HYDROLYSIS OF 4-METHYLUMBELLIFERYL N-ACETYL-CHITOTRIOSIDE CATALYZED BY HEN AND TURKEY LYSOZYMES - PH-DEPENDENCE OF THE KINETIC CONSTANTS

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  • 1 January 1980
    • journal article
    • research article
    • Vol. 87  (4) , 1003-1014
Abstract
The binding constants of 4-methylumbelliferyl-.beta.-glycosides of (GlcNAc)2 ((GlcNAc)2-MeU) and of (GlcNAc)3 ((GlcNAc)3-MeU) to hen lysozyme [EC 3.2.1.17] were determined by measuring changes in the fluorescence at 375 nm. (GlcNAc)2-MeU and (GlcNAc)3-MeU bind mainly at subsites B, C and D and A, B, C and D, respectively, with the terminal MeU group bound at subsite D. The rate of hydrolysis of (GlcNAc)3-MeU catalyzed by hen and turkey lysozymes was determined at 0.1 ionic strength and 42.degree. C in the pH range of 2-8. Release of 4-methylumbelliferone was followed fluorimetrically. The pH dependences of kcat, Kcat/Km, and Km were analyzed assuming that nonproductive binding occurs competitively and that the molecular species with ionized Asp 52 and protonated Glu 35 is active. Comparison of the pH dependences of the kinetic constants for hen lysozyme with those for turkey lysozyme, in which Asp 101 of hen lysozyme is replaced by Gly, made it possible to determine the pK values of Asp 52, Glu 35 and Asp 101. The pK values of Asp 52 and Glu 35 were 3.60 and 6.20, respectively, for hen and turkey lysozymes and 3.95 and 6.55, respectively, for their nonproductive complexes. The pK value of Asp 101 of hen lysozyme was 4.20 for the free enzyme, 3.30 for the nonproductive complex and 3.95 for the productive complex. These pK values, except for the pK value of Asp 52 of the nonproductive complex, are in excellent agreement with those determined by spectroscopic methods. Lysozyme-catalyzed hydrolysis can be fully explained in terms of the proposal based on the X-ray data with regard to the participation of Asp 52 and Glu 35.