Article Biochemistry, Vol. 48, No. 43, 2009 10393 FIGURE 4: Structure of the substrate spanning the active site, in the azide complex. For the region shown, the substrate conformation is similar in all three structures, except for the Ser 78 side chain torsion and residue 79. The catalytic triad (Ala 221, His 64, and Ala 32), the oxyanion ligand Asn 155, and the azide anion are indicated. The position of the catalytic nucleophile 221 OG atom has been modeled, based on the wild-type structure (PDB entry 1CSE). White lines represent selected interactions <3.3 A˚ in length. This image was created with Pymol and Image Magik. would functionally replace the deleted carboxylate by binding in or near this void. DISCUSSION To model the mechanism of the active (Ser 221) form of the anion-triggered protease, we utilized the wild-type subtilisin structure 1CSE to model the position of the Ser 221 OG atom (9, 10). Figure 5 shows the active site in the 3BGO azide complex, superimposed onto the wild-type active site of PDB entry 1CSE. Azide does not occupy the same location as the missing Asp 32 carboxylate (this would be prevented by steric overlap with the methyl group of Ala 32) but is in van der Waals contact with the methyl group (distance of 3.29 A˚ ). The N3 anion occupies a location that in many subtilisin structures accommodates a highly ordered water (at the location of the lower end of the azide) and often a second water (at the upper end of azide, the end nearest to His 64). The azide is within H-bonding distance of several nearby polar groups, including a 2.95 A˚ H-bond to the NE2 atom of His 64. The conformation of His 64 in 3BGO stands in comparison with the canonical active rotamer for His 64 as observed in 1CSE, where χ1 = -180 and the imidazole forms H-bonds both to Asp 32 and to Ser 221. In 3BGO, the His 64 χ1 = 124 , placing the imidazole 4.16 A˚ from Ala 221. This conformation is torsionally unfavorable but minimizes the dis- tance to the azide anion, suggesting that the interaction with azide is the primary force setting the position of the His 64 imidazole. In the wild-type active site of the 1CSE structure, all three interactions along the “charge-relay” chain (Asp 32-His 64-Ser 221-substrate) are shorter than 3 A˚ , and thus, it appears that electronic and H-ion motions alone can trigger the nucleophilic attack that initiates proteolysis. In contrast, in all three of these structures (anion-free, with fluoride, and with azide), the His 64 rotamer is swung somewhat away from Ala 221 and toward Ala 32 (shortest distance from imidazole ring to the modeled Ser 221 OG atom is 4.2 A˚ ). This torsional excursion is made possible by removing the Asp 32 side chain, which opens the space into which FIGURE 5: Superposition of the catalytic triad in the eglin complex (1CSE, cyan) onto the corresponding residues in the azide complex (3BGO, green). In the 1CSE structure, the three dashed lines (the three not involving azide) connecting substrate, Ser 221, His 64, and Asp 32 are all <3 A˚ in length. In the azide complex, the 2.9 A˚ H-bond between His 64 and azide is shown. the imidazole swings. Another factor governing His 64’s position appears to be a steric “push” from the substrate’s P10 side chain (Ser 780), which is 4.1 A˚ from His 64 (Cβ-Cβ distance) (34). The P10 side chain sterically blocks the His 64 imidazole from adopting the outward χ1 = -60 rotamer, constraining it toward its observed position where it is adjacent to Ala 32 and adjacent to the anion site. When substrate is omitted, His 64 is observed with χ1 in the -60 rotamer, pointing away from Ala 32 and placing the imidazole ring well away from contact with either Ala 32 or Ala 221. This has been observed in the substrate-free D32A active site in PDB entry 3F49. It thus appears that the P10 residue of the substrate helps to position His 64 for catalysis in D32A variants. Accordingly, once His 64 is pushed approximately into the active χ1 conformation by substrate binding, the imidazole’s ability to ionize makes the adjacent cavity favorable for binding anions. This is consistent with the inability of azide or fluoride to accelerate the cleavage rate of peptide substrates with p-nitroa- nilide or amino methyl coumarin leaving groups (21), which suggests that productive anion binding requires the presence of a P10 amino acid. Since the Ser 221 form of these structures is activated by anions, evidently His 64 is able to swing back toward Ser 221 sufficiently to accept its proton and thus stimulate its nucleophilic attack on the substrate. This is also consistent with the fact that, in all three of these structures, but especially in the two anion- bound structures, the electron density for the His 64 side chain is observed to be slightly smeared over a range of torsions. The angular extent of its local disorder appears to be ∼50-70 . This suggests that His 64 swings across a range of χ1 positions and transiently occupies an active position with a sufficient frequency to enable efficient proteolysis. Although the protonation state of His 64 is not known, and the observed torsional disorder could be associated with an oscillating protonation state, the imidazole must be deprotonated to be active; hence, we will focus on the mechanistic role of the deprotonated state. One additional uncertainty is generated by the unknown χ2 rotameric state of the imidazole (ring flip). While in the eglin structure the positions of the ring nitrogens are clearly inferred from the H-bonding interactions, in the azide complex the observed H-bond between azide and His 64 is consistent with either χ2 rotamer. The observed position of azide is not compatible, for any static His 64 rotamer, with a simple, fixed-atom proton shuttle as in