Human placental aldose reductase: role of Cys-298 in substrate and inhibitor binding.
Steady-state kinetic and inhibition properties of human placental aldose reductase carboxymethylated at Cys-298 were investigated. A comparison of the primary deuterium kinetic isotope effect on the reduced and the carboxymethylated enzymes suggests that carboxymethylation did not affect the reaction sequence of substrate binding and release. Values of DV/KD-glyceraldehyde greater than DV suggest that steps in the reaction scheme subsequent to hydride transfer, particularly the release of NADP may be rate limiting. Carboxymethylation of Cys-298 was also found to affect NADPH and aldehyde binding to the enzyme. Carboxymethylation had little effect on the secondary structure of the enzyme, but a comparison of the circular dichroic spectra of the reduced and carboxymethylated enzyme, suggests a weakened interaction between the nicotinamide and 2'-monophosphoadenosine 5'-diphosphoribose of NADPH, and the carboxymethylated enzyme. Interaction between Cys-298 and NADPH appears to determine the rate of isomerization of the E:NADP binary complex and carboxymethylation-induced decrease in kcat may be due to slower isomerization of the E:NADP binary complex. The carboxymethylated enzyme was less sensitive than the reduced enzyme to most aldose reductase inhibitors including sorbinil (d-6-fluoro-spiro[chroman-4,4'-imidazolidine]-2',5'-dione), except tolrestat (N-methyl-N-[(5-trifluromethyl-6-methoxy-1-naphthalenyl)- thiomethyl]glycine) and quercetin. On the basis of these observations it is suggested that Cys-298 may form a part of the 'S'-inhibitor binding site of the enzyme and may be responsible for tight binding of NADPH.
Bhatnagar
A
A
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77555.
Liu
S Q
SQ
Ueno
N
N
Chakrabarti
B
B
Srivastava
S K
SK
D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism*; D002942:Circular Dichroism; D003545:Cysteine / Q000378:metabolism; D003903:Deuterium; D005260:Female; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D048289:Imidazolidines; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007700:Kinetics; D009249:NADP / Q000378:metabolism*; D009281:Naphthalenes / Q000494:pharmacology; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011794:Quercetin / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet
D000447:Aldehydes; D007093:Imidazoles; D048289:Imidazolidines; D007461:Iodoacetates; D009281:Naphthalenes; C040550:tolrestat; D009249:NADP; D011794:Quercetin; D003903:Deuterium; D000449:Aldehyde Reductase; C026411:sorbinil; D003545:Cysteine; D019807:Iodoacetic Acid