ROLE OF TYROSINE RESIDUES IN HG(II) DETOXIFICATION BY MERCURIC REDUCTASE FROM BACILLUS SP STRAIN RC607

Two tyrosine residues of mercuric reductase (MerA), Tyr-264 and Tyr-60 5, which were shown by the X-ray crystal structure to be involved in m etal binding, were changed to phenylalanine residues by site-directed mutagenesis, both singly (Y264F, Y605F) and to form a double mutant (Y 264,605F). The effect of these mutations on Hg(II) reduction activity varied. While MerA Y605F has a similar apparent K(m) to the wild-type enzyme and an apparent k(cat) reduced by 6-fold, MerA Y264F has an app arent K(m) 5-fold lower than the wild type and apparent k(cat) 160-fol d lower. The double mutant MerA Y264,605F has the same apparent K(m) a s MerA Y264F, but its apparent k(cat) was reduced by a further 7-fold. These results show that the roles of the two tyrosine residues are no t equivalent and that Y264 is important for catalysis, possibly by des tabilizing the binding of Hg(II) to the two ligating thiolates at the active site of MerA.