Role of lysine residues in the nucleotides binding to bovine liver high-Km aldehyde reductase.
The inactivation of bovine liver high-Km aldehyde reductase (ALR) by heat (47 degrees C), 0.3 mM 2,4,6-trinitrobenzene sulfonate (TNBS) and 0.03 mM pyridoxal 5'-phosphate (PAL-P) followed pseudo-first-order kinetic as a function of incubation-time and concentration of TNBS. Nucleotides which have a 2'-phosphate group, especially beta-NADPH and beta-NADP+, showed effective protection on ALR-inactivation. However, typical substrates for ALR such as D,L-glyceraldehyde, D-erythrose, D-glucuronate and p-carboxybenzaldehyde could not protect the enzyme from inactivation. Completely inactivated enzyme was estimated to have 2.07 TNBS-modified lysine residues/mol enzyme from the determination of free amino group using fluorescamine (Ex = 390 nm, Em = 475 nm). Enzyme protected by beta-NADP+ (96.5% remaining activity) did not lose a significant number of lysine residues. Kd-values for beta-NADPH and beta-NADP+ were estimated to be 0.48 microM and 4.7 microM, respectively and TNBS-treated enzyme lost its ability to bind to these nucleotides.
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Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Osaka University, Japan.
D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D006358:Hot Temperature; D008099:Liver / Q000201:enzymology*; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011725:Pyridines / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D014302:Trinitrobenzenesulfonic Acid / Q000009:adverse effects
D009711:Nucleotides; D011725:Pyridines; D009249:NADP; D011732:Pyridoxal Phosphate; D014302:Trinitrobenzenesulfonic Acid; D000449:Aldehyde Reductase; D008239:Lysine