کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
5372029 1503972 2008 6 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Roles of two surface residues near the access channel in the substrate recognition by cytochrome P450cam
کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی تئوریک و عملی
پیش نمایش صفحه اول مقاله
Roles of two surface residues near the access channel in the substrate recognition by cytochrome P450cam
چکیده انگلیسی

Detailed stopped-flow kinetics of binding of 1R-camphor to cytochrome P450cam has been studied at different temperatures for the wild type as well as for two site specific mutants T192E and S190D of the enzyme, where the surface exposed Threonine and Serine residues were mutated by acidic amino acids. The near-UV and visible circular dichroism spectra as well as the intrinsic fluorescence spectra of the WT and mutant enzymes showed that the mutation of the enzyme did not affect the tertiary structure of the enzyme over the temperature range 4-30 °C. The S190D mutation did not show any significant change in the rate constants of the substrate association while they were much lower in the T192E mutant compared to the WT enzyme. The activation energies for substrate association and dissociation processes were determined from the analysis of temperature dependence of the rate constants by the Arrhenius equation over the temperature range 4-19 °C. The activation energy for the substrate association was found to be significantly higher in the T192E mutant compared to the S190D mutant or the WT enzyme. The results showed that the Threonine 192 that resides on the F-G loop and directed towards the putative substrate access channel of the enzyme, plays an important role in recognition of the substrate at the surface of the enzyme. These results showed that though the active site of the enzyme resides deep inside the protein matrix, the substrate is recognized at the surface of the enzyme and directed towards the active site through the access channel.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biophysical Chemistry - Volume 135, Issues 1–3, June 2008, Pages 1-6
نویسندگان
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