Specific and non-specific effects of potassium cations on substrate-protein interactions in cytochromes P450cam and P450lin.
Substrate binding to cytochrome P450cam is generally considered to be a two-step process. The first step corresponds to the entrance of the substrate, camphor, into the heme pocket. The second step corresponds to a spin transition (low spin-->high spin) of the iron in the protein-substrate complex. This spin transition is related to the mobility of the substrate inside the active site [Biochim Biophys Acta 1338 (1997) 77]. Potassium cations (K(+)) have a specific effect on the spin equilibrium. This is generally attributed to the K(+) ion-induced conformational change of tyrosine 96, the hydroxyl group of which is hydrogen bonded to the keto group of camphor and results in optimum substrate orientation and reduced mobility of this substrate in the active site. In the present paper, we show that K(+) not only affects the substrate-Tyr 96 couple, but acts more globally since K(+) effects are also observed in the Tyr96Phe mutant as well as in complexes with camphor-analogues. Large compounds, that fit well in the heme pocket and bind with higher affinity than camphor, display high spin contents that are less dependent on the presence of K(+). In contrast, K(+) has a significant effect on the high spin content of substrate-cytochrome P450cam complexes with looser interactions. We conclude that large compounds with higher affinities than camphor have more van der Waals contacts with the active site residues. Their mobilities are then reduced and less dependent on the presence of K(+). In this study, we also explored, for comparison, the K(+) effect on the spin transition state of another member of the P450 superfamily, cytochrome P450lin. This effect is not as strong as those observed for cytochrome P450cam. Even though the spin equilibrium does not change dramatically in the presence of K(+) or Na(+), the value of the dissociation constant (K(d)) for linalool binding is significantly affected by ionic strength. Analysis of the thermodynamic parameters for the linalool binding strongly suggests that, similarly to our previous finding for cytochrome P450cam, electrostatic gates participate in the control of substrate access.
Deprez
Eric
E
Laboratoire de Biotechnologies et Pharmacologie Génétique Appliquée (UMR-CNRS 8532), Ecole Normale Supérieure de Cachan, Cachan, France.
Gill
Edward
E
Helms
Volkhard
V
Wade
Rebecca C
RC
Hui Bon Hoa
Gaston
G
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002151:Calorimetry; D002164:Camphor / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D019475:Camphor 5-Monooxygenase / Q000737:chemistry / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D011188:Potassium / Q000494:pharmacology*; D011485:Protein Binding; D011487:Protein Conformation; D016958:Pseudomonas putida / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D002164:Camphor; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C059620:linalool 8-monooxygenase; D019475:Camphor 5-Monooxygenase; D011188:Potassium