Bovine lens aldose reductase. pH-dependence of steady-state kinetic parameters and nucleotide binding.
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 pKa of 5.3 and a pKb 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 pKi 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 pKi 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.
Liu
S Q
SQ
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77555.
Bhatnagar
A
A
Srivastava
S K
SK
D000449:Aldehyde Reductase / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D003067:Coenzymes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007908:Lens, Crystalline / Q000201:enzymology*; D008433:Mathematics; D008962:Models, Theoretical; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D013379:Substrate Specificity
D003067:Coenzymes; D009243:NAD; D009249:NADP; C005941:3-acetylpyridine adenine dinucleotide; D000449:Aldehyde Reductase