کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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1385154 | 982432 | 2008 | 9 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: The dual exo/endo-type mode and the effect of ionic strength on the mode of catalysis of chitinase 60 (CHI60) from Serratia sp. TU09 and its mutants The dual exo/endo-type mode and the effect of ionic strength on the mode of catalysis of chitinase 60 (CHI60) from Serratia sp. TU09 and its mutants](/preview/png/1385154.png)
Mutations of the tryptophan residues in the tryptophan-track of the N-terminal domain (W33F/Y and W69F/Y) and in the catalytic domain (W245F/Y) of Serratia sp. TU09 Chitinase 60 (CHI60) were constructed, as single and double point substitutions to either phenylalanine or tyrosine. The enzyme–substrate interaction and mode of catalysis, exo/endo-type, of wild type CHI60 and mutant enzymes on soluble (partially N-acetylated chitin), amorphous (colloidal chitin), and crystalline (β-chitin) substrates were studied. All CHI60 mutants exhibited a reduced substrate binding activity on colloidal chitin. CHI60 possesses a dual mode of catalysis with both exo- and endo-type activities allowing the enzyme to work efficiently on various substrate types. CHI60 preferentially uses the endo-type mode on soluble and amorphous substrates and the exo-type mode on crystalline substrate. However, the prevalent mode of hydrolysis mediated by CHI60 is regulated by ionic strength. Slightly elevated ionic strength, 0.1–0.2 M NaCl, which promotes enzyme–substrate interactions, enhances CHI60 hydrolytic activity on amorphous substrate and, interestingly, on partially N-acetylated chitin. High ionic strength, 0.5–2.0 M NaCl, prevents the enzyme from dissociating from amorphous substrate, occupying the enzyme in an enzyme–substrate non-productive complex. However, on crystalline substrates, the activity of CHI60 was only inhibited approximately 50% at high ionic strength, suggesting that the enzyme hydrolyzes crystalline substrates with an exo-type mode processively while remaining tightly bound to the substrate. Moreover, substitution of Trp-33 to either phenylalanine or tyrosine reduced the activity of the enzyme at high ionic strength, suggesting an important role of Trp-33 on enzyme processivity.
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Journal: Carbohydrate Research - Volume 343, Issue 16, 3 November 2008, Pages 2754–2762