Apoenzyme of Pseudomonas cepacia salicylate hydroxylase. Preparation, fluorescence property, and nature of flavin binding.
The apoenzyme of Pseudomonas cepacia salicylate hydroxylase was prepared by a dialysis method. The apoprotein retains a dimeric structure and binds one FAD per monomer. Flavin binding results in both 81 and 60% of quenching and 15- and 5-nm blue shifts of FAD and protein fluorescence, respectively. A hydrophobic environment for the flavin site and a conformational difference between apoprotein and holoenzyme are thus indicated. Prior binding of NADH markedly retards the holoenzyme activity development upon a subsequent FAD addition. Flavin 1,N6-ethenoadenine dinucleotide binds to the apoenzyme much more weakly than FAD but this reconstituted holoenzyme and the FAD X enzyme both exhibit similar activities. The adenine moiety appears to be important to binding. The formation of holoenzyme from apoprotein and FAD involves minimally a two-step reversible process, an initial flavin-binding step followed by a conformational transition. At both 6 and 23 degrees C, the rates of hydroxylase activity recovery can be correlated with the rates of FAD binding, indicating that the initial FAD X apoenzyme complex is fully active and the subsequent slow conformational change has no significant effect on the catalytic efficiency. Overall dissociation constants calculated based on kinetic data are essentially identical with those determined by equilibrium measurements.
D001051:Apoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D001059:Apoproteins / Q000302:isolation & purification*; D003956:Dialysis; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D046911:Macromolecular Substances; D006899:Mixed Function Oxygenases / Q000302:isolation & purification* / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D001051:Apoenzymes; D001059:Apoproteins; D046911:Macromolecular Substances; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D006899:Mixed Function Oxygenases; C022619:salicylate 1-monooxygenase