Imparl-Radosevich J M, Li P, Zhang L, McKean A L, Keeling P L, Guan H
ExSeed Genetics, L.L.C., Iowa State University, Food Science Building, Ames, Iowa 50011, USA.
Arch Biochem Biophys. 1998 May 1;353(1):64-72. doi: 10.1006/abbi.1998.0613.
Comparison of the protein sequences deduced from the cDNAs of maize granule-bound starch synthase, Escherichia coli glycogen synthase, and maize starch synthase I (SSI) reveals that maize SSI contains an N-terminal extension of 93 amino acids. In order to study the properties of maize SSI and to understand the functions of the maize SSI N-terminal extension, the gene coding for full-length SSI (SSI-1) and genes coding for N-terminally truncated SSI (SSI-2 and SSI-3) were individually expressed in E. coli. Here we describe for the first time the purification of a higher plant starch synthase to apparent homogeneity. Its kinetic properties were therefore studied in the absence of interfering amylolytic enzymes. The specific activities of the purified SSI-1, SSI-2, and SSI-3 were 22.5, 33.4, and 26.3 micromol Glc/min/mg of protein, respectively, which are eight times higher than those of partially purified SSI from developing maize endosperm. The full-length recombinant enzyme SSI-1 exhibited properties similar to those of the enzyme from maize endosperm. As observed for native maize enzyme, recombinant SSI-1 exhibited "unprimed" activity without added primer in the presence of 0.5 M citrate. Our results have clearly indicated that the catalytic center of SSI is not located in its N-terminal extension. However, N-terminal truncation decreased the enzyme affinity for amylopectin, with the Km for amylopectin of the truncated SSI-3 being about 60-90% higher than that of the full-length SSI-1. These results suggest that the N-terminal extension in SSI may not be directly involved in enzyme catalysis, but may instead regulate the enzyme binding of alpha-glucans. Additionally, the N-terminal extension may play a role in determining the localization of SSI to specific portions of the starch granule or it may regulate its interactions with other enzymes involved in starch synthesis.
对从玉米颗粒结合淀粉合酶、大肠杆菌糖原合酶和玉米淀粉合酶I（SSI）的cDNA推导的蛋白质序列进行比较，发现玉米SSI含有一个93个氨基酸的N端延伸。为了研究玉米SSI的特性并了解玉米SSI N端延伸的功能，编码全长SSI（SSI-1）的基因和编码N端截短的SSI（SSI-2和SSI-3）的基因分别在大肠杆菌中表达。在此，我们首次描述了将一种高等植物淀粉合酶纯化至表观均一性。因此，在不存在干扰性淀粉分解酶的情况下研究了其动力学特性。纯化的SSI-1、SSI-2和SSI-3的比活性分别为22.5、33.4和26.3微摩尔葡萄糖/分钟/毫克蛋白质，比从发育中的玉米胚乳中部分纯化的SSI高八倍。全长重组酶SSI-1表现出与来自玉米胚乳的酶相似的特性。正如在天然玉米酶中观察到的那样，重组SSI-1在存在0.5 M柠檬酸盐的情况下，在不添加引物的情况下表现出“无引物”活性。我们的结果清楚地表明，SSI的催化中心不在其N端延伸中。然而，N端截短降低了酶对支链淀粉的亲和力，截短的SSI-3对支链淀粉的Km比全长SSI-1高约60-90%。这些结果表明，SSI中的N端延伸可能不直接参与酶催化，而是可能调节酶与α-葡聚糖的结合。此外，N端延伸可能在确定SSI在淀粉颗粒特定部分的定位中起作用，或者它可能调节其与参与淀粉合成的其他酶的相互作用。
