Abstract
Chemical modification of pig muscle aldose reductase (ALR2) with the arginine specific reagent phenylglyoxal resulted in the inactivation of the enzyme. This inactivation exhibited pseudo-first order kinetics typical of active-site directed chemical modification. Inactivation of ALR2 by [7-C 14] phenylglyoxal in the absence of NADPH or NADP + followed by tryptic digestion resulted in the isolation by HPLC of one major and one minor radioactive peptide. Protein sequencing revealed that the major peptide contained a modified arg 268, a residue located in the coenzyme binding site. The minor radioactive peptide and the single radioactive peptide isolated from ALR2 inactivated in the presence of NADP + contained chemically modified arg 293. The arginine residue modified at the active site is positioned to bind the 2′-OH phosphate group of the ribose sugar of the adenine moiety of NADP +. Arg 293 is present on the C-terminal loop of ALR2. The enhancement of Arg 293 modification by phenylglyoxal in the presence of NADP + indicates that this C-terminal loop may be involved in the slow conformational change that occurs during the reaction sequence upon coenzyme binding.
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