Catalytic mechanism of S-ribosylhomocysteinase: ionization state of active-site residues.

S-Ribosylhomocysteinase (LuxS) catalyzes the cleavage of the thioether linkage in S-ribosylhomocysteine (SRH) to produce homocysteine (Hcys) and 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of type II bacterial autoinducer (AI-2). The proposed catalytic mechanism involves two consecutive ribose carbonyl migration steps via an intramolecular redox reaction and a subsequent beta-elimination step, all catalyzed by a divalent metal ion (e.g., Fe(2+) or Co(2+)) and two general acids/bases in the active site. Absorption and EPR spectroscopic studies were performed with both wild-type and various mutant forms of LuxS under a wide range of pH conditions. The studies revealed a pK(a) of 10.4 for the metal-bound water. The pK(a) value of Cys-83 was determined to be <6 by (13)C-(1)H HSQC NMR experiments with [3-(13)C]cysteine-labeled Zn(2+)-substituted Escherichia coli LuxS. The active form of LuxS contains a metal-bound water and a thiolate ion at Cys-83, consistent with the proposed roles of the metal ion (Lewis acid) and Cys-83 (general acid/base) during catalysis. Finally, an invariant Arg-39 in the active site was demonstrated to be at least partially responsible for stabilizing the thiolate anion of Cys-83.