PDBsum entry 4rko

Hydrolase/hydrolase inhibitor

PDB id: 4rko

Contents

Protein chains

250 a.a.

40 a.a.

Ligands

0G6

MES

GOL ×2

NAG

Metals

_NA

Waters ×145

PDB id:

4rko

Name:

Hydrolase/hydrolase inhibitor

Title:

Crystal structure of thrombin mutant s195t bound with ppack

Structure:

Thrombin heavy chain. Chain: b. Engineered: yes. Mutation: yes. Thrombin light chain. Chain: a. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: f2. Expressed in: cricetulus griseus. Expression_system_taxid: 10029. Expression_system_cell_line: bhk cells. Expression_system_cell_line: bhk cells

Resolution:

1.84Å R-factor: 0.168 R-free: 0.202

Authors:

A.L.Pelc,Z.Chen,D.W.Gohara,A.D.Vogt,N.Pozzi,E.Di Cera

Key ref:

L.A.Pelc et al. (2015). Why Ser and not Thr brokers catalysis in the trypsin fold. Biochemistry, 54, 1457-1464. PubMed id: 25664608 DOI: 10.1021/acs.biochem.5b00014

Date:

13-Oct-14 Release date: 11-Mar-15

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 250 a.a.*

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 40 a.a.

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

Enzyme reactions

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

DOI no: 10.1021/acs.biochem.5b00014

Biochemistry 54:1457-1464 (2015)

PubMed id: 25664608

Why Ser and not Thr brokers catalysis in the trypsin fold.

L.A.Pelc, Z.Chen, D.W.Gohara, A.D.Vogt, N.Pozzi, E.Di Cera.

ABSTRACT

Although Thr is equally represented as Ser in the human genome and as a nucleophile is as good as Ser, it is never found in the active site of the large family of trypsin-like proteases that utilize the Asp/His/Ser triad. The molecular basis of the preference of Ser over Thr in the trypsin fold was investigated with X-ray structures of the thrombin mutant S195T free and bound to an irreversible active site inhibitor. In the free form, the methyl group of T195 is oriented toward the incoming substrate in a conformation seemingly incompatible with productive binding. In the bound form, the side chain of T195 is reoriented for efficient substrate acylation without causing steric clash within the active site. Rapid kinetics prove that this change is due to selection of an active conformation from a preexisting ensemble of reactive and unreactive rotamers whose relative distribution determines the level of activity of the protease. Consistent with these observations, the S195T substitution is associated with a weak yet finite activity that allows identification of an unanticipated important role for S195 as the end point of allosteric transduction in the trypsin fold. The S195T mutation abrogates the Na(+)-dependent enhancement of catalytic activity in thrombin, activated protein C, and factor Xa and significantly weakens the physiologically important allosteric effects of thrombomodulin on thrombin and of cofactor Va on factor Xa. The evolutionary selection of Ser over Thr in trypsin-like proteases was therefore driven by the need for high catalytic activity and efficient allosteric regulation.