Involvement of a disulfide bridge in catalytic activity of camel lens xi-crystallin

Chemical modification studies were performed to elucidate the role of cysteine residues in the catalytic activity of camel lens xi-crystallin. 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) titration of the camel lens xi-crystallin revealed that it contained 3 SH-groups and a disulfide bridge per subunit. One of the three SH-groups was readily accessible, whereas the other two were buried. Inactivation of xi-crystallin by DTNB was caused by a modification of one cysteine residue per subunit. The resulting DTNB-modified enzyme was reactivated by dithiothreitol (DTT) or KCN. The inactivation was partially protected by NADPH, whereas 9,10-phenanthrenequinone (PQ) enhanced the modification of the enzyme. These results indicate that the SH- group being modified is located at/near the NADPH binding site which is not essential for catalysis. Incubation of the enzyme with DTT led to a substantial loss of the enzyme activity. However, DTT inhibition was prevented completely by preincubation of enzyme with PQ but not by NADPH indicating that an essential disulfide-bridge is present at the substrate binding site of xi-crystallin.