Scheme 2.

The two possible regiochemistries for hydrolytic cleavage of the proposed anhydride intermediate formed in the thioesterase-catalyzed hydrolysis of 4-HB-CoA carried-out in oxygen-18 enriched water under single turnover conditions. In “pathway a” the Thr77 directs the water nucleophile to attack the Glu73 carbonyl carbon and therefore the 4-hydroxybenzoate product is not labeled with oxygen-18. In “pathway b” the Thr77 directs the water nucleophile to attack the 4-hydroxybenzoyl carbonyl carbon and therefore the 4-hydroxybenzoate product is labeled with oxygen-18.