Cysteine 351 is an essential nucleophile in catalysis by Porphyromonas gingivalis peptidylarginine deiminase.
Peptidylarginine deiminase (PAD), which catalyzes the deimination of the guanidino group from peptidylarginine residues, belongs to a superfamily of guanidino group modifying enzymes that have been shown to produce an S-alkylthiouronium ion intermediate during catalysis. Thiol-directed reagents iodoacetamide and iodoacetate inactivate recombinant PAD, and substrate protects the enzyme from inactivation. Activity measurements together with peptide mapping by mass spectrometry of PAD modified in the absence and presence of substrate demonstrated that cysteine-351 is modified by iodoacetamide. The pK(a) value of the cysteine residue, 7.7±0.2 as determined by iodoacetamide modification, agrees well with a critical pK value identified in pH rate studies. The role of cysteine-351 in catalysis was tested by site-directed mutagenesis in which the cysteine was replaced with serine to eliminate the proposed nucleophilic interaction. Binding studies carried out using fluorescence spectrometry established the structural integrity of the C351S PAD. However, the C351S PAD variant was catalytically inactive, exhibiting <0.01% wild-type activity. These results indicate that Cys 351 is a nucleophile that initiates the enzymatic reaction.
Rodríguez
Sofía B
SB
Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA 19140, United States.
Stitt
Barbara L
BL
Ash
David E
DE
D002384:Catalysis; D003545:Cysteine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D007460:Iodoacetamide / Q000737:chemistry; D019807:Iodoacetic Acid / Q000737:chemistry; D008024:Ligands; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D016966:Porphyromonas gingivalis / Q000201:enzymology*; D011485:Protein Binding; D000076342:Protein-Arginine Deiminases; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry
D008024:Ligands; D011994:Recombinant Proteins; D006867:Hydrolases; D000076342:Protein-Arginine Deiminases; D003545:Cysteine; D019807:Iodoacetic Acid; D007460:Iodoacetamide