PDBsum entry 3fp7

Hydrolase/hydrolase inhibitor

PDB id: 3fp7

Contents

Protein chains

223 a.a. *

15 a.a. *

43 a.a. *

Ligands

EDO ×5

PG4

SO4 ×4

Metals

_CA

Waters ×371

* Residue conservation analysis

PDB id:

3fp7

Name:

Hydrolase/hydrolase inhibitor

Title:

Anionic trypsin variant s195a in complex with bovine pancreatic trypsin inhibitor (bpti) cleaved at the scissile bond (lys15-ala16) determined to the 1.46 a resolution limit

Structure:

Anionic trypsin-2. Chain: e. Synonym: anionic trypsin ii, pretrypsinogen ii, serine protease 2. Engineered: yes. Mutation: yes. Pancreatic trypsin inhibitor. Chain: i. Synonym: basic protease inhibitor, bpti, bpi, aprotinin. Engineered: yes.

Source:

Rattus norvegicus. Brown rat,rat,rats. Organism_taxid: 10116. Gene: prss2, try2. Expressed in: saccharomyces cerevisiae. Expression_system_taxid: 4932. Synthetic: yes. Other_details: bovine pancreatic trypsin inhibitor was obtained in its hydrolyzed form by partial reduction at the cys14-cys38

Resolution:

1.46Å R-factor: 0.175 R-free: 0.186

Authors:

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

Key ref:

E.Zakharova et al. (2009). Structure of a serine protease poised to resynthesize a peptide bond. Proc Natl Acad Sci U S A, 106, 11034-11039. PubMed id: 19549826 DOI: 10.1073/pnas.0902463106

Date:

04-Jan-09 Release date: 17-Feb-09

Protein chain

P00763  (TRY2_RAT) -  Anionic trypsin-2 from Rattus norvegicus

Seq: 246 a.a.

Struc: 223 a.a.*

Protein chain

P00974  (BPT1_BOVIN) -  Pancreatic trypsin inhibitor from Bos taurus

Seq: 100 a.a.

Struc: 15 a.a.

Protein chain

P00974  (BPT1_BOVIN) -  Pancreatic trypsin inhibitor from Bos taurus

Seq: 100 a.a.

Struc: 43 a.a.

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

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

DOI no: 10.1073/pnas.0902463106

Proc Natl Acad Sci U S A 106:11034-11039 (2009)

PubMed id: 19549826

Structure of a serine protease poised to resynthesize a peptide bond.

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

ABSTRACT

The serine proteases are among the most thoroughly studied enzymes, and numerous crystal structures representing the enzyme-substrate complex and intermediates in the hydrolysis reactions have been reported. Some aspects of the catalytic mechanism remain controversial, however, especially the role of conformational changes in the reaction. We describe here a high-resolution (1.46 A) crystal structure of a complex formed between a cleaved form of bovine pancreatic trypsin inhibitor (BPTI) and a catalytically inactive trypsin variant with the BPTI cleavage site ideally positioned in the active site for resynthesis of the peptide bond. This structure defines the positions of the newly generated amino and carboxyl groups following the 2 steps in the hydrolytic reaction. Comparison of this structure with those representing other intermediates in the reaction demonstrates that the residues of the catalytic triad are positioned to promote each step of both the forward and reverse reaction with remarkably little motion and with conservation of hydrogen-bonding interactions. The results also provide insights into the mechanism by which inhibitors like BPTI normally resist hydrolysis when bound to their target proteases.

Selected figure(s)

Figure 2.
Structures of the intact and cleaved trypsin-binding regions in complexes of rat trypsin with BPTI and BPTI*. (A and B) The enzyme active site is shown in a surface representation, and the primary binding residues of the inhibitors are represented as sticks. Carbon, nitrogen, oxygen, and sulfur atoms of the inhibitor are colored white, blue, red, and yellow respectively. The scissile peptide bond of the intact inhibitor in A is identified by an arrow. The surface of the side-chain oxygen of the catalytic Ser residue (Ser-195) of trypsin in A is colored red. Electron density maps (composite simulated annealing omit maps) corresponding to the inhibitor residues are represented as cages, contoured at the level of 1 σ. (C) The side chains of the residues making up the catalytic triad of rat trypsin in the complexes with BPTI and BPTI* (with the carbon atoms colored green and orange, respectively) are superimposed with those in structures of an acyl intermediate (PDB ID code 2AGE, with the carbon atoms colored gray) and a tetrahedral transition-state analog (PDB ID code 1BTZ, with the carbon atoms colored purple) formed with bovine trypsin.

Figure 3.
Structural reconstruction of the catalytic mechanism for peptide hydrolysis by serine proteases. (A) The enzyme–substrate complex, drawn from atomic coordinates of the structure of the BPTI–trypsin complex. (B) The tetrahedral intermediate for nucleophilic substitution of the P1′ amino group by Ser-195, based on the crystal structure of a boronate transition-state analog bound to bovine trypsin (PDB ID code 1BTZ). (C) The acyl-enzyme intermediate after its formation by nucleophilic attach by Ser-195, drawn from the superimposed structures of the BPTI*–trypsin complex (P1′ amino group and His-57) and an acyl-enzyme intermediate (acylated Ser-195). (D) The acyl-enzyme intermediate after dissociation of the P1′ amino group and entry of the hydrolytic water molecule, shown as a red sphere (PDB ID code 2AGE). (E) The tetrahedral intermediate for hydrolysis of the acyl intermediate (PDB ID code 1BTZ). (F) The final enzyme-product complex, drawn from the superimposed coordinates of the BPTI–trypsin (His-57 and Ser-195) and BPTI*–trypsin (P1 carboxyl group) complexes. In each drawing, the position of the Hε[2] hydrogen atom was modeled by using standard stereochemistry for a His side chain. In B and E, the oxyanion oxygen atom, which is not present in the boronate, has been added to the models of the tetrahedral intermediates assuming standard geometry, and the boron atom of the inhibitor has been colored green to indicate its correspondence to the carbonyl carbon atom in the other structures. In B, the hydroxyl oxygen of the boronate has been colored blue to indicate its correspondence to the P1′ nitrogen atoms in A and C, and the position of the P1′ Cα atom was added by modeling. The positions of the modeled atoms in the tetrahedral intermediates are indicated by semitransparent bonds. The arrows represent the direction of nucleophilic attack on the carbonyl carbon. Dashed lines indicate the geometry of the hydrogen bonds formed by the Hε[2] atom in each drawing. Distances shown in parentheses were measured between atoms in superimposed structures.

Figures were selected by the author.