Threonine Deaminase of Clostridium tetanomorphum

Threonine deaminase (Lthreonine hydrolyase (deaminating), EC 4.2.1.16) has been purified approximately 700-fold from extracts of Clostridium tetanomorphum. Both threonine and serine can serve as substrates, but threonine is deaminated 5 to 10 times more rapidly than serine. Pyridoxal phosphate, a reducing agent, and alkaline pH are required for the deamination of either amino acid. A plot of the rate of reaction at increasing substrate concentration gives a sigmoidal curve. Addition of adenosine diphosphate stimulates the reaction markedly at low substrate concentrations and yields a curve approaching a rectangular hyperbola. Kinetic analysis using the empirical Hill equation indicates the existence of cooperative effects between substrate sites in the absence of ADP and little interaction, if any, in the presence of ADP. ADP may be replaced by guanosine and inosine diphosphates and less effectively by uridine and cytosine diphosphates; dATP and thymidine diphosphates have little stimulatory activity. Experiments with 14C-ADP show that the enzyme binds ADP. The enzyme is inhibited by pyridoxal phosphate binding agents, by sulfhydryl reagents, urea, detergents, and substrate analogues. ADP protects the enzyme against low concentrations of sulfhydryl inhibitors and hydrogen bondbreaking agents but not against other inhibitors. ADP (and the other nucleotides mentioned above), phosphate buffer at pH 7.0, divalent cations, and, to a lesser extent, monovalent cations, protect the enzyme against inactivation by heat and against loss of activity on dilution. Dialysis of enzyme preparation at an alkaline pH decreases the ability of the enzyme to bind r4C-ADP and reduces the catalytic response to ADP. Conversely, addition of agents that combine with pyridoxal phosphate inhibits catalysis but does not inhibit the binding of 14C-ADP. These differential effects suggest that the substrate and allosteric effector are bound to separate sites on the enzyme.