PDBsum entry 9lpr

Hydrolase/hydrolase inhibitor

PDB id: 9lpr

Contents

Protein chain

198 a.a. *

Ligands

ALA-ALA-PRO-BLE

SO4

Waters ×184

* Residue conservation analysis

PDB id:

9lpr

Name:

Hydrolase/hydrolase inhibitor

Title:

Structural basis for broad specificity in alpha-lytic protease mutants

Structure:

Alpha-lytic protease. Chain: a. Engineered: yes. Methoxysuccinyl-ala-ala-pro-leucine boronic acid inhibitor. Chain: p. Engineered: yes

Source:

Lysobacter enzymogenes. Organism_taxid: 69. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes

Biol. unit:

Dimer (from PQS)

Resolution:

2.20Å R-factor: 0.129

Authors:

R.Bone,D.A.Agard

Key ref:

R.Bone et al. (1991). Structural basis for broad specificity in alpha-lytic protease mutants. Biochemistry, 30, 10388-10398. PubMed id: 1931963 DOI: 10.1021/bi00107a005

Date:

05-Aug-91 Release date: 15-Jan-93

Protein chain

P00778  (PRLA_LYSEN) -  Alpha-lytic protease from Lysobacter enzymogenes

Seq: 397 a.a.

Struc: 198 a.a.

Enzyme reactions

• Enzyme class: E.C.3.4.21.12 - alpha-lytic endopeptidase.
• Reaction: Hydrolysis of proteins, especially bonds adjacents to L-alanine and L-valine residues in bacterial cell walls, elastin and other proteins.

DOI no: 10.1021/bi00107a005

Biochemistry 30:10388-10398 (1991)

PubMed id: 1931963

Structural basis for broad specificity in alpha-lytic protease mutants.

R.Bone, A.Fujishige, C.A.Kettner, D.A.Agard.

ABSTRACT

Binding pocket mutants of alpha-lytic protease (Met 192----Ala and Met 213----Ala) have been constructed recently in an effort to create a protease specific for Met just prior to the scissile bond. Instead, mutation resulted in proteases with extraordinarily broad specificity profiles and high activity [Bone, R., Silen, J. L., & Agard, D. A. (1989) Nature 339, 191-195]. To understand the structural basis for the unexpected specificity profiles of these mutants, high-resolution X-ray crystal structures have been determined for complexes of each mutant with a series of systematically varying peptidylboronic acids. These inhibitory analogues of high-energy reaction intermediates provide models for how substrates with different side chains interact with the enzyme during the transition state. Fifteen structures have been analyzed qualitatively and quantitatively with respect to enzyme-inhibitor hydrogen-bond lengths, buried hydrophobic surface area, unfilled cavity volume, and the magnitude of inhibitor accommodating conformational adjustments (particularly in the region of another binding pocket residue, Val 217A). Comparison of these four parameters with the Ki of each inhibitor and the kcat and Km of the analogous substrates indicates that while no single structural parameter consistently correlates with activity or inhibition, the observed data can be understood as a combination of effects. Furthermore, the relative contribution of each term differs for the three enzymes, reflecting the altered conformational energetics of each mutant. From the extensive structural analysis, it is clear that enzyme flexibility, especially in the region of Val 217A, is primarily responsible for the exceptionally broad specificity observed in either mutant. Taken together, the observed patterns of substrate specificity can be understood to arise directly from interactions between the substrate and the residues lining the specificity pocket and indirectly from interactions between peripheral regions of the protein and the active-site region that serve to modulate active-site flexibility.