Direct determination of the protonation states of aspartic acid-102 and histidine-57 in the tetrahedral intermediate of the serine proteases: neutron structure of trypsin

摘要: A neutron structure analysis at 2.2-A resolution has been performed on bovine trypsin covalently inhibited by a transition-state analogue, the monoisopropylphosphoryl (MIP) group. The unique ability of diffraction to locate hydrogen atoms experimentally allowed determination protonation states catalytic site residues (Asp-102 and His-57). Since bound MIP group mimics tetrahedral intermediate structure, these correspond most crucial step hydrolysis. This resolved much debated mechanistic issue showing conclusively that base in transition state reaction is His-57, not Asp-102. finding important implications for understanding hydrolysis mechanism serine proteases. detailed examination stereochemical interaction among groups was also conducted identify their individual roles mechanism. Besides functioning as group, it found His-57 could effectively "steer" attacking water toward acyl during deacylation. Other aspects protein which are observable only discussed. These include orientation well-ordered amide side chains, made possible large scattering difference between nitrogen oxygen atoms, location molecules, exchange properties protein.