed along with homology exhibited between AKB CoA lyase and 5-aminolevulinate synthase. On the basis of assays performed during the half-life determinations of AKB, it was found that the lyase enzyme manifests threonine aldolase activity. This was proven by showing that a variety of purification and inactivation procedures affected both AKB CoA lyase and threonine aldolase activities identically. In mechanistic studies, scL-threonine dehydrogenase was found to be inactivated by the methylating agent, methyl p-nitrobenzenesulfonate, with concomitant incorporation of 1 methyl group per subunit. Substrate protection studies along with isolation and sequencing of the major radioactive peptide after inactivation with $\sp{14}$C-methyl p-nitrobenzenesulfonate showed that histidine residue-90 is at the active site of threonine dehydrogenase. Furthermore, examination and comparison with the primary and tertiary structures of LADH has allowed the tentative assignment of a hydrogen-bonding role for His-90 of TDH.