Studies on the Catalytic Mechanism of Lysozyme

Secondary α-tritium kinetic isotope effects have been utilized to probe the nature of the transition state in the lysozyme catalyzed hydrolysis of chitotriose. A general synthesis of specifically labeled chitin oligomers (in particular 1,1',1"-³H-chitotriose, the substrate used in these studies) is described. Injection of Drosophila melanogaster larvae with labeled N-acetyl-D-glucosamine yields chitin, which can be hydrolyzed to give a range of chitin oligomers from chitobiose to chitoheptose. The value of kH/kTobtained for the lysozyme catalyzed hydrolysis of chitotriose was found to be 1.19. This result indicates very considerable carbonium ion character in the transition state, and thus the mechanistic alternatives for lysozyme hydrolysis become distinguishable. There is evidence for the existence of a stabilized intermediate formed after the production of the carbonium ion in a post-rate limiting step. Three alternatives exist for this stabilized intermediate: 1) a carbonium ion stabilized to some degree by asp 52 (an ion pair) 2) an acylal formed with asp 52 (a covalent intermediate ) 3) an oxazoline Substrates of the form NAG-GLU- NO₂ have been found to undergo lysozyme catalyzed transglycosylation. Since these substrates are cleaved specifically between GLU and the aglycone, the possibility of an oxazoline intermediate can be eliminated. A study of the variation with pH of the ratio transfer / transfer + hydrolysis for lysozyme has yielded the titration curve of a group of pKa~6.5. This has been assigned to glu 35. Because its pKa is unchanged from that in the unbound enzyme, the possibility of a highly charged intermediate can be eliminated. Therefore the stabilized intermediate must be an acylal or a tight ion pair.