Substrate-induced changes in protease active site conformation impact on subsequent reactions with substrates

Area of Science:

• Biochemistry
• Enzymology

Background:

• Enzymatic catalysis is vital for life.
• Early models like "lock and key" and "induced fit" explain enzyme-substrate interactions.
• The conformational flexibility of enzymes post-substrate binding remains an area of investigation.

Purpose of the Study:

• To investigate the conformational dynamics of Eisenia fetida protease-I upon binding with different chromogenic substrates.
• To determine if substrate-induced enzyme conformations are rigid or flexible.
• To elucidate the mechanisms (lock and key vs. induced fit) employed in sequential enzymatic reactions.

Main Methods:

• Enzyme activity assays were performed on Eisenia fetida protease-I.
• Intrinsic fluorescence measurements were used to monitor enzyme conformation.
• The enzyme was studied with various chromogenic substrates (Chromozym-Th, Chromozym-Ch, Chromozym-U).
• Guanidine hydrochloride was used to perturb enzyme conformation.

Main Results:

• Eisenia fetida protease-I utilized both "lock and key" and "induced fit" mechanisms, varying with substrate.
• Substrates Chromozym-Th and Chromozym-Ch induced conformations that could not bind Chromozym-U.
• The Chromozym-U-induced conformation remained flexible and amenable to further induction.
• The substrate-bound enzyme exhibited relative rigidity compared to the native enzyme, as indicated by fluorescence changes upon guanidine HCl treatment.

Conclusions:

• Enzyme-substrate interactions are dynamic, employing multiple conformational models.
• The degree of conformational change dictates the binding mechanism and subsequent substrate interactions.
• Substrate-induced rigidity via a "lock and key" mechanism may enhance enzymatic reaction efficiency.