IDENTIFICATION OF THE REACTIVE CYSTEINE RESIDUE IN HUMAN PLACENTA ALDOSE REDUCTASE

Modification of human placental aldose reductase by iodoacetate (IAA) led to a mol/mol binding of IAA, a 40% decrease in the k(cat), a 3-5-f old increase in the K(m,NADPH) and K(m,glyceraldehyde) and a 600-fold increase in the K(i,sorbinil); determined at pH 6.0. NADPH and 2'-mono phosphoadenosine 5'-diphosphoribose but neither glyceraldehyde nor sor binil, prevented carboxymethylation-induced changes. Cleavage of [C-14 ]IAA-modified enzyme by trypsin resulted in two radiolabeled peptides: Val-297 to Lys-307 and Val-297 to Phe-315. In both these peptides Cys -298 was the only radiolabeled residue. It is suggested that Cys-298 r egulates the kinetic and inhibition properties of the enzyme, but does not participate in catalysis.