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 (PHMB) was found to be biphasic with second-order rate constants of 80 and 6 M-1s-1 for the fast (kf) and slow (ks) phase respectively. Spectroscopic studies indicated that the inactivation was due to modification of one Cys residue per molecule of XR and not due to subsequent disruption of the quaternary structure. The binding of NADPH to XR (Kd 0.9 microM) was depressed on modification of the enzyme by PHMB (Kd 2.3 microM). The dependence of PHMB induced inactivation of XR in the presence of alcohols and varying temperature revealed that the Cys residue is situated in a hydrophobic microenvironment and is not involved in hydrogen bonding. Our present investigation using o-phthalaldehyde (OPTA) as the chemical initiator for fluorescent chemo-affinity labeling and double inhibition studies indicates that Cys residues involved in the reaction with PHMB (SHI) and OPTA (SHII) are distinctly different. Experimental evidence presented here serves to implicate that SHI located in a hydrophobic microenvironment at the high affinity NADPH binding site of XR plays a role in the binding of the coenzyme to XR, whereas SHII serves to maintain the conformation of the active site essential for catalysis by interacting with the NH2 group of an essential lysine residue.
Rawat
U
U
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Rao
M
M
D000449:Aldehyde Reductase / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive; D002942:Circular Dichroism; D003545:Cysteine / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006902:Hydroxymercuribenzoates / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D009492:Neurospora crassa / Q000201:enzymology*; D011487:Protein Conformation / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D009764:o-Phthalaldehyde / Q000378:metabolism / Q000494:pharmacology
D006902:Hydroxymercuribenzoates; C030601:4-hydroxymercuribenzoate; D009764:o-Phthalaldehyde; D000449:Aldehyde Reductase; D008239:Lysine; D003545:Cysteine