CATALYTIC COMPETENCE OF O-ACETYLSERINE SULFHYDRYLASE IN THE CRYSTAL PROBED BY POLARIZED ABSORPTION MICROSPECTROPHOTOMETRY

The reactions of the pyridoxal 5'-phosphate-dependent enzyme O-acetyl- serine sulfhydrylase with the substrate O-acetyl-L-serine and substrat e analogs have been investigated in the crystalline state by single-cr ystal polarized absorption microspectrophotometry. This approach has a llowed us to examine the catalytic competence of the enzyme in differe nt crystalline states, one of which was used to determine the three-di mensional structure; experimental conditions were defined for the accu mulation of catalytic intermediates in the crystal suitable for crysta llographic analyses. O-Acetyl-L-serine reacts with the enzyme in one o f the crystal forms leading via a beta-elimination reaction to the acc umulation of the alpha-aminoacrylate Schiff base, absorbing maximally at 320 and 470 nm, as in solution. The dissociation constant for the a lpha-aminoacrylate Schiff base is in the millimolar range, 500-fold hi gher than in solution, suggesting that crystal lattice interactions ma y oppose functionally relevant conformational changes. The dissociatio n constant exhibits a bell-shaped dependence on pH centered at pH 7. A t this pH the alpha-aminoacrylate species slowly decays with time (30% decrease in 24 hours). The alpha-aminoacrylate intermediate readily r eacts with sodium azide, an analog of sulfide, the natural nucleophili c agent, to give a new amino acid and the native enzyme, indicating th at the crystalline enzyme catalyzes the overall beta-replacement react ion as in solution. In other crystal forms, including that used for th e X-ray investigation, O-acetyl-L-serine either has an even higher dis sociation constant or causes crystal damage upon binding. When the cry stalline enzyme reacts with either L-cysteine or L-serine, the externa l aldimine intermediate is formed. The dissociation constants for both substrate analogs are closer to those observed in solution and are mo dulated 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 alpha-aminoacrylate intermediate makes feasible their structural dete rmination and, therefore, the elucidation of the catalytic pathway at the molecular level. (C) 1998 Academic Press.