Kinetic studies of FR-1, a growth factor-inducible aldo-keto reductase.
Murine fibroblasts cultured in the presence of fibroblast growth factor-1 express relatively high levels of FR-1, a approximately 36 kDa protein related to the aldo-keto reductase superfamily [Donohue, P. J., Alberts, G. F., Hampton, B. S., Winkles, J. A. (1994) J. Biol. Chem. 269, 8604-8609]. While the crystal structure of FR-1 shows striking homology with human aldose reductase [Wilson, D. K., Nakano, T., Petrash, J. M., Quiocho, F. A. (1995) Biochemistry 34, 14323-14330], an enzyme linked to the pathogenesis of diabetic complications, the physiological role of FR-1 is not known. We show that FR-1 is capable of reducing a broad range of aromatic and aliphatic aldehydes, including the abundant and highly reactive lipid-derived aldehyde 4-hydroxy-2-nonenal (HNE; Km approximately 9 microM). However, in the absence of coenzyme, HNE caused a time-dependent inactivation of FR-1. Results from electrospray ionization-mass spectrometry and Edman-degradation of peptides derived from HNE-modified FR-1 were consistent with formation of a Michael adduct at Cys298. This was confirmed with a C298S mutant, which was resistant to HNE-induced inactivation. Since steady-state Km values determined with alkanals, alpha,beta-unsaturated alkenals, alkadienals, and 4-hydroxyalkenals fall within their physiological concentrations, lipid-derived aldehydes appear to be potential in vivo substrates for FR-1.
Srivastava
S
S
Department of Human Biological Chemistry & Genetics, University of Texas Medical Branch, Galveston, USA.
Harter
T M
TM
Chandra
A
A
Bhatnagar
A
A
Srivastava
S K
SK
Petrash
J M
JM
D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism / Q000494:pharmacology; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D003545:Cysteine / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor; D006133:Growth Substances / Q000494:pharmacology*; D007700:Kinetics; D013058:Mass Spectrometry; D051379:Mice; D016297:Mutagenesis, Site-Directed; D014176:Protein Biosynthesis; D011506:Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics; D013379:Substrate Specificity
D000447:Aldehydes; C464944:Emr4 protein, mouse; D006133:Growth Substances; D011506:Proteins; D012694:Serine; D004815:Epidermal Growth Factor; D000449:Aldehyde Reductase; C027576:4-hydroxy-2-nonenal; D003545:Cysteine