A thioester substrate binds to the enzyme Arthrobacter thioesterase in two ionization states: evidence from Raman difference spectroscopy

4-Hydroxybenzoyl-CoA (4-HB-CoA) thioesterase from Arthrobacter is the final enzyme catalyzing the hydrolysis of 4-HB-CoA to produce coenzyme A and 4-hydroxybenzoic acid in the bacterial 4-chlorobenzoate dehalogenation pathway. Using a mutation E73A that blocks catalysis, stable complexes of the enzyme and its substrate can be analyzed by Raman difference spectroscopy. Here we have used Raman difference spectroscopy, in the non-resonance regime, to characterize 4-HB-CoA bound in the active site of the E73A thioesterase. In addition, we have characterized complexes of the wild-type enzyme complexed with the unreactive substrate analog 4-hydroxyphenacyl-CoA (4-HP-CoA). Both sets of complexes show evidence for two forms of the ligand in the active site: one population has the 4-hydroxy group protonated, 4-OH; while the second has the group as the hydroxide, 4-O-. For bound 4-HP-CoA, X-ray data show that glutamate 78 is close to the 4-OH in the complex and it is likely that this is the proton acceptor for the 4-OH proton. Although the pKa of the 4-OH group on the free substrate in aqueous solution is 8.6, the relative populations of ionized and neutral 4-HB-CoA bound to E73A remain invariant between pH 7.3 and 9.8. The invariance with pH suggests that the 4-OH and the -COO- of E78 constitute a tightly coupled pair where their separate pKa s lose their individual qualities. Narrow band profiles are seen in the C?O double bond and C-S regions, suggesting that the hydrolyzable thioester group is rigidly bound in the active site in a syn gauche conformation. Copyright (c) 2011 John Wiley & Sons, Ltd.