A rapid reaction study of anthranilate hydroxylase. Evidence for a catalytically important conformational change during slow initial turnover with anthranilate

• Published in: The Journal of biological chemistry 1989-09, Vol.264 (27), p.16008-16016
• Main Authors: POWLOWSKI, J, BALLOU, D, MASSEY, V
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Binding, Competitive; Biological and medical sciences; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Kinetics; Ligands; Mathematics; Mitosporic Fungi - enzymology; Mixed Function Oxygenases - metabolism; Models, Theoretical; o-aminobenzoic acid; ortho-Aminobenzoates - metabolism; Oxidation-Reduction; Oxidoreductases; Protein Conformation; Salicylates - metabolism; Salicylic Acid; Spectrophotometry; Trichosporon - enzymology; Trichosporon cutaneum
• ISSN: 0021-9258; 1083-351X

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.