Cysteine 351 is an essential nucleophile in catalysis by Porphyromonas gingivalis peptidylarginine deiminase

Abstract

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 pKa 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.

Defining the role ofPorphyromonas gingivalispeptidylarginine deiminase (PPAD) in rheumatoid arthritis through the study of PPAD biology

“…Indeed, PPAD citrullination may be biologically unfavourable for P gingivalis . Rodríguez et al 30 reported that PPAD activity declines with enzyme autocitrullination, and citrullination of R352 adjacent to the active site of PPAD (C351) has been hypothesised to explain this reduction in enzyme activity 12 30 36. Mechanisms that block PPAD autocitrullination in P gingivalis may therefore conserve PPAD activity.…”

“…Enzymatically inactive PPAD was generated by site-directed mutagenesis to replace cysteine 351 at the active site of the protein with serine (PPAD C351S ) 36. N-terminal truncated PPAD (rPPAD Ntx ) was generated by amplifying the coding sequence for amino acids 44 to 556 of full-length PPAD with primers containing a 5′ Kozak sequence.…”

Periodontitis, Porphyromonas, and the pathogenesis of rheumatoid arthritis

“…20 It has been proposed that P. gingivalis may have an indirect role in promoting human PAD activity by increasing intracellular calcium concentrations through induction of defensive enzymatic activity. 64,65…”

Purification of enzymatically inactive peptidylarginine deiminase type 6 from mouse ovary that reveals hexameric structure different from other dimeric isoforms

“…Cys 645 near the C-terminal is necessary for catalysis. However, only one PAD occurs in prokaryotes [10]; the Porphyromonas gingivalis PAD also carries a reactive Cys residue that acts as a nucleophile essential for catalysis despite no requirement of Ca 2+ [11,12].…”