10392 Biochemistry, Vol. 48, No. 43, 2009 Scheme 1 Because the complex of pS169 and substrate is very tight (the dissociation constant for the ES complex is <1 nM) and is preformed in the kinetic experiment, the data can be fit to a simple two-step mechanism:where E is pS169, S is pG12, P1 consists of residues 1-77 of pG12, and P2 is the released GB domain. The apparent KD for azide is 50 mM. The scissile bond cleavage which releases the GB domain is concomitant with the acylation step in the enzymatic pathway. Because the complex of pS169 and P1 is also very tight (the dissociation constant for the EP1 complex is <1 nM), only one catalytic cycle is observed in this reaction. With no azide present, the rate of GB release is 0.0019 s-1 at 22 C. The rate of release in 100 mM azide is 4.1 s-1 at 22 C, and the maximum rate in saturating azide (k2) is 6.4 s-1 at 22 C, corresponding to an azide-dependent rate enhancement of ∼3300-fold. The anion-activated cleavage rate is on the same order as for the corresponding wild-type active site (Asp 32 version) of pS169, for which the rate of the acylation step is ∼20 s-1 (data not shown). Triggering by cyanate, formate, fluoride, and chloride was also examined for at least three concentrations of each anion. The apparent KD values and the rates of cleavage at saturation for each are given in Table 1. Cyanate (pKa = 3.46) is isosteric with azide (pKa = 4.72) but binds more weakly and triggers more weakly at saturation. Formate (pKa = 3.72) is a weaker trigger than cyanate. [We note that acetate (pKa = 4.76) exhibits no detectable triggering activity presumably because it is too large to fit into the anion binding site.] Fluoride (pKa = 3.16) strongly triggers pS169 (maximum rate of 11.2 s-1 in saturating fluoride) but with a KD that is ∼15-fold higher (apparent KD of 0.75 M) than that of azide. Chloride (pKa = -9) binds with an apparent KD of >1 M and has a maximum rate of ∼0.15 s-1 in saturating chloride. The ability of a particular anion to trigger proteolysis appears to be a complicated function of its size, shape, pKa, and ability to accept a H-bond from His 64. For example, a non-hydrogen bonding anion such as chloride binds to the enzyme substrate complex but is a poor trigger. In contrast, a good H-bonding anion, such as fluoride, binds with an affinity similar to that of chloride but triggers ∼100-fold faster. Inhibi- tion of azide triggering by chloride was analyzed by measuring cleavage rates as a function of azide in 0.25 M KCl and in 0.5 M KCl. An apparent KD of 1.2 M was calculated for chloride from the inhibition data, using a KD of 50 mM for azide (in the absence of chloride) and a maximum rate of 6.4 s-1 in saturating azide. The structures of the three pS164-substrate complexes with azide (3BGO), with fluoride (3CO0), and without added anions (3CNQ) are generally similar. Although the fluoride anion is a strong activator of the enzyme, its electron density is not distinguishable from water at this resolution, preventing identi- fication of a fluoride binding site. In contrast, azide has a unique structure, and its location in the electron density map is clear. Unless otherwise specified, the following descriptions apply to the azide complex structure. Figure 3 shows the electron density for the azide anion, the His 64 side chain, and the scissile region of the substrate. The anion site is buried under the substrate, adjacent to the mutated Ala 32, 8 A˚ from the scissile peptide. Table 1: Kinetic Parameters for Triggering Anionsa anion azide formate cyanate fluoride chloride pKa 4.72 3.72 3.46 3.16 -9.0 KD (M) 0.05 0.6 0.3 0.75 1.2 Gallagher et al. k2 (s-1) 6.4 1.2 2.7 11.2 ∼0.15 aRates were measured for at least three concentrations of each anion as described in Methods. KD values and maximum rates at saturation were calculated according to Scheme 1 using Kintek Global Explorer. FIGURE 3: Unbiased electron density map showing the azide anion, the His 64 imidazole, and the scissile bond region of the substrate. The difference map is contoured at 2.5σ and shows the positions of all atoms in Ser 780, and most atoms of Ala 790. At lower contour values, the map is less clean but includes all atoms of residue 790. A black triangle marks the scissile peptide. The two H-bonds from azide are both between 2.9 and 3.0 A˚ in length. The dumbbell-shaped electron density for azide is consistent with resonance theory that gives excess negative charge to its two terminal atoms. Figure 4 shows the substrate bound across the active site. Both Ser 780 (the prime symbol denotes the substrate) and Ala 790 are in the β conformation, and Ala 790 forms two H-bonds with Ser 218 of the enzyme, in a standard antiparallel β-sheet interaction. The side chain of Ser 780 appears in two different conformations, depending on the presence of anion. In the anion-free structure, the conformation is the χ1 = -60 rotamer, while in both the fluoride- and azide-soaked structures, the χ1 torsion shifts to the 180 rotamer. The dramatic stimulation of enzyme activity by azide and fluoride restores activity in otherwise marginally active D32A variants to near wild-type levels, suggesting that these anions functionally replace the Asp 32 carboxylate. In the generally accepted “catalytic triad” mechanism of wild-type subtilisin, the role of the Asp 32 carboxylate is to polarize the His 64 imidazole so that it removes the proton from the OG atom of Ser 221 and thus primes the OG atom for nucleophilic attack on the carbonyl carbon atom of the substrate’s P1 residue (e.g., see ref 33 for a useful review). The D32A mutation was expected to create a small void adjacent to His 64, and we hypothesized that azide