A rapid reaction study of anthranilate hydroxylase. Evidence for a catalytically important conformational change during slow initial turnover with anthranilate.
Rapid reaction kinetics of the flavoprotein anthranilate hydroxylase from Trichosporon cutaneum were examined for reactions involving anthranilate, the native substrate. As was reported earlier for the nonhydroxylated substrate analogue, salicylate, some reactions in the first turnover with anthranilate occur slower than those in subsequent turnovers (Powlowski, J., Massey, V., and Ballou, D. P. (1989) J. Biol. Chem. 264, 5606-5612). Evidence is presented for slow conformational changes that occur both on binding of the aromatic ligand and on reduction of the enzyme. These changes are apparently important for rapid anthranilate binding to occur in turnovers subsequent to the first. Moreover, bound anthranilate is required for rapid reduction of enzyme-bound FAD by NADPH. Studies to probe the accessibility of reagents to modified flavins that had been incorporated into the apoenzyme indicate that anthranilate binding causes a conformational change in the protein, allowing increased access to the benzene ring moiety of the flavin. An unusual isotope effect with (R)-NADPD (4(R)-2H] NADPH) is observed on Kd rather than on kred, which is consistent with a model involving slow interconversion of enzyme-substrate complexes before productive binding of NADPH and reduction of the enzyme flavin.
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606.
J
J
Powlowski
D
D
Ballou
V
V
Massey
D001667:Binding, Competitive; D007700:Kinetics; D008024:Ligands; D008433:Mathematics; D003904:Mitosporic Fungi / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008962:Models, Theoretical; D010084:Oxidation-Reduction; D011487:Protein Conformation; D012459:Salicylates / Q000378:metabolism; D020156:Salicylic Acid; D013053:Spectrophotometry; D014250:Trichosporon / Q000201:enzymology*; D062367:ortho-Aminobenzoates / Q000378:metabolism
D008024:Ligands; D012459:Salicylates; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; D006899:Mixed Function Oxygenases; C023440:anthranilate 2,3-dioxygenase(deaminating); D020156:Salicylic Acid