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J. Biochem, 2003, Vol. 133, No. 2 253-258
© 2003 Japanese Biochemical Society

BIOCHEMISTRY

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

Yuji Honda1,2, Motomitsu Kitaoka+,1, Ken Tokuyasu1, Chiye Sasaki3, Tamo Fukamizo3 and Kiyoshi Hayashi1

1 National Food Research Institute, 2-1-12, Kannondai, Tsukuba, Ibaraki 305-8642; 2 Japan Science and Technology Corporation, 4-1-8, Honmachi, Kawaguchi, Saitama 332-0012; and 3 Faculty of Agriculture, Kinki University, 3327-204, Nakamachi, Nara 631-8505

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 Km value relative to 1, indicating that the N-acetyl group was recognised at the –2 subsite. The kcat value obtained with ChiA was identical to the kcat value observed for 1. However, the kcat 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 Km values relative to 1, respectively, and 60- and 30-fold smaller kcat 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.

+ To whom correspondence should be addressed. Tel: +81-298-38-8071, Fax: +81-298-38-7321, E-mail: mkitaoka{at}nfri.affrc.go.jp


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