Optical activity and solvent perturbation of fumarase

Abstract

The circular dichroism (CD), optical rotatory dispersion (ORD), and the solvent perturbation difference spectra of fumarase in the far- and near-ultraviolet region have been investigated. The CD spectrum of fumarase is characterized by CD bands centering at 222, 209, and 193 nm with [ θ] λ = −18 700, −18 800, and 45 500 degrees·cm 2·dmole −1, respectively. The corresponding ORD spectrum has a trough and maximum at 233 and 198–199 nm, with [ m′] λ = −6880 and 34 900 degrees·cm 2·dmole −1. In the aromatic absorption region the CD spectrum is dominated by tryptophan contributions with a slightly negative band at about 300 nm and positive shoulders and maxima at 291–292, 283, and 275–278 nm, with [ θ] 283 = 30 degrees·cm 2·dmole −1. Phenylalanine contributions to the CD spectrum are observed at 259–261 and 265–268 nm. The analysis of the CD and ORD spectra based on the published model parameters for α-, β-, and random structures suggests the presence of approx. 50% α-helix, 10–30% β-structure, with the remaining portions of the enzyme in an unordered conformation. Inhibitors of fumarase were found to have little or no effect on the far-ultraviolet CD and ORD spectra. Interaction of the enzyme with malonate, succinate, glutarate, or adipate were found to cause a slight enhancement of the CD spectrum in the aromatic region. The solvent perturbation difference spectra of fumarase obtained with 20% ethylene glycol, glycerol, and 50% deuterium oxide as perturbants indicate that 4–6 of the 8 tryptophyls and 20–24 of the 40 tyrosyls occupy surface positions where they are accessible to solvent, with the remaining groups being buried in the interior folds of the enzyme.