Catalytic competence of O-acetylserine sulfhydrylase in the crystal probed by polarized absorption microspectrophotometry

Abstract

The reactions of the pyridoxal 5′-phosphate-dependent enzyme O-acetylserine sulfhydrylase with the substrate O-acetyl- l-serine and substrate analogs have been investigated in the crystalline state by single-crystal polarized absorption microspectrophotometry. This approach has allowed us to examine the catalytic competence of the enzyme in different crystalline states, one of which was used to determine the three-dimensional structure; experimental conditions were defined for the accumulation of catalytic intermediates in the crystal suitable for crystallographic analyses. O-Acetyl- l-serine reacts with the enzyme in one of the crystal forms leading via a β-elimination reaction to the accumulation of the α-aminoacrylate Schiff base, absorbing maximally at 320 and 470 nm, as in solution. The dissociation constant for the α-aminoacrylate Schiff base is in the millimolar range, 500-fold higher than in solution, suggesting that crystal lattice interactions may oppose functionally relevant conformational changes. The dissociation constant exhibits a bell-shaped dependence on pH centered at pH 7. At this pH the α-aminoacrylate species slowly decays with time (30% decrease in 24 hours). The α-aminoacrylate intermediate readily reacts with sodium azide, an analog of sulfide, the natural nucleophilic agent, to give a new amino acid and the native enzyme, indicating that the crystalline enzyme catalyzes the overall β-replacement reaction as in solution. In other crystal forms, including that used for the X-ray investigation, O-acetyl- l-serine either has an even higher dissociation constant or causes crystal damage upon binding. When the crystalline enzyme reacts with either l-cysteine or l-serine, the external aldimine intermediate is formed. The dissociation constants for both substrate analogs are closer to those observed in solution and are modulated by pH as in solution. Findings demonstrate that O-acetylserine sulfhydrylase is catalytically competent in the crystal although some regions of the molecule, likely involved in an open-closed transition induced by O-acetyl- l-serine binding, may have a limited flexibility. The accumulation in the crystal of both the external aldimine and the α-aminoacrylate intermediate makes feasible their structural determination and, therefore, the elucidation of the catalytic pathway at the molecular level.