PDBsum entry 1anb

Serine protease

PDB id: 1anb

Contents

Protein chain

223 a.a. *

Ligands

BEN ×2

Metals

_CA

Waters ×149

* Residue conservation analysis

PDB id:

1anb

Name:

Serine protease

Title:

Anionic trypsin mutant with ser 214 replaced by glu

Structure:

Anionic trypsin. Chain: a. Engineered: yes. Mutation: yes

Source:

Rattus rattus. Black rat. Organism_taxid: 10117. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

2.80Å R-factor: 0.167

Authors:

R.J.Fletterick,M.E.Mcgrath

Key ref:

M.E.McGrath et al. (1992). Perturbing the polar environment of Asp102 in trypsin: consequences of replacing conserved Ser214. Biochemistry, 31, 3059-3064. PubMed id: 1554694 DOI: 10.1021/bi00127a005

Date:

21-Dec-94 Release date: 01-Apr-97

Protein chain

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

Seq: 246 a.a.

Struc: 223 a.a.*

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

Enzyme reactions

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

DOI no: 10.1021/bi00127a005

Biochemistry 31:3059-3064 (1992)

PubMed id: 1554694

Perturbing the polar environment of Asp102 in trypsin: consequences of replacing conserved Ser214.

M.E.McGrath, J.R.Vásquez, C.S.Craik, A.S.Yang, B.Honig, R.J.Fletterick.

ABSTRACT

Much of the catalytic power of trypsin is derived from the unusual buried, charged side chain of Asp102. A polar cave provides the stabilization for maintaining the buried charge, and it features the conserved amino acid Ser214 adjacent to Asp102. Ser214 has been replaced with Ala, Glu, and Lys in rat anionic trypsin, and the consequences of these changes have been determined. Three-dimensional structures of the Glu and Lys variant trypsins reveal that the new 214 side chains are buried. The 2.2-A crystal structure (R = 0.150) of trypsin S214K shows that Lys214 occupies the position held by Ser214 and a buried water molecule in the buried polar cave. Lys214-N zeta is solvent inaccessible and is less than 5 A from the catalytic Asp102. The side chain of Glu214 (2.8 A, R = 0.168) in trypsin S214E shows two conformations. In the major one, the Glu carboxylate in S214E forms a hydrogen bond with Asp102. Analytical isoelectrofocusing results show that trypsin S214K has a significantly different isoelectric point than trypsin, corresponding to an additional positive charge. The kinetic parameter kcat demonstrates that, compared to trypsin, S214K has 1% of the catalytic activity on a tripeptide amide substrate and S214E is 44% as active. Electrostatic potential calculations provide corroboration of the charge on Lys214 and are consistent with the kinetic results, suggesting that the presence of Lys214 has disturbed the electrostatic potential of Asp102.