PDBsum entry 1b7x

Hydrolase/hydrolase inhibitor

PDB id: 1b7x

Contents

Protein chains

26 a.a. *

246 a.a. *

Ligands

PHE-PRO-ARG

Waters ×146

* Residue conservation analysis

PDB id:

1b7x

Name:

Hydrolase/hydrolase inhibitor

Title:

Structure of human alpha-thrombin y225i mutant bound to d-phe-pro-arg- chloromethylketone

Structure:

Protein (thrombin light chain). Chain: a. Engineered: yes. Protein (thrombin heavy chain). Chain: b. Engineered: yes. Mutation: yes. Protein (inhibitor). Chain: c.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: saccharomyces cerevisiae. Expression_system_taxid: 4932. Expression_system_cell_line: baby hamster kidney cells (bhk-21).

Biol. unit:

Trimer (from PQS)

Resolution:

2.10Å R-factor: 0.226 R-free: 0.282

Authors:

S.Caccia,K.Futterer,E.Di Cera,G.Waksman

Key ref:

E.R.Guinto et al. (1999). Unexpected crucial role of residue 225 in serine proteases. Proc Natl Acad Sci U S A, 96, 1852-1857. PubMed id: 10051558 DOI: 10.1073/pnas.96.5.1852

Date:

25-Jan-99 Release date: 02-Mar-99

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 26 a.a.

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 246 a.a.*

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

Enzyme reactions

• Enzyme class: Chains A, B: E.C.3.4.21.5 - thrombin.
• Reaction: Preferential cleavage: Arg-|-Gly; activates fibrinogen to fibrin and releases fibrinopeptide A and B.

DOI no: 10.1073/pnas.96.5.1852

Proc Natl Acad Sci U S A 96:1852-1857 (1999)

PubMed id: 10051558

Unexpected crucial role of residue 225 in serine proteases.

E.R.Guinto, S.Caccia, T.Rose, K.Fütterer, G.Waksman, E.Di Cera.

ABSTRACT

Residue 225 in serine proteases of the chymotrypsin family is Pro or Tyr in more than 95% of nearly 300 available sequences. Proteases with Y225 (like some blood coagulation and complement factors) are almost exclusively found in vertebrates, whereas proteases with P225 (like degradative enzymes) are present from bacteria to human. Saturation mutagenesis of Y225 in thrombin shows that residue 225 affects ligand recognition up to 60,000-fold. With the exception of Tyr and Phe, all residues are associated with comparable or greatly reduced catalytic activity relative to Pro. The crystal structures of three mutants that differ widely in catalytic activity (Y225F, Y225P, and Y225I) show that although residue 225 makes no contact with substrate, it drastically influences the shape of the water channel around the primary specificity site. The activity profiles obtained for thrombin also suggest that the conversion of Pro to Tyr or Phe documented in the vertebrates occurred through Ser and was driven by a significant gain (up to 50-fold) in catalytic activity. In fact, Ser and Phe are documented in 4% of serine proteases, which together with Pro and Tyr account for almost the entire distribution of residues at position 225. The unexpected crucial role of residue 225 in serine proteases explains the evolutionary selection of residues at this position and shows that the structural determinants of protease activity and specificity are more complex than currently believed. These findings have broad implications in the rational design of enzymes with enhanced catalytic properties.

Selected figure(s)

Figure 1.
Fig. 1. Effect of residue 225 on the Na^+ specificity of thrombin, measured as the ratio of the k[cat]/K[m] values for the hydrolysis of H-D-Phe-Pro-Arg-p-nitroanilide (FPR) in the presence of 200 mM NaCl or choline chloride, 5 mM Tris, 0.1% polyethylene glycol, pH 8.0 at 25°C (6). Binding of Na^+ to wild type (Tyr) enhances k[cat]/K[m] nearly 25-fold. A significant enhancement also is observed for Phe, His, Met, Gln, Trp, and Ser, in decreasing order. All other residues show no significant difference between Na^+ and choline.

Figure 3.
Fig. 3. Effect of residue 225 on the architecture of the water channel around the primary specificity site of thrombin. Shown is a cross section of the enzyme along the water channel that reveals the active site inhibitor PPACK (purple), D189 in the primary specificity site, residue 225 with the carbonyl O atom of residue 224 (red), buried water molecules (blue), and Na^+ (yellow). The surface of the enzyme is rendered as a net (black, above the plan of section; green, below it). The side chain of residue 225 points away from D189 and makes no contact with PPACK. The Y225F mutant is practically identical to wild type (11) and shows a bound Na^+ coordinated octahedrally by the carbonyl O atoms of K224 and R221a (not shown), and four water molecules (23, 26). In this mutant, the water channel connects the active site to an aperture at the bottom of the molecule (arrow). In the Y225P mutant, there is no evidence of bound Na^+; the carbonyl O atom of K224 is shifted 70° toward the interior of the channel and occludes it in the middle. In addition, the channel is shunted laterally (arrow) around residue 225 because of the Y225P replacement. In the Y225I mutant, the channel has three apertures.

Figures were selected by an automated process.