Active site studies of cytochrome P=450CAM. I. Specific cysteine labeling with the affinity reagent isobornyl bromoacetate as a model for substrate binding.
A model is presented suggesting a function of specific cysteine residue of cytochrome P-450CAM in binding the substrate camphor, via a thiohemiketal bond, for its correct orientation to the heme iron and for the subsequent transfer of nascent product to facilitate its release. This model was developed to explain the results of affinity labeling with isobornyl bromoacetate. This reagent couples to the proteins via a thioether bond to cysteine, eliciting a type I transition in the difference spectrum. Formation of this covalent complex, which is strongly inhibited by the substrate, can be monitored by quantitation of S-carboxymethylcysteine in acid hydrolyzates. While addition of one equivalent of label yields 0.3 equivalents of the cysteine derivative after 5 min, increasing to 0.8 equivalents after 24 h, the spectral shift decays with time. Kinetic analysis of the spectral decay and of covalent coupling strongly suggests that thioether bond formation occurs at the substrate binding-site, in a reaction step prior to, and distinct from, the step associated with the spectral decay. The P-450CAM derivative, when titrated with camphor, produced again a type I spectrum virtually identical with the spectrum of the native P-450CAM-substrate complex. While the model presented here is not the only possible interpretation of the results, it is fully consistent with them and provides an excellent framework for further study of the catalytic mechanism of P-450CAM.
D000345:Affinity Labels / Q000494:pharmacology*; D001892:Camphanes / Q000138:chemical synthesis / Q000494:pharmacology*; D003545:Cysteine; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D011549:Pseudomonas / Q000201:enzymology
D000345:Affinity Labels; D001892:Camphanes; C036205:isobornyl bromoacetate; D003577:Cytochrome P-450 Enzyme System; D003545:Cysteine