PDBsum entry 2fi3

Hydrolase/hydrolase inhibitor

PDB id: 2fi3

Contents

Protein chains

223 a.a. *

58 a.a. *

Ligands

SO4 ×8

EDO ×6

Metals

_NA

_CA ×2

Waters ×261

* Residue conservation analysis

PDB id:

2fi3

Name:

Hydrolase/hydrolase inhibitor

Title:

Crystal structure of a bpti variant (cys14->ser, cys38->ser) in complex with trypsin

Structure:

Cationic trypsin. Chain: e. Synonym: beta-trypsin. Pancreatic trypsin inhibitor. Chain: i. Synonym: basic protease inhibitor, bpi, bpti, aprotinin. Engineered: yes. Mutation: yes

Source:

Bos taurus. Cattle. Organism_taxid: 9913. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PQS)

Resolution:

1.58Å R-factor: 0.203 R-free: 0.218

Authors:

E.Zakharova,M.P.Horvath,D.P.Goldenberg

Key ref:

E.Zakharova et al. (2008). Functional and structural roles of the Cys14-Cys38 disulfide of bovine pancreatic trypsin inhibitor. J Mol Biol, 382, 998. PubMed id: 18692070

Date:

27-Dec-05 Release date: 24-Jan-06

Protein chain

P00760  (TRY1_BOVIN) -  Serine protease 1 from Bos taurus

Seq: 246 a.a.

Struc: 223 a.a.

Protein chain

P00974  (BPT1_BOVIN) -  Pancreatic trypsin inhibitor from Bos taurus

Seq: 100 a.a.

Struc: 58 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: Chain E: E.C.3.4.21.4 - trypsin.
• Reaction: Preferential cleavage: Arg-|-Xaa, Lys-|-Xaa.

J Mol Biol 382:998 (2008)

PubMed id: 18692070

Functional and structural roles of the Cys14-Cys38 disulfide of bovine pancreatic trypsin inhibitor.

E.Zakharova, M.P.Horvath, D.P.Goldenberg.

ABSTRACT

The disulfide bond between Cys14 and Cys38 of bovine pancreatic trypsin inhibitor lies on the surface of the inhibitor and forms part of the protease-binding region. The functional properties of three variants lacking this disulfide, with one or both of the Cys residues replaced with Ser, were examined, and X-ray crystal structures of the complexes with bovine trypsin were determined and refined to the 1.58-A resolution limit. The crystal structure of the complex formed with the mutant with both Cys residues replaced was nearly identical with that of the complex containing the wild-type protein, with the Ser oxygen atoms positioned to replace the disulfide bond with a hydrogen bond. The two structures of the complexes with single replacements displayed small local perturbations with alternate conformations of the Ser side chains. Despite the absence of the disulfide bond, the crystallographic temperature factors show no evidence of increased flexibility in the complexes with the mutant inhibitors. All three of the variants were cleaved by trypsin more rapidly than the wild-type inhibitor, by as much as 10,000-fold, indicating that the covalent constraint normally imposed by the disulfide contributes to the remarkable resistance to hydrolysis displayed by the wild-type protein. The rates of hydrolysis display an unusual dependence on pH over the range of 3.5-8.0, decreasing at the more alkaline values, as compared with the increased hydrolysis rates for normal substrates under these conditions. These observations can be accounted for by a model for inhibition in which an acyl-enzyme intermediate forms at a significant rate but is rapidly converted back to the enzyme-inhibitor complex by nucleophilic attack by the newly created amino group. The model suggests that a lack of flexibility in the acyl-enzyme intermediate, rather than the enzyme-inhibitor complex, may be a key factor in the ability of bovine pancreatic trypsin inhibitor and similar inhibitors to resist hydrolysis.