Protein arginine deiminase 4: evidence for a reverse protonation mechanism

Abstract

The presumed role of an overactive protein arginine deiminase 4 (PAD4) in the pathophysiology of rheumatoid arthritis (RA) suggests that PAD4 inhibitors could be used to treat an underlying cause of RA, potentially offering a mechanism to stop further disease progression. Thus, the development of such inhibitors is of paramount importance. Toward the goal of developing such inhibitors, we initiated efforts to characterize the catalytic mechanism of PAD4 and thereby identify important mechanistic features that can be exploited for inhibitor development. Herein we report the results of mutagenesis studies as well as our efforts to characterize the initial steps of the PAD4 reaction, in particular, the protonation status of Cys645 and His471 prior to substrate binding. The results indicate that Cys645, the active site nucleophile, exists as the thiolate in the active form of the free enzyme. pH studies on PAD4 further suggest that this enzyme utilizes a reverse protonation mechanism.

Figure 1

Reaction catalyzed by PAD4.

Figure 2

(A) Active site of PAD4. (B) Working model of PAD4 catalysis. A possible mechanism of PAD4 catalysis involves a nucleophilic thiolate (Step 1) and His 471 acts as a general acid, donating a proton to the departing amine during the collapse of the first tetrahedral intermediate (Step 2). This leads to the formation of an S-alkyl thiouronium intermediate. The exchange of a molecule of water for ammonia occurs and, as drawn, His471 acts as a general base to activate a water molecule for nucleophilic attack on the thiouronium intermediate (Step 3). This leads to the formation of the second tetrahedral intermediate that collapses to eliminate the Cys thiolate and in the process generate Cit. Asp473 is appropriately positioned to deprotonate the hydroxyl. Step 4 involves the exchange of product for substrate.

Figure 3

(A) Plot of log k_cat/K_m versus pH. (B) Plot of log k_cat versus pH.

Figure 4

Time and concentration dependent inactivation of PAD4 by iodoacetamide. (A) observed inactivation at pH 7.6 by different concentrations of iodoacetamide: 0 (◇), 250 (▼), 500 (●), 750 (▲), 1000 (○), and 1500 μM (■). (B) The pseudo first order rate constant of PAD4 inactivation is plotted versus iodoacetamide concentration and plots were fitted to equation 5 as described in materials and methods. (C) The pK_a of C645. Second order rate constants were plotted versus pH and fit to equation 6 as described in materials and methods.

Figure 5

Inactivation of PAD4 with iodoacetamide. (A) Iodoacetamide is a better PAD4 inactivator than iodoacetic acid. Percent activity remaining was plotted versus time and fit to equation 4 as described in materials and methods. (B) Substrate protects against iodoacetamide-induced inactivation of PAD4. Plots of product formation versus time are depicted for PAD4 in the absence and presence of iodoacetamide (1.25 mM) at two different concentrations of BAEE (2 and 10 mM).

Figure 6

Time and concentration dependent inactivation of PAD4 by 2-chloroacetamidine. (A) Observed inactivation at pH 7.6 by different concentrations of 2-chloroacetamidine: 0 (◇), 250 (●), 500 (▲), 1000 (○), and 1500 μM (■). (B) The pseudo first order rate constant of PAD4 inactivation is plotted versus 2-chloroacetamidine concentration and plots were fitted to equation 5 as described in materials and methods. (C) The pKa of C645. Second order rate constants were plotted versus pH and fit to equation 6 as described in materials and methods. (D) Substrate protects against 2-chloroacetamidine-induced inactivation of PAD4. Plots of product formation versus time are depicted for PAD4 in the absence or presence of 2-chloroacetamidine (5 mM) at two different concentrations of PAD4: 10 mM BAEE (●), 2 mM BAEE (■), 10 mM BAEE with 2-chloroacetamidine (○), and 2 mM BAEE with 2-chloroacetamidine (□).

Figure 7

Solvent Isotope Effect (SIE). Plots of log k_cat/K_m versus pL in H₂O (●) or D₂O (○).

Figure 8

Fraction of enzyme in the active form (yellow) with C645 deprotonated and H471 protonated, i.e., E-S⁻H⁺.

Scheme 1

Binding of inactivator to either the E-SH (upper pathway) or E-S⁻ (lower pathway) complexes.