MULTIPLE ROLE OF HYDROPHOBICITY OF TRYPTOPHAN-108 IN CHICKEN LYSOZYME - STRUCTURAL STABILITY, SACCHARIDE BINDING ABILITY, AND ABNORMAL PKA OF GLUTAMIC ACID-35

Trp108 of chicken lysozyme is in van der Waals contact with Glu35, one of two catalytic carboxyl groups. The role of Trp108 in lysozyme function and stability was investigated by using mutant lysozymes secreted from yeast. By the replacement of Trp108 with less hydrophobic residues, Tyr (W108Y lysozyme) and Gln (W108Q lysozyme), the activity, saccharide binding ability, stability, and pK(a) of Glu35 were all decreased with a decrease in the hydrophobicity of residue 108. Namely, at pH 5.5 and 40-degrees-C, the activities of W108Y and W108Q lysozymes against glycol chitin were 17.3 and 1.6% of that of wild-type lysozyme, and their dissociation constants for the binding of a trimer of N-acetyl-D-glucosamine were 7.4 and 309 times larger than that of wild-type lysozyme, respectively. For the reversible unfolding at pH 3.5 and 30-degrees-C W108Y and W108Q lysozymes were less stable than wild-type lysozyme by 1.4 and 3.6 kcal/mol, respectively. As for the pK(a) of Glu35,the values for W108Y and W108Q lysozymes were found to be lower than that for wild-type lysozyme by 0.2 and by 0.6 pK(a) unit, respectively. The pK(a) of Glu35 in lysozyme was also decreased from 6.1 to 5.4 by the presence of 1-3 M guanidine hydrochloride, or to 5.5 by the substitution of Asn for Asp52, another catalytic carboxyl group. Thus, both the hydrophobicity of Trp108 and the electrostatic interaction with Asp52 are equally responsible for the abnormally high pK(a) (6.1) of Glu35, compared with that (4.4) of a normal glutamic acid residue. A high pK(a) of Glu35 is considered to be critical for this residue to serve as general-acid catalyst in the glycosidase activity of lysozyme at higher pHs. Therefore, it is concluded that the hydrophobicity of Trp108 plays multiple important roles in lysozyme's function and stability, such as the maintenance of the tertiary structure, effective substrate binding, and keeping the pK(a) of Glu35 abnormally high.