Neutron diffraction identifies His 57 as the catalytic base in trypsin

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• The precise role of specific amino acid residues in the active site of serine proteases, such as trypsin, remains a subject of ongoing scientific discussion.
• Previous investigations using techniques like NMR and quantum mechanical calculations have yielded conflicting results regarding the identity of the catalytic base.

Purpose of the Study:

• To definitively identify the chemical base responsible for catalysis in the active site of trypsin.
• To leverage the unique capabilities of neutron diffraction for precise atomic localization, particularly of hydrogen atoms.

Main Methods:

• Covalent inhibition of bovine trypsin using a transition-state analogue, specifically the monoisopropylphosphoryl (MIP) group.
• Acquisition and analysis of a 2.2-Å resolution neutron diffraction dataset of the inhibited trypsin complex.

Main Results:

• Experimental localization of hydrogen atoms within the trypsin active site.
• Clear identification of Histidine 57 (His 57) as the amino acid residue functioning as the catalytic base during the enzymatic reaction.

Conclusions:

• Neutron diffraction provides a direct and unambiguous method for resolving mechanistic questions in enzyme catalysis.
• Histidine 57 is confirmed as the essential catalytic base in the mechanism of trypsin action.