IMPORTANCE OF GLUTAMIC ACID 35-TRYPTOPHAN 109 CONTACT IN THE MAINTENANCE OF THE CATALYTIC CLEFT GEOMETRY OF HUMAN LYSOZYME. M. Muraki, S. Goda, H. Nagahora and K. Harata, National Institute of Bioscience and Human- Technology, Tsukuba, Ibaraki 305, Japan

Human lysozyme catalyses the hydrolysis of glycosidic linkages in the peptidoglycan of the bacterial cell wall. In the crystal structure of wildtype human lysozyme, the side-chain of Glu35 that acts as a general acid in the catalytic action and the side-chain of Trp109 are located very close to each other within the distance of van der Waals contact. Mutations of human lysozyme replacing Glu35 with Asp or Trp109 with Phe reduced the lytic activity against M.luteus cells to ca. 0.3% and ca. 20% of that of wild-type enzyme, respectively. In order to investigate the structural effect of these mutations and to clarify the cause of the functional change, the structures of the mutant enzymes were analysed by X-ray crystallography. The refinements were carried out at 1.66 Å resolution (Glu35Asp mutant) and at 1.60 Å resolution (Trp109Phe mutant). The coordinate errors were estimated to be 0.16 Å-0.17 Å for either mutant. The RMS values of the distance between the corresponding [[alpha]]-carbon atoms of the mutant enzyme and that of wild-type enzyme were 0.12 Å (Glu35Asp mutant) and 0.13 Å (Trp109Phe mutant), indicating no significant change in the global conformation of the molecule. However, a remarkable change in the local conformation was detected in either mutant as compared with wild-type enzyme. The difference of 0.4 Å-0.7 Å in the distance between the corresponding [[alpha]]-carbon atoms from that of wild-type enzyme were found in the region from residue 110 to 118 (Glu35Asp mutant) and from residue 104 to 111 (Trp109Phe mutant). The above result together with the results of kinetic analysis suggests the existence of the contact between Glu35 and Trp109 is important not only in making the hydrophobic environment around the carboxylate group of Glu35 but also in the maintenance of the catalytic cleft geometry, which is responsible for the formation of the "productive" complex between the enzyme and the substrate.