Kinetic studies on the hydrolysis of N-acetylated and N-deacetylated derivatives of 4-methylumbelliferyl chitobioside by the family 18 chitinases ChiA and ChiB from Serratia marcescens.
Kinetic analyses of the hydrolysis reactions of N-acetylated and N-deacetylated derivatives of 4-methylumbelliferyl chitobioside [(GlcNAc)(2)-UMB (1), GlcN-GlcNAc-UMB (2), GlcNAc-GlcN-UMB (3), and (GlcN)(2)-UMB (4)] by ChiA and ChiB from Serratia marcescens were performed. Both enzymes released UMB from all compounds apart from 4. The S-v curves of the hydrolyses of 1 by ChiA and ChiB both exhibited atypical kinetic patterns, and the shapes of the two S-v curves were different from one another. However, both curve shapes were explained by assuming some of the enzyme present formed complexes with multiple molecules of the substrate. Conversely, the S-v curves generated in the cleavage of 2 and 3 by ChiA exhibited typical Michaelis-Menten profiles. Both enzymes hydrolysed 2 with an approximately 14-fold higher K(m) value relative to 1, indicating that the N-acetyl group was recognised at the -2 subsite. The k(cat) value obtained with ChiA was identical to the k(cat) value observed for 1. However, the k(cat) value for ChiB was one-fourth that of 1, suggesting that the removal of the N-acetyl group caused an increase in the formation of a non-productive ES-complex. ChiA and ChiB hydrolysed 3 with 5- and 20-fold greater K(m) values relative to 1, respectively, and 60- and 30-fold smaller k(cat) values relative to 1, respectively. The reaction mechanism of family 18 chitinases is discussed based upon the results obtained from the hydrolysis of these compounds.
Honda
Yuji
Y
National Food Research Institute, 2-1-12, Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
Kitaoka
Motomitsu
M
Tokuyasu
Ken
K
Sasaki
Chiye
C
Fukamizo
Tamo
T
Hayashi
Kiyoshi
K
D000107:Acetylation; D001426:Bacterial Proteins / Q000378:metabolism; D002688:Chitinases / Q000378:metabolism*; D004187:Disaccharides / Q000378:metabolism*; D005456:Fluorescent Dyes; D006868:Hydrolysis; D006923:Hymecromone / Q000031:analogs & derivatives*; D007700:Kinetics; D012706:Serratia marcescens / Q000201:enzymology*
D001426:Bacterial Proteins; D004187:Disaccharides; D005456:Fluorescent Dyes; D006923:Hymecromone; C032438:chitobiose; D002688:Chitinases; C094590:chitinase ChiA