Kinetic and structural studies on roles of the serine ligand and a strictly conserved tyrosine residue in nitrile hydratase

摘要

Nitrile hydratases (NHase), which catalyze the hydration of nitriles to amides, have an unusual Fe~(3+) or Co~(3+) center with two modified Cys ligands: cysteine sulfininate (Cys-SO_2~-) and either cysteine sulfenic acid or cysteine sulfenate [Cys-SO(H)]. Two catalytic mechanisms have been proposed. One is that the sulfenyl oxygen activates a water molecule, enabling nucleophilic attack on the nitrile carbon. The other is that the Ser ligand ionizes the strictly conserved Tyr, activating a water molecule. Here, we characterized mutants of Fe-type NHase from Rhodococcus erythropolis N771, replacing the Ser and Tyr residues, αS113A and βY72F. The αS113A mutation partially affected catalytic activity and did not change the pH profiles of the kinetic parameters. UV-vis absorption spectra indicated that the electronic state of the Fe center was altered by the αS113A mutation, but the changes could be prevented by a competitive inhibitor, n-butyric acid. The overall structure of the αS113A mutant was similar to that of the wild type, but significant changes were observed around the catalytic cavity. Like the UV-vis spectra, the changes were compensated by the substrate or product. The Ser ligand is important for the structure around the catalytic cavity, but is not essential for catalysis. The βY72F mutant exhibited no activity. The structure of the βY72F mutant was highly conserved but was found to be the inactivated state, with αCys114-SO(H) oxidized to Cys-SO_2~-, suggesting that βTyr72 affected the electronic state of the Fe center. The catalytic mechanism is discussed on the basis of the results obtained.