Sucrose 6-alpha-D-glucosyltransferase from Streptococcus sobrinus: characterization of a glucosyl-enzyme complex.
Sucrose 6-alpha-D-glucosyltransferase from Streptococcus sobrinus: characterization of a glucosyl-enzyme complex.
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来自远缘链球菌的蔗糖 6-α-D-葡萄糖基转移酶：葡萄糖基酶复合物的表征。
DOI：
10.1021/bi00428a006
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Iwaoka,KR
中科院分区：
文献类型：
--
作者：
Mooser,G;Iwaoka,KR
南加州大学牙科学院生物化学系，洛杉矶，加州90089接收于1988年6月23日;修订的Mandarin pt接收于1988年8月29日摘要：从远缘链球菌蔗糖6-α D-葡糖基转移酶和放射性标记蔗糖的淬灭反应中分离出一种共价葡糖基酶。没有复杂的观察与热灭活的酶或当蔗糖被替换为放射性标记的麦芽糖或葡萄糖。该复合物在pH 2的1%十二烷基硫酸钠，6.0 M尿素和4.0 M盐酸胍中是稳定的，但随着pH值的增加（32分钟半衰期，pH 7.0）变得越来越不稳定。当在温和碱性条件下释放所有放射性时，D-葡萄糖是唯一鉴别的放射性标记化合物。葡萄糖基-酶水解速率与氢氧离子浓度呈线性关系，二级速率常数为2.15 X 105 M-1 min-1。当与已知糖基氨基酸衍生物的碱不稳定性相比时，葡糖基-酶的pH依赖性最接近于葡糖基与羧基的连接。本文提出了一种新的应用--碳水化合物高效液相色谱柱在水溶液中鉴定变性葡萄糖基酶碱性水解和蔗糖天然酶水解释放的D-葡萄糖的端基异构体。在前一种情况下鉴定出β-端基异构体，在后一种情况下鉴定出β-端基异构体。变性葡糖基-酶的结果与在酯碳处水解并保留异头构型的天冬氨酸或谷氨酸的β-葡糖基酯键一致;对于天然葡糖基转移酶催化，数据也与β-葡糖基共价中间体一致，其中通过在缩醛碳处攻击并发生异头转化而发生脱葡糖基化。然而，葡萄糖基-酶复合物不能复性以证明催化能力，留下了在酶活性位点塌陷期间形成共价键的可能性。虽然葡糖基键是极端碱不稳定的，但发现其在低pH下足够稳定以经受胃蛋白酶蛋白水解裂解。该家族的口腔细菌糖基转移酶的活性位点标签几乎是未知的，并且该复合物可能是研究酶活性位点结构的有用探针。
Department of Biochemistry, School of Dentistry, University of Southern California, Los Angeles, California 90089 Received June 23, 1988; Revised Manuscript Received August 29, 1988 abstract: A covalent glucosyl-enzyme was isolated from a quenched reaction of Streptococcus sobrinus sucrose 6-aD-glucosyltransferase and radiolabeled sucrose. No complex was observed with heat-inactivated enzyme or when sucrose was replaced with radiolabeled maltose or glucose. The complex was stable at pH 2 in 1% sodium dodecyl sulfate, 6.0 M urea, and 4.0 M guanidine hydrochloride, but became increasingly labile with increased pH (32-min half-life at pH 7.0). D-Glucose was the exclusive radiolabeled compound identified when all radioactivity was released under mild alkaline conditions. Glucosyl-enzyme hydrolysis rates were linearly dependent on hydroxide ion concentration, giving a second-order rate constant of 2.15 X 105 M" 1 min" 1. When compared to the base lability of known glycosyl amino acid derivatives, the pH dependency of the glucosyl-enzyme most closely paralleled a glucosyl linkage to a carboxyl group. A novel application of a carbohydrate high-performance liquid chromatography column in aqueous solution was used to identify the anomericform of D-glucose released on (i) alkalinehydrolysis of denatured gluco-syl-enzyme and (ii) native enzyme hydrolysis of sucrose. The/3-anomer was identified in the former case and the-anomer in the latter. The results with the denatured glucosyl-enzyme are consistent with a j3-glucosyl ester linkage to an aspartic or glutamic acid that hydrolyzesat the ester carbon with retention of anomeric configuration; for native glucosyltransferase catalysis, the data are consistent with a/3-glucosyl covalent intermediate as well, where deglucosylation occurs by attack at the acetal carbon with anomeric inversion. However, the glucosyl-enzyme complex could not be renatured to demonstrate catalytic com-petence, leaving open the possibility that the covalent bond formed during collapse of the enzyme active site. While the glucosyl bond was extremely base labile, it was found to be sufficiently stable at low pH to survive pepsin proteolytic cleavage. Active-site labels for this family of oral bacterial glycosyltransferases are virtually unknown, and the complex may be a useful probe to study the structure of the enzyme active site.
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