کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
9882192 | 1536546 | 2005 | 10 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Mechanistic analysis of wheat chlorophyllase
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
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چکیده انگلیسی
Chlorophyllase catalyzes the initial step in the degradation of chlorophyll and plays a key role in leaf senescence and fruit ripening. Here, we report the cloning of chlorophyllase from Triticum aestivum (wheat) and provide a detailed mechanistic analysis of the enzyme. Purification of recombinant chlorophyllase from an Escherichia coli expression system indicates that the enzyme functions as a dimeric protein. Wheat chlorophyllase hydrolyzed the phytol moiety from chlorophyll (kcat = 566 minâ1; Km = 63 μM) and was active over a broad temperature range (10-75 °C). In addition, the enzyme displays carboxylesterase activity toward p-nitrophenyl (PNP)-butyrate, PNP-decanoate, and PNP-palmitate. The pH-dependence of the reaction showed the involvement of an active site residue with a pKa of â¼6.5 for both kcat and kcat/Km with chlorophyll, PNP-butyrate, and PNP-decanoate. Using these substrates, solvent kinetic isotope effects ranging from 1.5 to 1.9 and from 1.4 to 1.9 on kcat and kcat/Km, respectively, were observed. Proton inventory experiments suggest the transfer of a single proton in the rate-limiting step. Our analysis of wheat chlorophyllase indicates that the enzyme uses a charge-relay mechanism similar to other carboxylesterases for catalysis. Understanding the activity and mechanism of chlorophyllase provides insight on the biological and chemical control of senescence in plants and lays the groundwork for biotechnological improvement of this enzyme.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Archives of Biochemistry and Biophysics - Volume 438, Issue 2, 15 June 2005, Pages 146-155
Journal: Archives of Biochemistry and Biophysics - Volume 438, Issue 2, 15 June 2005, Pages 146-155
نویسندگان
Kiani A.J. Arkus, Edgar B. Cahoon, Joseph M. Jez,