Inhibition of camel lens zeta-crystallin/NADPH:quinone oxidoreductase by pyridoxal-5'-phosphate.
Camel lens zeta-crystallin was inhibited by pyridoxal-5'-phosphate (PAL-P) 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 constant of 91 M-1 s-1. The modified enzyme showed the characteristic absorption peak at 325 nm indicative of the formation of phosphopyridoxallysine. Quantitative analysis suggested the incorporation of 1 mole of PAL-P/subunit of enzyme. NADPH was able to substantially protect zeta-crystallin against PAL-P inactivation, whereas the substrate 9,10-phenanthrenequinone (PQ) did not provide any protection. Inhibition of zeta-crystallin by PAL-P was uncompetitive with NADPH (Ki=37 microM) and non-competitive with respect to the substrate (Ki=57 microM). Inhibition of zeta-crystallin by o-phthalaldehyde was used to establish the location of an essential lysine residue. Incubation of zeta-crystallin with o-phthalaldehyde resulted in the formation of an isoindole derivative that had a characteristic fluorescence spectrum. This suggested that a lysine residue is located within 3 A of a cysteine residue at the NADPH binding region. SDS-PAGE showed the o-phthalaldehyde-modified enzyme remained largely monomer (approx. 80%), although bands corresponding to dimer and tetramer forms were also present. These results suggested that an essential lysine residue is located in the vicinity of the NADPH binding site. This residue may simply ensure the proper binding of NADPH to the active site of zeta-crystallin.
Rabbani
N
N
Department of Biochemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia.
Duhaiman
A S
AS
D000818:Animals; D001665:Binding Sites; D002162:Camelus; D003459:Crystallins / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000037:antagonists & inhibitors*; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D009764:o-Phthalaldehyde / Q000494:pharmacology; D038226:zeta-Crystallins
D003459:Crystallins; D004791:Enzyme Inhibitors; D038226:zeta-Crystallins; D011732:Pyridoxal Phosphate; D009764:o-Phthalaldehyde; D016660:NAD(P)H Dehydrogenase (Quinone); D008239:Lysine