Characterization of retinaldehyde dehydrogenase 3

RALDH3 (retinal dehydrogenase 3) was characterized by kinetic and binding studies, protein engineering, homology modelling, ligand clocking and electrostatic-potential calculations. The major recognition determinant of an RALDH3 substrate was shown to be all eight-carbon chain bonded to the aldehyde group Whose kinetic influence (k(cat)/K-m at pH 8.5) decreases when shortened or lengthened. Surprisingly, the beta-ionone ring of all-trans-retinal is not a major recognition site. The dissociation constants (K-d) Of the complexes of RALDH3 with octanal, NAD(+) and NADH were determined by intrinsic tryptophan fluorescence. The similarity of the Kd values for the complexes with NAD(+) and with octanal suggests a random kinetic mechanism for RALDH3, ill contrast with the ordered sequential mechanism often associated with aldehyde dehydrogenase enzymes. Inhibition of RALDH3 by tri-iodothyronine binding ill competition with NAD(+), predicted by the modelling, was established kinetically and by immunoprecipitation. Mechanistic implications of the kinetically influential ionizations with macroscopic pK(a) values of 5.0 and 7.5 revealed by the pH-dependence of are discussed. Analogies with data for non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans, together with the present modelled structure OF the thioacyl RALDH3, Suggest (a) that k(cat) characterizes deacylation of this intermediate for specific substrates and (b) the assignment of the pK(a) of the major ionization (approximating to 7.5) to the perturbed carboxy group of Glu(280) whose Conjugate base is envisaged as Supplying general base catalysis to attack of a water-molecule. The macroscopic pK(a) of the minor ionization (5.0) is considered to approximate to that of the carboxy group of Glu(488)..