PDBsum entry 2f91

Hydrolase/hydrolase inhibitor

PDB id: 2f91

Contents

Protein chains

237 a.a. *

33 a.a. *

Metals

_CL ×2

_CD ×6

Waters ×343

* Residue conservation analysis

PDB id:

2f91

Name:

Hydrolase/hydrolase inhibitor

Title:

1.2a resolution structure of a crayfish trypsin complexed with a peptide inhibitor, sgti

Structure:

Hepatopancreas trypsin. Chain: a. Serine protease inhibitor i/ii. Chain: b. Fragment: protease inhibitor sgpi-1, residues 20-54. Synonym: schistocerca gregaria trypsin inhibitor, sgti. Engineered: yes

Source:

Pontastacus leptodactylus. Narrow-clawed crayfish. Organism_taxid: 6717. Tissue: hepatopancreas. Synthetic: yes. Other_details: the protein was chemically synthesized, this sequence occurs naturally in schistocerca gregaria (desert locust)

Biol. unit:

Dimer (from PQS)

Resolution:

1.20Å R-factor: 0.139 R-free: 0.182

Authors:

K.Fodor,V.Harmat,C.Hetenyi,J.Kardos,J.Antal,A.Perczel,A.Patthy, G.Katona,L.Graf

Key ref:

K.Fodor et al. (2006). Enzyme:substrate hydrogen bond shortening during the acylation phase of serine protease catalysis. Biochemistry, 45, 2114-2121. PubMed id: 16475800 DOI: 10.1021/bi0517133

Date:

05-Dec-05 Release date: 18-Apr-06

Supersedes:

1yr4

Protein chain

Q52V24  (Q52V24_ASTLP) -  Hepatopancreas trypsin (Fragment) from Astacus leptodactylus

Seq: 237 a.a.

Struc: 237 a.a.*

Protein chain

O46162  (SGP1_SCHGR) -  Serine protease inhibitor I/II from Schistocerca gregaria

Seq: 92 a.a.

Struc: 33 a.a.

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

Enzyme reactions

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

DOI no: 10.1021/bi0517133

Biochemistry 45:2114-2121 (2006)

PubMed id: 16475800

Enzyme:substrate hydrogen bond shortening during the acylation phase of serine protease catalysis.

K.Fodor, V.Harmat, R.Neutze, L.Szilágyi, L.Gráf, G.Katona.

ABSTRACT

Atomic resolution (<or=1.2 A) serine protease intermediate structures revealed that the strength of the hydrogen bonds between the enzyme and the substrate changed during catalysis. The well-conserved hydrogen bonds of antiparallel beta-sheet between the enzyme and the substrate become significantly shorter in the transition from a Michaelis complex analogue (Pontastacus leptodactylus (narrow-fingered crayfish) trypsin (CFT) in complex with Schistocerca gregaria (desert locust) trypsin inhibitor (SGTI) at 1.2 A resolution) to an acyl-enzyme intermediate (N-acetyl-Asn-Pro-Ile acyl-enzyme intermediate of porcine pancreatic elastase at 0.95 A resolution) presumably synchronously with the nucleophilic attack on the carbonyl carbon atom of the scissile peptide bond. This is interpreted as an active mechanism that utilizes the energy released from the stronger hydrogen bonds to overcome the energetic barrier of the nucleophilic attack by the hydroxyl group of the catalytic serine. In the CFT:SGTI complex this hydrogen bond shortening may be hindered by the 27I-32I disulfide bridge and Asn-15I of SGTI. The position of the catalytic histidine changes slightly as it adapts to the different nucleophilic attacker during the transition from the Michaelis complex to the acyl-enzyme state, and simultaneously its interaction with Asp-102 and Ser-214 becomes stronger. The oxyanion hole hydrogen bonds provide additional stabilization for acyl-ester bond in the acyl-enzyme than for scissile peptide bond of the Michaelis complex. Significant deviation from planarity is not observed in the reactive bonds of either the Michaelis complex or the acyl-enzyme. In the Michaelis complex the electron distribution of the carbonyl bond is distorted toward the oxygen atom compared to other peptide bonds in the structure, which indicates the polarization effect of the oxyanion hole.