Regulation of oxidation-reduction potentials of anthranilate hydroxylase from Trichosporon cutaneum by substrate and effector binding.
The pH dependence of the redox behavior of anthranilate hydroxylase from Trichosporon cutaneum in its uncomplexed and anthranilate-complexed forms, as well as the effects on the reduction potential, at pH 7.4, of enzyme in complex with 3-methylanthranilate, salicylate, 3-acetylpyridine adenine dinucleotide phosphates, and azide plus anthranilate, is described. At pH 7.4 the midpoint potential of uncomplexed enzyme (EFlox/EFlredH-) is -0.229 V vs SHE, close to that of free flavin. The aromatic substrates and effector all shift the midpoint potential value in a positive direction by 0.068-0.100 V. This shift results in thermodynamically more favorable reduction of the substrate/effector-complexed enzyme by NADPH. Consistent with thermodynamic considerations, the aromatic substrates (or effector) are bound to the reduced enzyme 2-4 orders of magnitude more tightly than to the oxidized enzyme. The tighter binding of the substrate to the two-electron-reduced enzyme may be related to the double hydroxylation reaction performed by this enzyme, which is a more complex reaction than is carried out by typical flavoprotein hydroxylases. The acetylpyridine nucleotides appear to have no significant regulatory role.
Department of Chemistry, University of Minnesota, Minneapolis 55455.
G H
GH
Einarsdottir
M T
MT
Stankovich
J
J
Powlowski
D P
DP
Ballou
V
V
Massey
D001386:Azides; D001665:Binding Sites; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D003904:Mitosporic Fungi / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009249:NADP; D010084:Oxidation-Reduction; D012459:Salicylates; D020156:Salicylic Acid; D013816:Thermodynamics; D014250:Trichosporon / Q000378:metabolism*; D062367:ortho-Aminobenzoates
D001386:Azides; D012459:Salicylates; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; D009249:NADP; D006899:Mixed Function Oxygenases; C023440:anthranilate 2,3-dioxygenase(deaminating); D020156:Salicylic Acid