Lin F P, Chen H C, Lin C S
Institute of Marine Biotechnology, National Taiwan Ocean University, Keelung.
IUBMB Life. 1999 Aug;48(2):199-204. doi: 10.1080/713803487.
Site-directed mutagenesis was used to explore the roles of amino acid residues involved in the activity of chitinase from Aeromonas caviae. Kinetic parameters for 4-methylumbelliferyl-N,N'-diacetyl-chitobiose or 4-methylumbelliferyl-N,N',N"-triacetylchitotriose hydrolysis were determined with wild-type and mutant chitinases. Chitinases with the mutations E315D (or Q) and D391E (or N) were severely impaired and had dramatically decreased kcat. However, the effect of the these mutations on the Km values were different. The function of the carboxyl group of Asp313 was partially replaced by the amide of Asn when the 4-methylumbelliferyl-N,N',N"-triacetylchitotriose substrate was used. Results indicated that Asp313, Glu315, and Asp391 might be the best candidates for the catalytic residues of chitinase A from Aeromonas caviae.
利用定点诱变技术探究了豚鼠气单胞菌几丁质酶活性相关氨基酸残基的作用。用野生型和突变型几丁质酶测定了4-甲基伞形酮基-N,N'-二乙酰壳二糖或4-甲基伞形酮基-N,N',N''-三乙酰壳三糖水解的动力学参数。具有E315D（或Q）和D391E（或N）突变的几丁质酶严重受损，kcat显著降低。然而，这些突变对Km值的影响不同。当使用4-甲基伞形酮基-N,N',N''-三乙酰壳三糖底物时，Asp313羧基的功能被Asn的酰胺部分取代。结果表明，Asp313、Glu315和Asp391可能是豚鼠气单胞菌几丁质酶A催化残基的最佳候选者。
