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PMID:19912053

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Contents

Citation

McCrudden, MT, Ryan, LA, Turkington, P and Timson, DJ (2009) The contribution of key hydrophobic residues in ecotin to enzyme-inhibitor complex stability. J Enzyme Inhib Med Chem 24:1207-10

Abstract

The Escherichia coli protease inhibitor ecotin is believed to be implicated in the evasion of host defenses during infection. The protein has also attracted attention as a scaffold for the design of novel, specific protease inhibitors. Ecotin interacts with its targets through two sites. Key hydrophobic residues in both sites (Leu-64, Trp-67, Tyr-69, Met-84, and Met-85) were mutated to alanine and the effects on the inhibition of trypsin, chymotrypsin, and elastase were assessed. Each of these mutant ecotin proteins tested in kinetic assays with these enzymes exerted less inhibitory potency compared to wild-type ecotin. However, these effects were relatively small and not additive.

Links

PubMed Online version:10.3109/14756360902779458

Keywords

Alanine/chemistry; Alanine/genetics; Alanine/metabolism; Amino Acid Sequence; Amino Acid Substitution/genetics; Amino Acid Substitution/physiology; Binding Sites; Chymotrypsin/antagonists & inhibitors; Chymotrypsin/genetics; Chymotrypsin/metabolism; Enzyme Stability; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Hydrophobic and Hydrophilic Interactions; Kinetics; Molecular Sequence Data; Mutation; Pancreatic Elastase/antagonists & inhibitors; Pancreatic Elastase/genetics; Pancreatic Elastase/metabolism; Periplasmic Proteins/chemistry; Periplasmic Proteins/genetics; Periplasmic Proteins/metabolism; Serine Proteinase Inhibitors/genetics; Serine Proteinase Inhibitors/metabolism; Trypsin Inhibitors/genetics; Trypsin Inhibitors/metabolism

Significance

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This work looks at the roles of selected non-polar residues in ecotin. These residues were selected because of their known role in contacting proteases and/or because mutations at these sites has previously been shown to alter the specificity of ecotin.

Typically mutagenic studies of protein function begin by substituting alanine for key residues. However, in the case of ecotin most previously identified mutations were isolated by high-throughput techniques such as phaeg display. This work complements the phage display work by investigating what happens when the side chains of these key residues are (in effect) removed by mutation to alanine. It turns out that the protein is quite tolerant to single, or even multiple changes. Although each alanine mutation tended to reduce the affinity of ecotin for the proteases tested, none abolished this activity completely.

Useful Materials and Methods

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Expression of ecotin was carried out in an ecotine-deleted strain. This was necessary as ecotin is a dimer and is constituitively expressed. Therefore in a non-ecotin-deleted background any mutant forms would be purified as a mixture of mutant homdimers (the desired product), wild type homodimers and mutant/wild type heterodimers.

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