Kuroki R, Yamada H, Moriyama T, Imoto T
J Biol Chem. 1986 Oct 15;261(29):13571-4.
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.
在一个两步过程中，即酯化和氨解过程，溶菌酶中的Glu-35和Asp-52被酰胺化成为谷氨酰胺和天冬酰胺残基。由于谷氨酰胺和天冬酰胺的侧链大小与谷氨酸和天冬氨酸的侧链几乎相等，这些转化将提供合适的衍生物来阐明这些残基的催化作用。在3.4 - 8.0的pH范围内，发现所得的[Gln35]溶菌酶和[Asn52]溶菌酶的酶活性不到天然溶菌酶的4%。由于这些衍生物没有活性，我们能够确定β-1,4-连接的n-聚体（一种N-乙酰-D-葡萄糖胺的六糖）与[Gln35]溶菌酶和[Asn52]溶菌酶结合的解离常数（Ks值）。在pH 5.5和40℃时，[Gln35]溶菌酶的Ks值为1.6×10⁻⁵ M，[Asn52]溶菌酶的Ks值为2.7×10⁻⁵ M。这些值与天然溶菌酶的值相似。这些结果直接证明了Glu35和Asp52参与了溶菌酶的催化作用。本文描述了一种在液氨中对蛋白质中的酯基进行氨解的方法，该方法将有助于蛋白质羧基的酰胺化。
