Conformation and microenvironment of the active site of xylose reductase inferred by fluorescent chemoaffinity labeling.
Conformation and microenvironment at the active site of xylose reductase (XR) from Neurospora crassa was probed with fluorescent chemoaffinity labeling (FCAL) using o-phthalaldehyde as a chemical initiator. Formation of a single isoindole derivative resulted in complete inactivation of the enzyme as judged by spectroscopic and fluorescence studies. Kinetic analysis of the 2,4,6-trinitrobenzenesulfonic-acid-modified XR implicated the presence of an essential lysine residue at the active site of XR. Modification of lysine in XR abolished the ability of the enzyme to form isoindole derivative, indicating that the lysine residue involved in the reaction with 2,4,6-trinitrobenzenesulfonic acid and o-phthalaldehyde is the same and that the probe o-phthalaldehyde is directed to the active site. Fluorescence studies revealed that inactivation of XR by Gdn/HCl precedes gross conformational change and the possibility of secondary-conformational change was eliminated by acrylamide quenching studies. The enzyme inactivated by low concentrations of Gdn/HCl retained its ability to form the fluorescent XR-isoindole derivative indicating that inactivation is not due to conformational changes at or near the active site of XR. Gdn/HCl also had no effect on the high-affinity and low-affinity NADPH-binding sites of XR. Energy-transfer experiments further revealed structural integrity at the active site of the Gdn/HCl-inactivated XR. Changes in the fluorescence emission maximum of 1-(beta-hydroxyethylthio)-2-beta hydroxyethyl isoindole (EA adduct) in solvents of varying polarity was studied, the data obtained were utilized to interpret the fluorescence behaviour of XR-isoindole derivative and assess the polarity at the active site. Experimental evidence presented here serves to suggest that the inactivation of XR by Gdn/HCl precedes conformational changes at the active site located in a microenvironment of low polarity.
Rawat
U B
UB
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Rao
M B
MB
D000345:Affinity Labels; D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D009249:NADP / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D009764:o-Phthalaldehyde / Q000494:pharmacology
D000345:Affinity Labels; D006146:Guanidines; D009249:NADP; D009764:o-Phthalaldehyde; D000449:Aldehyde Reductase; D019791:Guanidine