The Catalytic Serine of meta-Cleavage Product Hydrolases Is Activated Differently for C–O Bond Cleavage Than for C–C Bond Cleavage

meta-Cleavage product (MCP) hydrolases catalyze C–C bond fission in the aerobic catabolism of aromatic compounds by bacteria. These enzymes utilize a Ser-His-Asp triad to catalyze hydrolysis via an acyl–enzyme intermediate. BphD, which catalyzes the hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) in biphenyl degradation, catalyzed the hydrolysis of an ester analogue, p-nitrophenyl benzoate (pNPB), with a k_cat value (6.3 ± 0.5 s^–1) similar to that of HOPDA (6.5 ± 0.5 s^–1). Consistent with the breakdown of a shared intermediate, product analyses revealed that BphD catalyzed the methanolysis of both HOPDA and pNPB, partitioning the products to benzoic acid and methyl benzoate in similar ratios. Turnover of HOPDA was accelerated up to 4-fold in the presence of short, primary alcohols (methanol > ethanol > n-propanol), suggesting that deacylation is rate-limiting during catalysis. In the steady-state hydrolysis of HOPDA, k_cat/K_m values were independent of methanol concentration, while both k_cat and K_m values increased with methanol concentration. This result was consistent with a simple model of nucleophilic catalysis. Although the enzyme could not be saturated with pNPB at methanol concentrations of >250 mM, k_obs values from the steady-state turnover of pNPB at low methanol concentrations were also consistent with a nucleophilic mechanism of catalysis. Finally, transient-state kinetic analysis of pNPB hydrolysis by BphD variants established that substitution of the catalytic His reduced the rate of acylation by more than 3 orders of magnitude. This suggests that for pNPB hydrolysis, the serine nucleophile is activated by the His-Asp dyad. In contrast, rapid acylation of the H265Q variant during C–C bond cleavage suggests that the serinate forms via a substrate-assisted mechanism. Overall, the data indicate that ester hydrolysis proceeds via the same acyl–enzyme intermediate as that of the physiological substrate but that the serine nucleophile is activated via a different mechanism.