The pH-dependence of nucleotide binding and steady-state kinetic parameters of aldehyde reduction and alcohol oxidation catalyzed by bovine lens aldose reductase were studied. The maximal velocity of aldehyde reduction with NADPH and p-chlorobenzaldehyde was pH independent at low pH but decreased at high pH with a pK of 7.6. The V/K of NADPH displayed a bell- shaped dependence on pH and decreased with a pK(a) of 5.3 and a pK(b) of 7.5. The dissociation constant of NADPH and 3-acetylpyridine adenine dinucleotide phosphate (3-APADP) increased at low pH with a pK of 5.6-5.8 and at high pH with a pK of 9.4-9.7. The pK(i) of NADP and NADPH decreased below a pH of 5 and 6.7 and above a pH of 8.5 and 9.7, respectively. The pK of 8.5-9.7 appears to be due to the interaction of the 2'-phosphate of the nucleotide with a protonated base, possibly a lysine residue. The maximal velocity of alcohol oxidation was pH independent at high pH but decreased at low pH with a pK of 6.5-7.0, when p-chlorobenzyl alcohol or benzyl alcohol and 3-APADP were used. The amino acid residue for alcohol binding has a pK of 7.5-8.2 and also appears in pK(i) profiles of sorbinil, a competitive inhibitor versus the alcohol. Large (3-3.5) isotope effects on maximal velocity obtained with benzyl alcohol and 3-APADP suggest that with these substrates the hydride transfer step is rate-limiting and a pK of 6.5-7.0 may be the true pK of the acid-base catalyst, possibly a histidine.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - Jan 1 1993|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology