Abstract
5-Oxoprolinase catalyzes the ATP-dependent decyclization of 5-oxo-L-proline to L-glutamate. Previous studies provided evidence for the intermediate formation of a phosphorylated form of 5-oxoproline (Seddon, A. P., and Meister, A. (1986) J. Biol. Chem. 261, 11538-11541) and of a tetrahedral intermediate (Li, L., Seddon, A. P., and Meister, A. (1987) J. Biol. Chem. 262, 11020-11025). A new approach to the study of the reaction mechanism using the 18O isotope effect on the 13C NMR signals for 5-oxoproline and glutamate is reported here. The 13C chemical shifts induced by 18O substitution for the carbonyl group of 5-oxoproline and the gamma-carboxyl group of glutamate are about 0.03 ppm with respect to the corresponding 16O-compounds. Using 5-[18O]oxo[5-13C]proline (97 and 79.5 atom % excess, 13C and 18O, respectively), the disappearance of the 18O-labeled and unlabeled 5-oxoproline and formation of the corresponding glutamate species were followed in the reactions catalyzed by purified preparations of 5-oxoprolinase isolated from Pseudomonas putida and from rat kidney. This procedure permits simultaneous determinations of the rates of 18O exchange and of the overall decyclization reaction. The ratios of 18O exchange rates to the overall reaction rates for the bacterial and kidney enzyme catalyzed-reactions were 0.28 and 0.14, respectively. The findings support the view that the coupling of ATP hydrolysis to 5-oxoproline decyclization involves formation of a phosphorylated tetrahedal intermediate. Although the exchange phenomena are consistent with the mechanistic interpretations, they seem not to be required for catalysis.
Highlights
Previous studies [4] showed that, in the reactioncatalyzed by the bacterial enzyme complex, "0 from Hk80is incorporated into the amide carbonyl oxygen of the residual 5-oxoproline
Consistent with previous observations [4], the amide carbonyl oxygen of 5-oxoproline and the y-carboxyl oxygen atoms of glutamate were shown to undergo replacement by oxygen atoms derived from water
The reaction is initiated by phosphorylation of the carbonyl oxygen atom of 5-oxoproline, followedby formation of the tetrahedal intermediate
Summary
The 13Cchemical shifts induced by "0 formation of a phosphorylated form of 5-oxoproline [3] This substitution for the carbonyl gorfo5u-poxoproline and the y-carboxyl groupof glutamate are about 0.03 ppm with respect to the correspondin"g0-compounds. ComponentB interthe "0-labeled and unlabeled 5-oxoproline and for- acts with ComponentA in a substrate-dependent manner to mation of the corresponding glutamate species were followed in the reactions catalyzedby purified preparations of 5-oxoprolinase isolated from Pseudomonas putida and from rat kidney. This procedure permits simultaneous determinations of the rates of "0 exchange and of the overall decyclization reaction. Thesefindings led totheproposal of a mechanismthat involves phosphorylation of the amide carbonyloxygen of 5oxoproline [3] and formation of a tetrahedral intermediate [4].Ring opening of the latter gives y-glutamyl phosphate, H which undergoes hydrolysis.Randomization of the phosphoryl
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