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Identification of an Acyl-Enzyme Intermediate in a meta-Cleavage Product Hydrolase Reveals the Versatility of the Catalytic Triad

Ruzzini, Antonio C., Ghosh, Subhangi, Horsman, Geoff P., Foster, Leonard J., Bolin, Jeffrey T., Eltis, Lindsay D.
Journal of the American Chemical Society 2012 v.134 no.10 pp. 4615-4624
acylation, biphenyl, crystal structure, histidine, hydrogen bonding, hydrolysis, mass spectrometry, oxyanions, oxygen, serine, serine proteinases, stable isotopes
Meta-cleavage product (MCP) hydrolases are members of the α/β-hydrolase superfamily that utilize a Ser-His-Asp triad to catalyze the hydrolysis of a C–C bond. BphD, the MCP hydrolase from the biphenyl degradation pathway, hydrolyzes 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to 2-hydroxypenta-2,4-dienoic acid (HPD) and benzoate. A 1.6 Å resolution crystal structure of BphD H265Q incubated with HOPDA revealed that the enzyme’s catalytic serine was benzoylated. The acyl-enzyme is stabilized by hydrogen bonding from the amide backbone of ‘oxyanion hole’ residues, consistent with formation of a tetrahedral oxyanion during nucleophilic attack by Ser112. Chemical quench and mass spectrometry studies substantiated the formation and decay of a Ser112-benzoyl species in wild-type BphD on a time scale consistent with turnover and incorporation of a single equivalent of ¹⁸O into the benzoate produced during hydrolysis in H₂¹⁸O. Rapid-scanning kinetic studies indicated that the catalytic histidine contributes to the rate of acylation by only an order of magnitude, but affects the rate of deacylation by over 5 orders of magnitude. The orange-colored catalytic intermediate, ESʳᵉᵈ, previously detected in the wild-type enzyme and proposed herein to be a carbanion, was not observed during hydrolysis by H265Q. In the newly proposed mechanism, the carbanion abstracts a proton from Ser112, thereby completing tautomerization and generating a serinate for nucleophilic attack on the C6-carbonyl. Finally, quantification of an observed pre-steady-state kinetic burst suggests that BphD is a half-site reactive enzyme. While the updated catalytic mechanism shares features with the serine proteases, MCP hydrolase-specific chemistry highlights the versatility of the Ser-His-Asp triad.