PDBsum entry 1f5r

Hydrolase/hydrolase inhibitor

PDB id: 1f5r

Contents

Protein chains

216 a.a. *

57 a.a. *

Metals

_CA

Waters ×270

* Residue conservation analysis

PDB id:

1f5r

Name:

Hydrolase/hydrolase inhibitor

Title:

Rat trypsinogen mutant complexed with bovine pancreatic trypsin inhibitor

Structure:

Trypsin ii, anionic. Chain: a. Engineered: yes. Mutation: yes. Pancreatic trypsin inhibitor. Chain: i. Synonym: bpti

Source:

Rattus norvegicus. Norway rat. Organism_taxid: 10116. Expressed in: saccharomyces cerevisiae. Expression_system_taxid: 4932. Bos taurus. Cattle. Organism_taxid: 9913. Tissue: pancreas

Biol. unit:

Tetramer (from PQS)

Resolution:

1.65Å R-factor: 0.189 R-free: 0.220

Authors:

A.Pasternak,A.White,M.Cahoon,D.Ringe,L.Hedstrom

Key ref:

A.Pasternak et al. (2001). The energetic cost of induced fit catalysis: Crystal structures of trypsinogen mutants with enhanced activity and inhibitor affinity. Protein Sci, 10, 1331-1342. PubMed id: 11420435

Date:

15-Jun-00 Release date: 04-Jul-01

Protein chain

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

Seq: 246 a.a.

Struc: 216 a.a.*

Protein chain

P00974  (BPT1_BOVIN) -  Pancreatic trypsin inhibitor from Bos taurus

Seq: 100 a.a.

Struc: 57 a.a.

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

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

Protein Sci 10:1331-1342 (2001)

PubMed id: 11420435

The energetic cost of induced fit catalysis: Crystal structures of trypsinogen mutants with enhanced activity and inhibitor affinity.

A.Pasternak, A.White, C.J.Jeffery, N.Medina, M.Cahoon, D.Ringe, L.Hedstrom.

ABSTRACT

The contribution of induced fit to enzyme specificity has been much debated, although with little experimental data. Here we probe the effect of induced fit on enzyme specificity using the trypsin(ogen) system. BPTI is known to induce trypsinogen to assume a trypsinlike conformation. Correlations are observed between BPTI affinity and the values of k(cat)/K(m) for the hydrolysis of two substrates by eight trypsin(ogen) variants. The slope of both correlations is -1.8. The crystal structures of the BPTI complexes of four variant trypsinogens were also solved. Three of these enzymes, K15A, DeltaI16V17/D194N, and DeltaI16V17/Q156K trypsinogen, are 10- to 100-fold more active than trypsinogen. The fourth variant, DeltaI16V17 trypsinogen, is the lone outlier in the correlations; its activity is lower than expected based on its affinity for BPTI. The S1 site and oxyanion hole, formed by segments 184A-194 and 216-223, are trypsinlike in all of the enzymes. These structural and kinetic data confirm that BPTI induces an active conformation in the trypsin(ogen) variants. Thus, changes in BPTI affinity monitor changes in the energetic cost of inducing a trypsinlike conformation. Although the S1 site and oxyanion hole are similar in all four variants, the N-terminal and autolysis loop (residues 142-152) segments have different interactions for each variant. These results indicate that zymogen activity is controlled by a simple conformational equilibrium between active and inactive conformations, and that the autolysis loop and N-terminal segments control this equilibrium. Together, these data illustrate that induced fit does not generally contribute to enzyme specificity.