On the mechanism of activation of L-threonine deaminase from Clostridium tetanomorphum by adenosine diphosphate.

In order to clarify the mechanism of activation of L-threonine deaminase by adenosine diphosphate, the enzyme was purified about 700.fold from sonic extracts of Closlridium tetanomorphum, and kinetic and ADP-binding studies were carried out. Plots of reaction rates against L-threonine concentrations gave a sigmoid curve in the absence of ADP, whereas a hyperbolic curve was obtained in the presence of low3 M ADP. Double reciprocal plots were parabolic in the former and linear in the latter case. Different pH profiles of the apparent k=, and maximal velocity were observed with and without ADP. A linear Arrhenius plot was obtained in the absence of ADP, whereas a discontinuity in the slope was found in the presence of ADP. Values of the activation energy were calculated to be 13.9 and 11.4 kcal per mole over the temperature range of 4-37’ in the absence and presence of ADP, respectively. The Michaelis constant for ADP as an activator was 2.3 X 10-K M at a threonine concentration of 10B3 M. No mutual effect of ADP and threonine on K,,, and K, was observed. Adenosine triphosphate offset activation of the enzyme by ADP, and increased the sigmoid nature of the rate-substrate concentration curve. D-Threonine and semicarbazine inhibited the deaminase activity competitively toward L-threonine both in the presence and in the absence of ADP. p-Chloromercuribenzoate inhibited the enzyme competitively toward L-threonine in the absence of ADP, but noncompetitively in the presence of ADP. The dissociation constant for ADP, estimated by equilibrium dialysis, was 3.0 x 10v6 M, which agreed reasonably well with the value obtained from reaction kinetics. DThreonine did not interfere with the binding of ADP. On the basis of kinetic analysis, two substrate sites are postulated, a catalytic site with a dissociation constant for substrate (KS) of 3.5 x low3 M, and an activating site with a K, of 3.3 x 1O-2 M.