Mooser G, Hefta S A, Paxton R J, Shively J E, Lee T D
Department of Basic Sciences, School of Dentistry, University of Southern California, Los Angeles 90089-0641.
J Biol Chem. 1991 May 15;266(14):8916-22.
An active-site peptide containing an aspartic acid implicated in catalysis has been isolated and sequenced from two Streptococcus sobrinus extracellular glucosyltransferases: sucrose:1,3-alpha-D-glucan 3-alpha-D-glucosyltransferase (GTase-I) and sucrose:1,6-alpha-D-glucan 6-alpha-D-glucosyltransferase (GTase-S). The sequenced peptides, tagged with radiolabeled glucose, were isolated from a pepsin digest of a stabilized glucosylenzyme complex prepared by rapidly denaturing a reaction of enzyme and radiolabeled sucrose. The glucosyl linkage had previously been characterized as a beta-anomer bound to an active-site carboxyl group. Purified GTase-I and GTase-S glucosyl-peptides had the following similar but not identical sequences: GTase-I, Asp-Ser-Ile-Arg-Val-Asp-Ala-Val-Asp; and GTase-S, Asp-Gly-Val-Arg-Val-Asp-Ala-Val-Asp. Each has 3 aspartic acids as potential sites of glucose conjugation, but the relevant residue was not identified in sequence analysis because the highly base-labile glucosyl bond was cleaved in the first sequence cycle. As an alternative, the GTase-I glucosyl-peptide was partially digested at the N terminus with cathepsin C and at the C terminus with carboxypeptidase P. Analysis of the truncated products by fast atom bombardment mass spectrometry localized the glucosyl group to Asp-6 i the GTase-I peptide. In the native enzyme, this sequence is found near the N terminus, well-removed from the glucan-binding site located on a 60-kDa domain at the C terminus. The catalysis-dependent method of incorporating a glucosyl label implicates the aspartic acid as the residue involved in stabilizing an oxocarbonium ion transition state. The peptide segment is highly conserved and homologous to a peptide from sucrase-isomaltase labeled by site-directed irreversible inhibition and peptide segments common to a broad array of alpha-glucosidases and related transferases.
蔗糖：1,3-α-D-葡聚糖 3-α-D-葡糖基转移酶（GTase-I）和蔗糖：1,6-α-D-葡聚糖 6-α-D-葡糖基转移酶（GTase-S）。将用放射性标记葡萄糖标记的测序肽，从通过使酶与放射性标记蔗糖的反应快速变性而制备的稳定化葡糖基酶复合物的胃蛋白酶消化物中分离出来。葡糖基连接先前已被表征为与活性位点羧基结合的β-异头物。纯化的 GTase-I 和 GTase-S 葡糖基肽具有以下相似但不完全相同的序列：GTase-I，天冬氨酸-丝氨酸-异亮氨酸-精氨酸-缬氨酸-天冬氨酸-丙氨酸-缬氨酸-天冬氨酸；和 GTase-S，天冬氨酸-甘氨酸-缬氨酸-精氨酸-缬氨酸-天冬氨酸-丙氨酸-缬氨酸-天冬氨酸。每种都有 3 个天冬氨酸作为葡萄糖缀合的潜在位点，但在序列分析中未鉴定出相关残基，因为高度对碱不稳定的葡糖基键在第一个序列循环中被裂解。作为替代方法，GTase-I 葡糖基肽在 N 端用组织蛋白酶 C 部分消化，在 C 端用羧肽酶 P 部分消化。通过快原子轰击质谱对截短产物的分析将葡糖基定位到 GTase-I 肽中的天冬氨酸-6。在天然酶中，该序列位于 N 端附近，与位于 C 端 60-kDa 结构域上的葡聚糖结合位点相距较远。掺入葡糖基标记的催化依赖性方法表明天冬氨酸是参与稳定氧鎓离子过渡态的残基。该肽段高度保守，并且与通过定点不可逆抑制标记的蔗糖酶-异麦芽糖酶的肽以及广泛的α-葡糖苷酶和相关转移酶共有的肽段同源。
