Isolation and sequence of an active-site peptide containing a catalytic aspartic acid from two Streptococcus sobrinus alpha-glucosyltransferases.
Isolation and sequence of an active-site peptide containing a catalytic aspartic acid from two Streptococcus sobrinus alpha-glucosyltransferases.
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DOI：
10.1016/s0021-9258(18)31531-x
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发表时间：
1991-05
期刊：
影响因子：
--
通讯作者：
G. Mooser;S. Hefta;R. Paxton;J. Shively;Terry D. Lee
中科院分区：
文献类型：
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作者：
G. Mooser;S. Hefta;R. Paxton;J. Shively;Terry D. Lee
已从两种远缘链球菌胞外葡糖基转移酶（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））中分离并测序了一种含有参与催化作用的天冬氨酸的活性位点肽。用放射性标记的葡萄糖标记的测序肽，从通过快速变性酶和放射性标记的蔗糖的反应制备的稳定的葡萄糖基酶复合物的胃蛋白酶消化物中分离。葡糖基键先前被表征为与活性位点羧基结合的β-端基异构体。纯化的GTase-I和GTase-S葡糖基肽具有以下相似但不相同的序列：GTase-I，Asp-Ser-Ile-Arg-Val-Asp-Ala-Val-Asp;和GTase-S，Asp-Gly-Val-Arg-Val-Asp-Ala-Val-Asp。每一个都有3个天冬氨酸作为葡萄糖结合的潜在位点，但是在序列分析中没有鉴定出相关的残基，因为高度碱基不稳定的葡萄糖键在第一个序列循环中被切割。作为替代方案，将GTase-I葡糖基肽在N末端用组织蛋白酶C部分消化，在C末端用羧肽酶P部分消化。通过快速原子轰击质谱法对截短产物进行分析，将葡糖基定位于GTase-I肽中的Asp-6。在天然酶中，该序列位于N末端附近，与位于C末端60 kDa结构域上的葡聚糖结合位点相距很远。将葡萄糖基标记的催化依赖性方法暗示天冬氨酸作为参与稳定氧碳鎓离子过渡态的残基。该肽段是高度保守的，并且与通过定点不可逆抑制标记的来自蔗糖酶-异麦芽糖酶的肽和广泛的α-葡糖苷酶和相关转移酶共有的肽段同源。
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.