Detailed studies on substrate structure requirements of glycoamidases A and F
Detailed studies on substrate structure requirements of glycoamidases A and F
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DOI：
10.1074/jbc.272.43.27058
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发表时间：
1997-10-24
影响因子：
4.8
通讯作者：
Lee, YC
中科院分区：
文献类型：
--
作者：
Fan, JQ;Lee, YC
糖酰胺酶(peptide-N-4-(N-acetyl-beta-glucosaminyl)-asparagine酰胺酶，EC 3.5.1.52；也称为多肽：N-糖苷酶(PNGase)通过水解天冬酰胺侧链的糖基化的β-酰胺键，从糖肽和/或糖蛋白中释放N-连接的寡糖。最广泛使用的糖酰胺酶是来自脑膜败血黄杆菌的那些糖胺酶(糖酰胺酶F或PNGase F)和杏仁乳蛋白(糖酰胺酶A或PNGase A)。在系统研究这些酶的底物结构要求的基础上，我们合成了含有纤维二糖、乳糖、GlcNAc和二-N，N‘-乙酰基壳二糖(CTB)的糖肽>；30。多肽的长度从1到5个氨基酸不等，糖基胺连接到位于多肽不同位置的天冬氨酸或谷氨酰胺，包括NH2和COOH末端。这两种酶都不能切割纤维二糖和乳糖糖肽，这表明天冬氨酸连接的GlcNAc上的2-乙酰氨基在糖胺酶A和F的识别中都是重要的。GlcNAc多肽可以被这两种酶切割，尽管不如CTB糖肽有效。这两种酶都不需要天冬氨酸-xaa-(丝氨酸/苏氨酸)序列(N-糖基化所需)才能发挥活性。糖酰胺酶A甚至可以作用于谷氨酰胺结合的CTB糖肽，而糖酰胺酶A作用于CTB二肽，糖酰胺酶A作用于CTB二肽，糖酰胺酶F优先作用于三肽或更长的多肽。T-丁氧羰基-(CTB)-天冬氨酸-丙氨酸-丝氨酸糖胺酶A的K-m和V-max分别为2.1 nM和0.66 mU·mol/min/mg。天然糖二肽Man(9)-GlcNAc(2)-Asn-Phe也被糖氨酸酶A完全降解。
Glycoamidases (peptide-N-4-(N-acetyl-beta-glucosaminyl)-asparagine amidase, EC 3.5.1.52; also known as peptide: N-glycanases (PNGases) release N-linked oligosaccharides from glycopeptides and/or glycoproteins by hydrolyzing the glycosylated beta-amide bond of the asparagine side chain. The most widely used glycoamidases are those from Flavobacterium meningosepticum (glycoamidase F or PNGase F) and almond emulsin (glycoamidase A or PNGase A). The study the substrate structure requirement of these enzymes systematically, we synthesized >30 glycopeptides containing cellobiose, lactose, GlcNAc, and di-N,N'-acetylchitobiose (CTB). The length of the peptide was varied from one to five amino acids, and glycosylamines were linked to either Asn or Gln located at different positions in the peptide, including NH2 and COOH termini. Neither enzyme could cleave cellobiose and lactose glycopeptides, indicating that the 2-acetamido group on the Asn-linked GlcNAc is important in the recognition by both glycoamidases A and F. GlcNAc peptides could be cleaved by both enzymes, albeit not as effectively as CTB glycopeptides. Neither enzyme requires the Asn-Xaa-(Ser/Thr) sequence (required for N-glycosylation) for activity. Glycoamidase A could even hydrolyze a Gln-bound CTB glycopeptide, whereas the action of glycoamidase A could act on CTB dipeptides, glycoamidase A could act on CTB dipeptides, glycoamidase F preferred a tripeptide or longer. The K-m and V-max values of glycoamidase A for t-butoxycarbonyl-(CTB)-Asn-Ala-Ser-OMe were 2.1 nM and 0.66 mu mol/min/mg, respectively. A natural glycodipeptide, Man(9)-GlcNAc(2)-Asn-Phe, was also completely hydrolyzed by glycoamidase A.