PDBsum entry 1q5p

Hydrolase

PDB id: 1q5p

Contents

Protein chain

269 a.a. *

Ligands

SO4

Metals

_CA ×2

Waters ×103

* Residue conservation analysis

PDB id:

1q5p

Name:

Hydrolase

Title:

S156e/s166d variant of bacillus lentus subtilisin

Structure:

Serine protease. Chain: a. Engineered: yes. Mutation: yes

Source:

Bacillus lentus. Organism_taxid: 1467. Expressed in: bacillus subtilis. Expression_system_taxid: 1423

Resolution:

1.60Å R-factor: 0.169

Authors:

R.R.Bott,G.Chan,B.Domingo,G.Ganshaw,C.Y.Hsia,M.Knapp,C.J.Murray

Key ref:

R.R.Bott et al. (2003). Do enzymes change the nature of transition states? Mapping the transition state for general acid-base catalysis of a serine protease. Biochemistry, 42, 10545-10553. PubMed id: 12962477 DOI: 10.1021/bi034773m

Date:

08-Aug-03 Release date: 11-Nov-03

Protein chain

P29600  (SUBS_LEDLE) -  Subtilisin Savinase from Lederbergia lenta

Seq: 269 a.a.

Struc: 269 a.a.*

* PDB and UniProt seqs differ at 6 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.4.21.62 - subtilisin.
• Reaction: Hydrolysis of proteins with broad specificity for peptide bonds, and a preference for a large uncharged residue in P1. Hydrolyzes peptide amides.

DOI no: 10.1021/bi034773m

Biochemistry 42:10545-10553 (2003)

PubMed id: 12962477

Do enzymes change the nature of transition states? Mapping the transition state for general acid-base catalysis of a serine protease.

R.R.Bott, G.Chan, B.Domingo, G.Ganshaw, C.Y.Hsia, M.Knapp, C.J.Murray.

ABSTRACT

The properties of the transition state for serine protease-catalyzed hydrolysis of an amide bond were determined for a series of subtilisin variants from Bacillus lentus. There is no significant change in the structure of the enzyme upon introduction of charged mutations S156E/S166D, suggesting that changes in catalytic activity reflect global properties of the enzyme. The effect of charged mutations on the pK(a) of the active site histidine-64 N(epsilon)(2)-H was correlated with changes in the second-order rate constant k(cat)/K(m) for hydrolysis of tetrapeptide anilides at low ionic strength with a Brønsted slope alpha = 1.1. The solvent isotope effect (D)2(O)(k(cat)/K(m))(1) = 1.4 +/- 0.2. These results are consistent with a rate-limiting breakdown of the tetrahedral intermediate in the acylation step with hydrogen bond stabilization of the departing amine leaving group. There is an increase in the ratio of hydrolysis of succinyl-Ala-Ala-Pro-Phe-anilides for p-nitroaniline versus aniline leaving groups with variants with more basic active site histidines that can be described by the interaction coefficient p(xy) = delta beta(lg)/delta pK(a) (H64) = 0.15. This is attributed to increased hydrogen bonding of the active site imidazolium N-H to the more basic amine leaving group as well as electrostatic destabilization of the transition state. A qualitative characterization of the transition state is presented in terms of a reaction coordinate diagram that is defined by the structure-reactivity parameters.