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Crystal structures of alpha-lytic protease complexes with irreversibly bound phosphonate esters
R Bone1, N S Sampson, P A Bartlett
1Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
Biochemistry
|February 26, 1991
Summary
Two phosphorus stereoisomers of a peptide analogue were studied using X-ray crystallography. One isomer mimics the enzyme
Area of Science:
- Biochemistry
- Structural Biology
- Enzyme Inhibition
Background:
- Alpha-lytic protease is a serine protease.
- Peptide analogues are used to study enzyme mechanisms.
- Phosphorus stereoisomers can exhibit different biological activities.
Purpose of the Study:
- Determine the high-resolution structures of alpha-lytic protease complexes with two phosphorus stereoisomers of a peptide analogue.
- Understand the mechanism of enzyme inactivation by these analogues.
- Compare the binding modes and interactions of the two stereoisomers.
Main Methods:
- X-ray crystallography at 1.9 A resolution.
- Analysis of enzyme-inhibitor interactions, including hydrogen bonding and hydrophobic contacts.
- Kinetic analysis of enzyme inactivation (second-order rate constants).
Main Results:
- Both stereoisomers inactivate alpha-lytic protease, with differing rates.
- Isomer B forms a stable tetrahedral adduct, displacing the phosphonate phenyl ester and interacting across seven substrate recognition sites.
- Isomer A forms a mixture of adducts, with one identical to isomer B and another resulting from hydrolysis of two residues.
- Histidine 57 (H57) rotates out of the active site in isomer B complex but interacts with the phosphonate in one isomer A adduct.
Conclusions:
- The complex with isomer B serves as an excellent mimic of the enzyme's transition state.
- The stereochemistry of the phosphorus analogue significantly influences the binding mode and inactivation mechanism.
- The study provides detailed structural insights into serine protease inhibition.