Crystal structure of bovine duodenase, a serine protease, with dual trypsin and chymotrypsin-like specificities

Abstract

The three‐dimensional structure of duodenase, a serine protease from bovine duodenum mucosa, has been determined at 2.4Å resolution. The enzyme, which has both trypsin‐like and chymotrypsin‐like activities, most closely resembles human cathepsin G with which it shares 57% sequence identity and similar specificity. The catalytic Ser195 in duodenase adopts the energetically favored conformation typical of serine proteinases and unlike the strained state typical of lipase/esterases. Of several waters in the active site of duodenase, the one associated with Ser214 is found in all serine proteinases and most lipase/esterases. The conservation of the Ser214 residue in serine proteinase, its presence in the active site, and participation in a hydrogen water network involving the catalytic triad (His57, Asp107, and Ser195) argues for its having an important role in the mechanism of action. It may be referred to as a fourth member of the catalytic triad. Duodenase is one of a growing family of enzymes that possesses trypsin‐like and chymotrypsin‐like activity. Not long ago, these activities were considered to be mutually exclusive. Computer modeling reveals that the S1 subsite of duodenase has structural features compatible with effective accommodation of P1 residues typical of trypsin (Arg/Lys) and chymotrypsin (Tyr/Phe) substrates. The determination of structural features associated with functional variation in the enzyme family may permit design of enzymes with a specific ratio of trypsin and chymotrypsin activities. Proteins 2000;41:8–16. © 2000 Wiley‐Liss, Inc.

How Immune Peptidases Change Specificity: Cathepsin G Gained Tryptic Function but Lost Efficiency during Primate Evolution

“…In classic tryptic peptidases, the anionic Asp 189 side chain carboxylate at the base of the primary specificity pocket forms a charge-charge complex with the substrate Lys or Arg side chain. Probably the closest parallel with the human cathepsin G configuration is that of bovine duodenase, which also has dual tryptic and chymotryptic specificity but with specificity triad Asn 189 /Gly 216 /Asp 226 , with Asp 226 proposed to serve the function of Asp 189 in classic tryptic serine peptidases (51). Duodenase is related to the chymase-cathepsin G-granzyme B family but forms its own clade, is absent in humans and mice, and is prominent in ruminant mammals, which also contain chymase and cathepsin G. The specificity triad of both types of cattle ( Bos taurus ) cathepsin G is identical to that of mouse and predicted ancestral cathepsin G, and thus is expected to lack tryptic activity.…”

Molecular markers of serine protease evolution

“…The backbone of Ser214 in chymotrypsin‐like proteases contributes to the S1 binding pocket (Perona and Craik, 1995). The side chain of the same residue helps generate a polar environment for the catalytic Asp102 (McGrath et al ., 1992), and both oxygens of Ser214 form hydrogen bonds with waters located in the active site cleft (Pletnev et al ., 2000). Ser125 (subtilisin BPN′ numbering) in subtilisin‐type proteases plays a similar role; its backbone contributes to the S1 binding pocket (Perona and Craik, 1995), whereas its side chain is directly adjacent to the catalytic residues (Siezen and Leunissen, 1997) so that the hydroxyl is within hydrogen‐bonding distance of the catalytic Asp32.…”

The influence of putrescine on the structure, enzyme activity and stability of α-chymotrypsin

“…4 a-Chy was folded into two domains with very little a-helix content and extensive regions of anti-parallel b-sheets, along with two interchain and three intrachain disulphide bonds. 32 Spectral deconvolution of a-Chy spectrum in the buffer showed 11.9% of a-helix, 20.5% of b structures, 27.7% of turns and 40% of the unordered structure. The data proposed that the secondary structure change and the unfolding of the protein skeleton by putrescine were low.…”