Structure-based design of a lysozyme with altered catalytic activity

Area of Science:

• Enzymology
• Structural Biology
• Biochemistry

Background:

• T4 lysozyme is a well-studied enzyme involved in breaking down bacterial cell walls.
• Understanding the catalytic mechanisms of enzymes like lysozyme is crucial for various biological and biotechnological applications.
• Previous studies suggest evolutionary links between different lysozymes, but mechanistic details remain debated.

Purpose of the Study:

• To investigate the impact of a specific active site mutation (Threonine 26 to Histidine) in T4 lysozyme.
• To elucidate the catalytic mechanism of wild-type T4 lysozyme and a mutant variant (T26H).
• To compare the enzymatic mechanism of T4 lysozyme with that of hen egg-white lysozyme.

Main Methods:

• Site-directed mutagenesis to create the T26H mutant of T4 lysozyme.
• Analysis of the reaction product anomeric configuration (alpha vs. beta).
• Examination of relevant crystal structures to infer mechanistic details.

Main Results:

• The Thr 26 --> His substitution in T4 lysozyme shifted the product from the alpha-anomer to the beta-anomer.
• Wild-type T4 lysozyme operates via an anomer-inverting, single displacement mechanism, with water attacking from the alpha-side.
• The T26H mutant functions as an anomer-retaining enzyme, suggesting a double displacement mechanism with water attacking from the opposite side.

Conclusions:

• The T26H mutation fundamentally alters the catalytic mechanism of T4 lysozyme.
• Wild-type T4 lysozyme employs a distinct catalytic mechanism compared to hen egg-white lysozyme, despite presumed common ancestry.
• This study provides key insights into enzyme reaction pathways and the evolution of lysozyme function.