IN-VIVO AND IN-VITRO EFFECTS OF MUTAGENESIS OF ACTIVE-SITE TYROSINE RESIDUES OF MERCURIC REDUCTASE

X-ray crystal structure analysis of mercuric reductase suggested that the binding site for Hg2+ consisted of two tyrosine residues, Tyr(264) and Tyr(605), as well as two cysteine residues, Cys(207) and Cys(628) . We have previously shown that mutagenesis of Tyr(605) to Phe lowered the k(cat) of the enzyme 6-fold, whereas the same mutation of Tyr(264 ) resulted in a reduction of 160-fold [(1993) Biochemistry 32, 7475-74 78]. Tyr(605) occupies the same position in mercuric reductase as the active site His residue in the related enzyme glutathione reductase. T he mutation of Tyr(605) of mercuric reductase to a His residue produce d a 24-fold decrease in k(cat) and a 15-fold decrease in K-m. The in v ivo resistance to Hg2+ of E. coli strains carrying wild type or mutant merA genes correlated with the in vitro measurements of k(cat)/K-m fo r mercuric reductase activity.