Exfoliative toxin A (ETA) causes staphylococcal scalded skin syndrome which is characterized by a specific intraepidermal separation of layers of the skin. The mechanism by which ETA causes skin separation is unknown although protease or superantigen activity has been implicated. The X-ray crystal structure of ETA has been solved in two crystal forms to 2.1 and 2.3 A resolution and R-factors of 17% and 19%, respectively. The structures indicate that ETA belongs to the chymotrypsin-like family of serine proteases and cleaves substrates after acidic residues. The conformation of a loop adjacent to the catalytic site is suggested to be key in regulating the proteolytic activity of ETA through controlling whether the main chain carbonyl group of Pro192 occupies the oxyanion hole. A unique amino-terminal domain containing a 15-residue amphipathic alpha helix may also be involved in protease activation through binding a specific receptor. Substitution of the active site serine residue with cysteine abolishes the ability of ETA to produce the characteristic separation of epidermal layers but not its ability to induce T cell proliferation.

Arylamine N-acetyltransferases (NATs) catalyze an acetyl group transfer from acetyl coenzyme A (AcCoA) to arylamines, hydrazines, and their N-hydroxylated arylamine metabolites. The recently determined three-dimensional structures of prokaryotic NATs have revealed a cysteine protease-like Cys-His-Asp catalytic triad, which resides in a deep and hydrophobic pocket. This catalytic triad is strictly conserved across all known NATs, including hamster NAT2 (Cys-68, His-107, and Asp-122). Treatment of NAT2 with either iodoacetamide (IAM) or bromoacetamide (BAM) at neutral pH rapidly inactivated the enzyme with second-order rate constants of 802.7 +/- 4.0 and 426.9 +/- 21.0 M(-1) s(-1), respectively. MALDI-TOF and ESI mass spectral analysis established that Cys-68 is the only site of alkylation by IAM. Unlike the case for cysteine proteases, no significant inactivation was observed with either iodoacetic acid (IAA) or bromoacetic acid (BAA). Pre-steady state and steady state kinetic analysis with p-nitrophenyl acetate (PNPA) and NAT2 revealed a single-exponential curve for the acetylation step with a second-order rate constant of (1.4 +/- 0.05) x 10(5) M(-1) s(-1), followed by a slow linear rate of (7.85 +/- 0.65) x 10(-3) s(-1) for the deacetylation step. Studies of the pH dependence of the rate of inactivation with IAM and the rate of acetylation with PNPA revealed similar pK(a)(1) values of 5.23 +/- 0.09 and 5.16 +/- 0.04, respectively, and pK(a)(2) values of 6.95 +/- 0.27 and 6.79 +/- 0.25, respectively. Both rates reached their maximum values at pH 6.4 and decreased by only 30% at pH 9.0. Kinetic studies in the presence of D(2)O revealed a large inverse solvent isotope effect on both inactivation and acetylation of NAT2 [k(H)(inact)/k(D)(inact) = 0.65 +/- 0.02 and (k(2)/K(m)(acetyl))(H)/(k(2)/K(m)(acetyl))(D) = 0.60 +/- 0.03], which were found to be identical to the fractionation factors (Phi) derived from proton inventory studies of the rate of acetylation at pL 6.4 and 8.0. Substitution of the catalytic triad Asp-122 with either alanine or asparagine resulted in the complete loss of protein structural integrity and catalytic activity. From these results, it can be concluded that the catalytic mechanism of NAT2 depends on the formation of a thiolate-imidazolium ion pair (Cys-S(-)-His-ImH(+)). However, in contrast to the case with cysteine proteases, a pH-dependent protein conformational change is likely responsible for the second pK(a), and not deprotonation of the thiolate-imidazolium ion. In addition, substitutions of the triad aspartate are not tolerated. The enzyme appears, therefore, to be engineered to rapidly form a stable acetylated species poised to react with an arylamine substrate.

The exfoliative toxins (ETs) cause staphylococcal scalded skin syndrome, a disease characterized by specific separation of layers of the skin. Evidence suggests that the toxins act as serine proteases, though the specific substrate and mode of action are not known for certain. The crystal structure of exfoliative toxin A (ETA) was reported earlier and shown to be similar to that of the chymotrypsin-like serine proteases. Here, we report the 2.4 A resolution crystal structure of the other exfoliative toxin, ETB, which is 40% identical to ETA. The overall structures of ETA and ETB are similar including the positions of key residues within the active site. The structure of ETB supports the previous findings that the ETs are serine proteases that cleave substrates after glutamic acid residues. In this study we also discuss a number of structural differences including a large 14 residue loop insertion which may be a key feature involved in the differing biological properties of the ETs, particularly the pyrogenic and lethal activities of ETB not shared by ETA.