Aromatic residues within the substrate-binding cleft of Bacillus circulans chitinase A1 are essential for hydrolysis of crystalline chitin.
Bacillus circulans chitinase A1 (ChiA1) has a deep substrate-binding cleft on top of its (beta/alpha)8-barrel catalytic domain and an interaction between the aromatic residues in this cleft and bound oligosaccharide has been suggested. To study the roles of these aromatic residues, especially in crystalline-chitin hydrolysis, site-directed mutagenesis of these residues was carried out. Y56A and W53A mutations at subsites -5 and -3, respectively, selectively decreased the hydrolysing activity against highly crystalline beta-chitin. W164A and W285A mutations at subsites +1 and +2, respectively, decreased the hydrolysing activity against crystalline beta-chitin and colloidal chitin, but enhanced the activities against soluble substrates. These mutations increased the K(m)-value when reduced (GlcNAc)5 (where GlcNAc is N -acetylglucosamine) was used as the substrate, but decreased substrate inhibition observed with wild-type ChiA1 at higher concentrations of this substrate. In contrast with the selective effect of the other mutations, mutations of W433 and Y279 at subsite -1 decreased the hydrolysing activity drastically against all substrates and reduced the kcat-value, measured with 4-methylumbelliferyl chitotrioside to 0.022% and 0.59% respectively. From these observations, it was concluded that residues Y56 and W53 are only essential for crystalline-chitin hydrolysis. W164 and W285 are very important for crystalline-chitin hydrolysis and also participate in hydrolysis of other substrates. W433 and Y279 are both essential for catalytic reaction as predicted from the structure.
Watanabe
Takeshi
T
Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, 8050 Ikarashi-2, Niigata 950-2181, Japan. wata@agr.niigata-u.ac.jp
Ariga
Yumiko
Y
Sato
Urara
U
Toratani
Tadayuki
T
Hashimoto
Masayuki
M
Nikaidou
Naoki
N
Kezuka
Yuichiro
Y
Nonaka
Takamasa
T
Sugiyama
Junji
J
D024322:Amino Acids, Aromatic / Q000235:genetics / Q000502:physiology*; D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D020134:Catalytic Domain; D002686:Chitin / Q000737:chemistry / Q000378:metabolism*; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003460:Crystallization; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D014364:Tryptophan / Q000235:genetics; D014443:Tyrosine / Q000235:genetics
D024322:Amino Acids, Aromatic; D002686:Chitin; D014443:Tyrosine; D014364:Tryptophan; D002688:Chitinases