Differential behavior of the sub-sites of cytochrome 450 active site in binding of substrates, and products (implications for coupling/uncoupling).
The cytochrome P450 catalyzes hydroxylation of many substrates in the presence of O(2) and specific electron transport system. The ternary complex S-Fe(+)O(2) with substrate and O(2) bound to their respective sites on the reduced enzyme is an important intermediate in the formation of the hydroxylating species. Then the active site may be considered as having two sub-sites geared for entirely different types of functionally relevant interactions. The two sites are the substrate binding site, the specific protein residues (Site I), and the L(6) position of the iron (Site II) to which O(2) binds upon reduction. In the ferric enzyme, when substrate binds to Site I, the low spin six-coordinated P450 is converted to the readily reducible high spin five coordinated state. Certain amines and OH compounds, such as products of P450-catalyzed reactions, can bind to Site II resulting in six coordinated inhibited complexes. Then the substrate and product interactions with the two sub-sites can regulate the functional state of the enzyme during catalysis. Product interactions have received very little attention. CYP101 is the only P450 in which X-ray and spectroscopic data on all three structures, the substrate-free, camphor-bound and the 5-exo-OHcamphor-bound are available. The substrate-free CYP101 is low spin and six-coordinated with a water molecule ligated at the L(6) position of the iron. The substrate camphor binds to Site I, and releases the L(6) water despite its inability to bind to this site, indicating that Site I binding can inhibit Site II ligation. The product 5-exo-OHcamphor in addition to binding to Site I, binds to Site II through its -OH group forming Fe-O bond, resulting in the low spin six-coordinated complex. New temperature-jump relaxation kinetic data indicating that Site II ligation inhibits Site I binding are presented. It appears that the Site I and Site II function as interacting sub-sites. The inhibitory allosteric interactions between the two sub-sites are also reflected in the data on binding of the substrate camphor (S) in the presence of the product 5-exo-OH camphor (P) to CYP101 (E). The data are in accordance with the two-site model involving the ternary complex ESP. The affinity of the substrate to the product-bound enzyme as well as the affinity of the product to the substrate-bound enzyme decreased with increase in product concentration, which is consistent with mixed inhibition indicative of inhibitory allosteric interactions between the two sub-sites. Implications of these observations for coupling/uncoupling mechanisms are discussed in the light of the published findings consistent with the two-site behavior of the P450 active site. In addition, kinetic data indicating that the transient high spin intermediate may have to be taken into account for understanding how some P450s have been able to express appreciable hydroxylation activities in the absence of substrate-induced low to high spin transition, observable by the traditional static spectroscopy, are presented.
Narasimhulu
Shakunthala
S
Harrison Department For Surgical Research, University of Pennsylvania, Philadelphia, PA 19104, USA. Shaknar@comcast.net
D000494:Allosteric Regulation; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D001665:Binding Sites; D002164:Camphor / Q000378:metabolism; D019475:Camphor 5-Monooxygenase / Q000378:metabolism; D002384:Catalysis; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D004791:Enzyme Inhibitors / Q000378:metabolism; D006900:Hydroxylation; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D013379:Substrate Specificity
D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D002164:Camphor; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit); D019475:Camphor 5-Monooxygenase