Kinetic Studies of FR-1, a Growth Factor-Inducible Aldo-Keto Reductase

Abstract

Murine fibroblasts cultured in the presence of fibroblast growth factor-1 express relatively high levels of FR-1, a ∼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; K_m ≈ 9 μM). 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 K_m values determined with alkanals, α,β-unsaturated alkenals, alkadienals, and 4-hydroxyalkenals fall within their physiological concentrations, lipid-derived aldehydes appear to be potential in vivo substrates for FR-1.