INHIBITION OF CAMEL LENS ZETA-CRYSTALLIN NADPH-QUINONE OXIDOREDUCTASEBY PYRIDOXAL-5'-PHOSPHATE/

Camel lens zeta-crystallin was inhibited by pyridoxal-5'-phosphate (PA LP) and o-phthalaldehyde. PAL-P inactivated zeta-crystallin in a time- and concentration-dependent manner. The initial rate of inactivation followed pseudo-first-order kinetics with the second-order rate consta nt of 91 M-1 s(-1). The modified enzyme showed the characteristic abso rption peak at 325 nm indicative of the formation of phosphopyridoxall ysine. Quantitative analysis suggested the incorporation of 1 mole of PALP/subunit of enzyme. NADPH was able to substantially protect zeta-c rystallin against PAL-P inactivation, whereas the substrate 9,10-phena nthrenequinone (PQ) did not provide any protection. Inhibition of zeta -crystallin by PAL-P was uncompetitive with NADPH (K-i = 37 mu M) and non-competitive with respect to the substrate (K-i = 57 mu M) Inhibiti on of zeta-crystallin by o-phthalaldehyde was used to establish the lo cation of an essential lysine residue. Incubation of zeta-crystallin w ith o-phthalaldehyde resulted in the formation of an isoindole derivat ive that had a characteristic fluorescence spectrum. This suggested th at a lysine residue is located within 3 Angstrom of a cysteine residue at the NADPH binding region. SDS-PAGE showed the a-phthalaldehyde-mod ified enzyme remained largely monomer (approx. 80%), although bands co rresponding to dimer and tetramer forms were also present. These resul ts suggested that an essential lysine residue is located in the vicini ty of the NADPH binding site, This residue may simply ensure the prope r binding of NADPH to the active site of zeta-crystallin. (C) 1998 Els evier Science B.V. All rights reserved.