Interaction of camel lens zeta-crystallin with quinones: portrait of a substrate by fluorescence spectroscopy.
Interaction of camel lens zeta-crystallin, an NADPH:quinone oxidoreductase, with several quinone derivatives was examined by fluorescence spectroscopy and activity measurements. Fluorescence of zeta-crystallin was quenched to different levels by the different quinones:juglone (5-OH, 1,4 naphthoquinone), 1,4 naphthoquinone (1,4-NQ), and 1,2 naphthoquinone (1,2-NQ) considerably quenched the fluorescence of zeta-crystallin, where as the commonly used substrate, 9,10-phenanthrenequinone (PQ) did not induce significant quenching. Activity measurements showed only PQ served as a substrate for camel lens zeta-crystallin, while juglone, 1,4-NQ, and 1,2-NQ were inhibitors. Thus quinones that interacted with zeta-crystallin directly inhibited the enzyme, whereas the substrate had very low affinity for the enzyme in the absence of NADPH. Another substrate, dichlorophenol indophenol (DCIP), conformed to the same pattern; DCIP did not quench the fluorescence of the enzyme significantly, but served as a substrate. This pattern is consistent with an ordered mechanism of catalysis with quinone being the second substrate. All three naphthoquinones were uncompetitive inhibitors with respect to NADPH and noncompetitive with respect to PQ. These kinetics are similar to those exhibited by cysteine- and/or lysine-modifying agents. Juglone, 1,4-NQ, and 1,2-NQ interacted with and quenched the fluorescence of camel lens alpha-crystallin, but to lesser extent than that of zeta-crystallin.
Bazzi
M D
MD
Department of Biochemistry, College of Science, King Saud University, Riyadh 11451, Saudi Arabia. mbazzi@ksu.edu.sa
D015086:2,6-Dichloroindophenol / Q000494:pharmacology; D000818:Animals; D002162:Camelus; D002384:Catalysis; D003459:Crystallins / Q000737:chemistry* / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D007908:Lens, Crystalline / Q000737:chemistry*; D008024:Ligands; D009249:NADP / Q000378:metabolism; D009285:Naphthoquinones / Q000494:pharmacology; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D003459:Crystallins; D008024:Ligands; D009285:Naphthoquinones; D009249:NADP; C040756:1,2-naphthoquinone; D015086:2,6-Dichloroindophenol; D003545:Cysteine; C035342:1,4-naphthoquinone; C005134:juglone