Deconstructing the Catalytic Efficiency of Peroxiredoxin-5 Peroxidatic Cysteine

Abstract

Human peroxiredoxin-5 (PRDX5) is a thiol peroxidase that reduces H2O2 10(5) times faster than free cysteine. To assess the influence of two conserved residues on the reactivity of the critical cysteine (C47), we determined the reaction rate constants of PRDX5, wild type (WT), T44V and R127Q with one substrate electrophile (H2O2) and a nonspecific electrophile (monobromobimane). We also studied the corresponding reactions of low molecular weight (LMW) thiolates in order to construct a framework against which we could compare our proteins. To obtain a detailed analysis of the structural and energetic changes involved in the reaction between WT PRDX5 and H2O2, we performed ONIOM quantum mechanics/molecular mechanics (QM/MM) calculations with a QM region including 60 atoms of substrate and active site described by the B3LYP density functional and the 6-31+G(d,p) basis set; the rest of the protein was included in the MM region. Brønsted correlations reveal that the absence of T44 can increase the general nucleophilicity of the C47 but decreases the specific reactivity toward H2O2 by a factor of 10(3). The R127Q mutation causes C47 to behave like a LMW thiolate in the two studied reactions. QM/MM results with WT PRDX5 showed that hydrogen bonds in the active site are the cornerstone of two effects that make catalysis possible: the enhancement of thiolate nucleophilicity upon substrate ingress and the stabilization of the transition state. In both effects, T44 has a central role. These effects occur in a precise temporal sequence that ensures that the selective nucleophilicity of C47 is available only for peroxide substrates.

The thiol of human serum albumin: Acidity, microenvironment and mechanistic insights on its oxidation to sulfenic acid

“…5 and Table 2 contrasts with our previous findings [20] for the analogous reaction of a cysteine with H2O2 embedded in a different protein environment, that of human peroxiredoxin 5 (hPrx5), where a sulfenic acid derivative is formed instead as stable product, after a second post-TS proton transfer occurs from a highly conserved protonated Arg residue present in such active-site [20].…”

“…Both thiol environments lack a positively charged entity proximal to stabilize the anionic TS by electrostatic interaction, a crucial factor largely recognized to enhance Cys nucleophilicity in different protein environments, in particular in more reactive ones present in other mammalian albumins [34] and peroxiredoxins [19,20,22,67]. Thus, enthalpy barriers are expected to be significantly higher for HSA−SH and free Cys in solution [66] than for peroxiredoxin active sites (10 3 -10 7 times faster than free cysteine) where wellconserved protonated Arg residues are proximal to the peroxidatic Cys.…”

“…Thus, enthalpy barriers are expected to be significantly higher for HSA−SH and free Cys in solution [66] than for peroxiredoxin active sites (10 3 -10 7 times faster than free cysteine) where wellconserved protonated Arg residues are proximal to the peroxidatic Cys. Details from calculated and experimental thermochemistry corresponding to hPrx5 [20] and MtAhpE [19] peroxiredoxins are also collected in Table 3. This data enables a more extended comparison, evidencing differences in the contributions that underlie a very similar outcome in terms of free-energy barriers, probably attributable to the absence/presence of water molecules at each active site and to differences in the nature and strength of the HB networks established at the corresponding Michaelis complex (RC) and TS pairs.…”

“…This data enables a more extended comparison, evidencing differences in the contributions that underlie a very similar outcome in terms of free-energy barriers, probably attributable to the absence/presence of water molecules at each active site and to differences in the nature and strength of the HB networks established at the corresponding Michaelis complex (RC) and TS pairs. In the particular case of hPrx5, examined in depth by some of us [20], the catalytic efficiency displayed by this peroxiredoxin is ascribed to a combination of effects including: (a) differential electrostatic stabilization of the anionic TS by protonated Arg127, also responsible of lowering Cys47 pKa, (b) strengthening the H2O2 HB network in going from RC to TS, and (c) the pivotal role played by a well-conserved Thr44 at the active site, that rotates its side-chain to modulate Cys47 nucleophilicity by the progressive release of a Sγ⋯HOT44 H-bond formed at RC while driving the substrate (tethered through HB to OHT44 as acceptor) into the right place at the right time for the reaction to proceed in terms of activation of the thiolate. Most of these effects are not found in the reaction with HSA.…”

“…Following the recognition that the reactions of thiols with H2O2 can participate in catalysis and regulation [9,10], these processes have received considerable interest in the last decade, both from experimental and theoretical points of view. Within the latter, a wide variety of computational strategies have been applied to address the reactivity of thiols and the mechanisms of H2O2 reactions with different LMWTs [11][12][13][14], with reduced models mimicking protein environments [15][16][17], and within whole proteins under different conditions [18][19][20][21]. A consensus on the SN2-character of Cys thiolate oxidation by H2O2 emerges from the studies conducted on LMWTs [11,12,14] and proteins [18][19][20][21][22][23].…”