PDBsum entry 1utm

Hydrolase

PDB id: 1utm

Contents

Protein chain

222 a.a. *

Ligands

PEA

Metals

_CA

Waters ×109

* Residue conservation analysis

PDB id:

1utm

Name:

Hydrolase

Title:

Trypsin specificity as elucidated by lie calculations, x-ray structures and association constant measurements

Structure:

Trypsin i. Chain: a. Ec: 3.4.21.4

Source:

Salmo salar. Atlantic salmon. Organism_taxid: 8030. Organ: pyloric caeca

Resolution:

1.50Å R-factor: 0.189 R-free: 0.217

Authors:

H.-K.S.Leiros,B.O.Brandsdal,O.A.Andersen,V.Os,I.Leiros,R.Helland, J.Otlewski,N.P.Willassen,A.O.Smalas

Key ref:

H.K.Leiros et al. (2004). Trypsin specificity as elucidated by LIE calculations, X-ray structures, and association constant measurements. Protein Sci, 13, 1056-1070. PubMed id: 15044735 DOI: 10.1110/ps.03498604

Date:

09-Dec-03 Release date: 09-Jan-04

Protein chain

P35031  (TRY1_SALSA) -  Trypsin-1 from Salmo salar

Seq: 242 a.a.

Struc: 222 a.a.*

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

Enzyme reactions

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

DOI no: 10.1110/ps.03498604

Protein Sci 13:1056-1070 (2004)

PubMed id: 15044735

Trypsin specificity as elucidated by LIE calculations, X-ray structures, and association constant measurements.

H.K.Leiros, B.O.Brandsdal, O.A.Andersen, V.Os, I.Leiros, R.Helland, J.Otlewski, N.P.Willassen, A.O.Smalås.

ABSTRACT

The variation in inhibitor specificity for five different amine inhibitors bound to CST, BT, and the cold-adapted AST has been studied by use of association constant measurements, structural analysis of high-resolution crystal structures, and the LIE method. Experimental data show that AST binds the 1BZA and 2BEA inhibitors 0.8 and 0.5 kcal/mole more strongly than BT. However, structural interactions and orientations of the inhibitors within the S1 site have been found to be virtually identical in the three enzymes studied. For example, the four water molecules in the inhibitor-free structures of AST and BT are channeled into similar positions in the S1 site, and the nitrogen atom(s) of the inhibitors are found in two cationic binding sites denoted Position1 and Position2. The hydrophobic binding contributions for all five inhibitors, estimated by the LIE calculations, are also in the same order (-2.1 +/- 0.2 kcal/mole) for all three enzymes. Our hypothesis is therefore that the observed variation in inhibitor binding arises from different electrostatic interactions originating from residues outside the S1 site. This is well illustrated by AST, in which Asp 150 and Glu 221B, despite some distance from the S1 binding site, lower the electrostatic potential of the S1 site and thus enhance substrate binding. Because the trends in the experimentally determined binding energies were reproduced by the LIE calculations after adding the contribution from long-range interactions, we find this method very suitable for rational studies of protein-substrate interactions.

Selected figure(s)

Figure 1.
Figure 1. Structural formulas of the synthetic trypsin inhibitors included in the study. The numbers 1-5 refer roughly to the strength of the inhibitors.

Figure 2.
Figure 2. Structural arrangements, 2F[o]-F[c] (cyan) electron density maps and Fourier difference maps (Fo-F[c]) at +4 (green) and -4 (red) of the active site of AST (A-D) and BT (E-H). The inhibitors are (A) benzamidine AST-1BZA, (B) benzylamine AST-2BEA, (C) phenylethylamine AST-3PEA, (D) phenylpropylamine AST-4PPA, (E) aniline BT-ANL, (F) benzylamine BT-2BEA, (G) phenylethylamine BT-3PEA, and (H) phenylbutylamine BT-5PBA. Ser 190 is not covered by electron density and only selected hydrogen bonds are included to simplify the figure created by BobScript (Esnouf 1997). The sigma levels of the 2F[o]-F[c] maps are 1.1-1.5 (AST) and 1.5-1.7 (BT).

The above figures are reprinted by permission from the Protein Society: Protein Sci (2004, 13, 1056-1070) copyright 2004.

Figures were selected by an automated process.