SITE AND SIGNIFICANCE OF CYSTEINE RESIDUES IN XYLOSE REDUCTASE FROM NEUROSPORA-CRASSA AS DEDUCED BY FLUORESCENCE STUDIES

Inactivation of xylose reductase (XR) by p-hydroxy-mercury benzoate (P HMB) was found to be biphasic with second order rate constants of 80 a nd 6 M-1 s(-1) for the fast (K-f) and slow (K-s) phase respectively. S pectrescopic studies indicated that the inactivation was due to modifi cation of one Cys residue per molecule of XR and not due to subsequent disruption of the quaternary structure. The binding of NADPH to XR (K -d 0.9 mu M) was depressed on modification of the enzyme by PHMB (K-d 2.3 mu M). The dependence of PHMB induced inactivation of XR in the pr esence of alcohols and varying temperature revealed that the Cys resid ue is situated in a hydrophobic microenvironment and is not involved i n hydrogen bonding, Our present investigation using o-phthalaldehyde ( OPTA) as the chemical initiator for fluorescent chemo-affinity labelin g and double inhibition studies indicates that Cys residues involved i n the reaction with PHMB (SHI) and OPTA (SHII) are distinctly differen t, Experimental evidence presented here serves to implicate that SHI l ocated in a hydrophobic microenvironment at the high affinity NADPH bi nding site of XR plays a role in the binding of the coenzyme to XR, wh ereas SHII serves to maintain the conformation of the active site esse ntial for catalysis by interacting with the NH2 group of an essential lysine residue. (C) 1997 Academic Press.