| Attributs | Valeurs |
|---|
| type
| |
| Is Part Of
| |
| Subject
| |
| Title
| - Role of Protein and Substrate Dynamics in Catalysis by Pseudomonas putidaCytochrome P450cam
|
| has manifestation of work
| |
| related by
| |
| Author
| |
| Abstract
| - The role of protein structural flexibility and substrate dynamics in catalysis by cytochromeP450 enzymes is an area of current interest. We have addressed these in cytochrome P450cam (P450cam)and its Y96A mutant with camphor and its related compounds using fluorescence spectroscopy. Previously[Prasad et al. (2000) FEBSLett. 477, 157−160], we provided experimental support to dynamic fluctuationsin P450cam, and substrate access into the active site region via the channel next to the flexible F−Ghelix−loop−helix segment. In the investigation described here, we show that the dynamic fluctuations inthe enzyme are substrate dependent as reflected by tryptophan fluorescence quenching experiments. Theorientation of tryptophan relative to heme (κ2) for W42 obtained from time-resolved tryptophan fluorescencemeasurements show variation with type of substrate bound to P450cam suggesting regions distant fromheme-binding site are affected by physicochemical and steric characteristics/protein−substrate interactionsof P450cam active site. We monitored substrate dynamics in the active site region of P450cam by time-resolved substrate anisotropy measurements. The anisotropy decay of substrates bound to P450cam indicatethat mobility of substrates is modulated by physicochemical and steric characteristics/protein−substrateinteractions of local active site structure, and provides an understanding of factors controlling observedhydroxylated products for substrate bound P450cam complexes. The present study shows that P450camlocal and peripheral structural flexibility and heterogeneity along with substrate mobility play an importantrole in regulating substrate binding orientation during catalysis and accommodating diverse range ofsubstrates within P450cam heme pocket.
|
| article type
| |
| is part of this journal
| |
