L78對L型澱粉磷解酶活性調控及 催化機制之角色

Abstract

低澱粉親和型之澱粉磷解脢 (L-SP) 廣泛存在於植物澱粉貯藏組織中，在in vitro情況下，發現有三種不同催化方式︰(1) 需醣引子的直鏈醣合成反應、(2) 不需醣引子之合成直鏈醣 (primer-independent activity, PI activity)、(3) 磷解澱粉反應。L-SP也有幾種不同的降解型式︰(1) 完整的L-SP含有特殊的L78片段、(2) L78部份斷裂的L-SP (L-SP’)、(3) L78完全斷裂的L-SP (L-SP*)。本論文釐清各種修飾形式的L-SP各自扮演何種功能︰當L-SP為完整的110 kDa時，不需醣引子直接以Glc-1-P合成直鏈醣，而SP* 就失去此種PI活性。另外，完整的L-SP及L78斷裂不完全的L-SP’，都可使用直鏈寡葡萄醣 (G2, G3, G5, G7, 數字代表葡萄糖單位) 進行直鏈醣合成，但L78若完全去除成為SP*，就無法使用較短的寡醣 (G2, G3)；而其中L-SP利用短鏈寡醣 (G2, G3) 與長鏈寡醣 (G5, G7) 合成直鏈醣的機制也不相同。在L-SP合成直鏈醣的反應中，速率決定步驟為從單醣Glc-1-P合成雙醣 (G2)。另一方面，L-SP藉由LSK磷酸化，加速L78的斷裂，可對上述L-SP三種反應巧妙調節；造成此斷裂的最後因子為蛋白脢，但此種降解模式為可調節性，並且不影響L-SP的構型，仍維持著完整活性。另外，也利用native-PAGE/SDS-PAGE及LC-MS/MS分析LSK，進行LSK peptide合成以製備LSK的單株抗體，將可進一步了解L-SP的催化機制及澱粉代謝上所扮演的生理角色。

The low-affinity type of starch phosphorylase (L-SP) is widely found in the starch-accumulating tissues of plant. In the test tube, it shows three types of catalysis: (1) the biosynthesis of oligo-glucan in the presence of a primer; (2) the same biosynthetic reaction in the absence of a primer (primer-independent activity, PI activity); and (3) the degradation of starch by phosphorolysis. The purified L-SP might have three modified forms: (1) the intact 110 kDa molecule containing the L78 insertion in the middle of L-SP; (2) the modified L-SP in which the L78 is nicked by proteolysis (L-SP’); and (3) the L78 on L-SP is essentially removed (L-SP*). Our study found that the modification on the L78 might cause the change of the catalytic behavior of L-SP. The intact L-SP can synthesize amylose directly from Glc-1-P in the absence of a prime. However, the SP* lost this PI activity completely. For amylose synthesis, the intact L-SP and SP’ can utilize various oligo-glucan (G2, G3, G5 and G7, numbers indicate units of glucose) as the primer; nevertheless, the SP* can not take G2 and G3 as the substrates. Furthermore, the catalytic mechanism of L-SP toward the shorter glucan (G2 and G3) was different from the longer glucan, G5 and G7. The rate-limiting step in the primer-independent reaction of L-SP was the formation of one disaccharide from two molecules of Glc-1-P. On the other hand, the L-SP was reported to be phosphorylated at a Ser residue on the L78 (Young et al, 2006). The phosphorylation of L-SP then enhanced the proteolytic modification of L78, which might accordingly regulate the enzyme behavior of L-SP between the three types of catalysis. The proteolysis of the phosphorylated L-SP might be regulated by an unknown mechanism. The specific kinase for the phosphorylation of L-SP was isolated by native/PAGE and SDS/PAGE, and its partial amino acid sequence was determined by LC/MS/MS. The antibody against a peptide from this kinase sequence was prepared by hybridoma technique, which might be useful for the exploration of the role of L-SP in starch biosynthesis.