Effect of mutation of lysine-120, located at the entry to the active site of O-acetylserine sulfhydrylase-A from Salmonella typhimurium.
O-Acetylserine sulfhydrylase catalyzes the final step of the biosynthesis of L-cysteine, the replacement of the beta-acetoxy group of O-acetyl-L-serine (OAS) by a thiol. The enzyme undergoes a conformational change to close the site upon formation of the external Schiff base (ESB) with OAS. Mutation of K120 to Q was predicted to destabilize the closed form of the ESB and decrease the rate. The K120Q mutant enzyme was prepared and characterized by UV-visible absorbance, fluorescence, visible CD, and 31P NMR spectral studies, as well as steady state and pre-steady state kinetic studies. Spectra suggest a shift in the tautomeric equilibrium toward the neutral enolimine and an increase in the rate of interconversion of the open and closed forms of the enzyme. A decrease in the rate of both half reactions likely reflects the stabilization of the ESB as a result of the increased rate of equilibration of the open and closed forms of the enzyme along the reaction pathway. Data suggest a role of K120 in helping to stabilize the closed conformation by participating in a new hydrogen bond to the backbone carbonyl of A231.
Tai
Chia-Hui
CH
Department of Chemical and Materials Engineering, Chenshiu University, No. 840 Chengcing Rd., Kaohsiung, Taiwan, 83347.
Rabeh
Wael M
WM
Guan
Rong
R
Schnackerz
Klaus D
KD
Cook
Paul F
PF
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D003547:Cysteine Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012486:Salmonella typhimurium / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D001426:Bacterial Proteins; D003547:Cysteine Synthase; D008239:Lysine