Rat intestinal trehalase. Studies of the active site

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Trehalase is a key enzyme in carbohydrate metabolism.
• Understanding trehalase's active site is crucial for enzyme mechanism studies.

Purpose of the Study:

• To elucidate the structural and functional characteristics of the rat intestinal trehalase active site.
• To identify key amino acid residues and binding regions within the trehalase active site.

Main Methods:

• Solubilization and purification of rat intestinal trehalase using octyl glucoside.
• Sodium dodecyl sulfate polyacrylamide-gel electrophoresis (SDS-PAGE) for molecular mass determination.
• Kinetic studies, site-directed mutagenesis, and chemical modification (e.g., N-ethylmaleimide, iodoacetate) to probe active site properties.
• Inhibition studies with various compounds (e.g., phlorizin, Tris-like inhibitors) and pH-dependent analyses.

Main Results:

• Purified trehalase exhibited a molecular mass of 67 kDa.
• The active site comprises a binding site (pKa 4.8) and a catalytic site (pKa 7.2).
• A functional thiol group in the catalytic site is sensitive to specific inhibitors.
• Sucrose and phlorizin protect the thiol group, while Tris-like inhibitors do not.
• A separate Tris binding site with a carboxyl group was identified.

Conclusions:

• A detailed model of the rat intestinal trehalase active site, including distinct binding and catalytic regions, has been proposed.
• The findings highlight the importance of a thiol group in catalysis and a carboxyl group in substrate/inhibitor binding.
• This study provides insights into trehalase's catalytic mechanism and potential for targeted inhibition.