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 homogenei ty and was found to be specific to NADPH (nicotinamide adenine dinucle otide phosphate). The purified enzyme showed M(r) of 60 and 29 kDa by gel filtration and SDS-PAGE indicating the presence of two subunits. T he kinetic mechanism of xylose reductase is 'iso-ordered bi bi', inact ivation of XR by N-bromosuccinimide (NBS) was found to be biphasic wit h second-order rate constants of 2.5 . 10(2) and 80 M(-1) s(-1) for th e fast (k(f)) and slow phase (k(s)), respectively. NADPH protected 90% of XR activity against inhibition by NBS. The fluorescence and circul ar dichroism (CD) studies revealed that inactivation was not due to gr oss conformational change in the enzyme. Analysis of the modified Ster n-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 bin ding site, Experimental evidence presented here serves to implicate th e involvement of a tryptophan residue at the low-affinity NADPH bindin g site and the nature of this site has been assessed by using the hydr ophobic probe ANS.