Abstract
In a two-step process, esterification and ammonolysis, Glu-35 and Asp-52 in lysozyme were amidated to glutamine and asparagine residues. Since the side chains of glutamine and asparagine are almost equal in size to those of glutamic acid and aspartic acid, these conversions would provide appropriate derivatives to elucidate the catalytic participations of these residues. The enzymatic activities of the resulting [Gln35]lysozyme and [Asn52]lysozyme were found to be less than 4% of that of native lysozyme in a pH range of 3.4-8.0. As these derivatives were inactive, we could determine the dissociation constants (Ks values) for the binding of beta-1,4-linked n-mer, a hexasaccharide of N-acetyl-D-glucosamine, to [Gln35]lysozyme and [Asn52] lysozyme. The values of Ks at pH 5.5 and 40 degrees C were 1.6 X 10(-5) M for [Gln35]lysozyme and 2.7 X 10(-5) M for [Asn52]lysozyme. These values are similar to that for native lysozyme. The results are direct proof for the involvements of Glu35 and Asp52 in the catalytic action of lysozyme. A method for ammonolysis of ester groups in proteins in liquid ammonia is described and will be useful for amidation of carboxyl groups of proteins.
Highlights
Ina two-step process, esterification andammono- sessed one-tenth of the activity of native lysozyme, presumlysis, Glu-35 and Asp-52 in lysozyme were amidated ably due to contamination by native-like enzyme [6]
Chemical modification is a powerful tool for identification of the catalytically essential residue(s) of an enzyme
If activity is lost on chemical modification of a certain residue, the residue modified is usually considered a possible catalytic residue
Summary
Uinsepfurlotfeoinrsianmliiqduaitdionamomf ocnairabiosxdyelscgrriboeudpsanodf wpirllobteeins.doMnaatetedribayls-QFiPveCo.tim(Jeasparenc)r.y4s-taDliliazzeodmheetnhyelg-gNw,Nh-itdeilmyestohzyymlbeewnzaesnesulfonamide was purchased from Wako Pure Chemical Industries. Hen egg white lysozyme (EC 3.2.1.17)is a carbohydrate Affinity chromatography of lysozyme derivatives was performed on a hydrolase with an acidic pH optimum for the certain sub- 7 X 13-cm or 2.5 X IO-cm column of chitin-coated Celite at 4 "C. strates andis one of the best characterized proteins [1].Since two of the carboxyl groups (Gld andAsps2)of lysozyme were implicated in catalysisfrom x-ray crystallographic studies [2], many chemical modifications of these carboxyls have been. Developed and all of the resulting derivatives found to be Enzymatic activities of lysozyme and itsderivatives against glycol almost inactive [3,4,5,6,7] Most of these modifications chitin were determined at various pH values and 40 " C (Table 11).
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