IMPORTANCE OF VAN-DER-WAALS CONTACT BETWEEN GLU-35 AND TRP-109 TO THECATALYTIC ACTION OF HUMAN LYSOZYME

The importance of van der Waals contact between Glu 35 and Trp 109 to the active-site structure and the catalytic properties of human lysozy me (HL) has been investigated by site-directed mutagenesis. The X-ray analysis of mutant HLs revealed that both the replacement of Glu 35 by Asp or Ala, and the replacement of Trp 109 by Phe or Ala resulted in a significant but localized change in the active-site cleft geometry. A prominent movement of the backbone structure was detected in the reg ion of residues 110 to 120 and in the region of residues 100 to 115 fo r the mutations concerning Glu 35 and Trp 109, respectively. Accompani ed by the displacement of the main-chain atoms with a maximal deviatio n of C alpha atom position ranging from 0.7 Angstrom to 1.0 Angstrom, the mutant HLs showed a remarkable change in the catalytic properties against Micrococcus luteus cell substrate as compared with native HL. Although the replacement of Glu 35 by Ala completely abolished the lyt ic activity, HL-Asp 35 mutant retained a weak but a certain lytic acti vity, showing the possible involvement of the side-chain carboxylate g roup of Asp 35 in the catalytic action. The kinetic consequence derive d from the replacement of Trp 109 by Phe or Ala together with the resu lt of the structural change suggested that the structural detail of th e cleft lobe composed of the residues 100 to 115 centered at Ala 108 w as responsible for the turnover in the reaction of HL against the bact erial cell wall substrate. The results revealed that the van der Waals contact between Glu 35 and Trp 109 was an essential determinant in th e catalytic action of HL.