Purification, kinetic characterization and involvement of tryptophan residue at the NADPH binding site of xylose reductase from Neurospora crassa.
Xylose reductase (XR) from Neurospora crassa was purified to homogeneity and was found to be specific to NADPH (nicotinamide adenine dinucleotide phosphate). The purified enzyme showed M(r) of 60 and 29 kDa by gel filtration and SDS-PAGE indicating the presence of two subunits. The kinetic mechanism of xylose reductase is 'iso-ordered bi bi'. Inactivation of XR by N-bromosuccinimide (NBS) was found to be biphasic with second-order rate constants of 2.5 x 10(2) and 80 M-1S-1 for the fast (kf) and slow phase (ks), respectively. NADPH protected 90% of XR activity against inhibition by NBS. The fluorescence and circular dichroism (CD) studies revealed that inactivation was not due to gross conformational change in the enzyme. Analysis of the modified Stern-Volmer plot indicated that 49% of the tryptophanyl fluorescence was available for quenching which was completely abolished in the presence of NADPH confirming the involvement of tryptophan at the coenzyme binding site. Experimental evidence presented here serves to implicate the involvement of a tryptophan residue at the low-affinity NADPH binding site and the nature of this site has been assessed by using the hydrophobic probe ANS.
Rawat
U B
UB
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Rao
M B
MB
D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001665:Binding Sites; D001974:Bromosuccinimide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism* / Q000494:pharmacology; D009492:Neurospora crassa / Q000201:enzymology*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism*
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D009249:NADP; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D000449:Aldehyde Reductase; D001974:Bromosuccinimide