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.
Zhu
Jinge
J
Department of Chemistry and Ohio State Biochemistry Program, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, USA.
Knottenbelt
Sushilla
S
Kirk
Martin L
ML
Pei
Dehua
D
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D013437:Carbon-Sulfur Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002712:Chlorides / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D011789:Quantum Theory; D013056:Spectrophotometry, Ultraviolet
D001426:Bacterial Proteins; D002712:Chlorides; D007477:Ions; D001120:Arginine; D013437:Carbon-Sulfur Lyases; C117753:LuxS protein, Bacteria; D003545:Cysteine