Interaction of the protein components of 5-oxoprolinase. Substrate-dependent enzyme complex formation.
5-Oxo-L-prolinase from Pseudomonas putida is composed of two reversibly dissociable proteins: Component A catalyzes 5-oxoproline-dependent cleavage of ATP, but does not catalyze the decyclization of 5-oxoproline; Component B is required for the coupling of ATP cleavage to ring-opening of 5-oxoproline to form glutamate (Seddon, A. P., Li, L., and Meister, A. (1984) J. Biol. Chem. 259, 8091-8094). We describe here the purifications of Components A and B to apparent homogeneity and the interactions between these two proteins. The cellular content of Component B activity is significantly greater than that of Component A. By gel filtration, Component A is a hexamer; but in the presence of substrates, it is a dimer. Component B can exist as an aggregate, an octamer, or a tetramer, depending upon the conditions used. Gel filtration of a mixture of Components A and B in the presence of substrates gives a unique protein species that exhibits 5-oxoprolinase activity. The Mr of this Component A-Component B complex indicates that it probably has an A2-B2 structure. The molar ratio of Component A to Component B in the complex was determined to be 1:1 by the continuous variation method (Job). Titrations of each component by the other suggest that phosphorylated 5-oxoproline-bound Component A is the entity that interacts with Component B. These studies indicate that the binding of phosphorylated 5-oxoproline-bound Component A to Component B to form a complex proceeds by a cooperative type mechanism. This is supported by the observed shifts of the intersection points of the Job curves (see Appendix).
Li
L Y
LY
Department of Biochemistry, Cornell University Medical College, New York, New York 10021.
Seddon
A P
AP
Meister
A
A
D000581:Amidohydrolases / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008962:Models, Theoretical; D008970:Molecular Weight; D011549:Pseudomonas / Q000201:enzymology*; D011752:Pyroglutamate Hydrolase / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D046911:Macromolecular Substances; D000581:Amidohydrolases; D011752:Pyroglutamate Hydrolase