Interaction of 5-oxo-L-prolinase with nucleoside triphosphates. Evidence suggesting substrate-dependent conformational change.

5-Oxoprolinase catalyzes the coupled hydrolysis of ATP and 5-oxoproline to yield glutamate, ADP, and Pi; the reaction may be partially or completely uncoupled by structural modification of either substrate. In the present work, we found slow 5-oxoproline-dependent changes in the rates of hydrolysis of ITP, GTP, and UTP. For example, in the absence of 5-oxoproline, the enzyme catalyzes the hydrolysis of UTP at a rapid and constant rate. Following addition of 5-oxo-L-proline, the rate of hydrolysis decreases slowly; after about 25 min, a much slower and constant rate of hydrolysis is attained. This change in rate is associated with a decrease in Vmax and an increase in the Km for UTP. In similar studies with ATP, both Vmax and Km increase over a much shorter time period (less than 10 s). The findings indicate that 5-oxoprolinase is a hysteretic enzyme, and are consistent with the hypothesis that in the normal catalytic reaction, the binding of both ATP and 5-oxo-proline to the enzyme induces a conformational change that brings the substrates into a juxtaposition that facilitates the reaction.