EFFECT OF SITE-DIRECTED MUTATIONS ON PROCESSING AND ACTIVITY OF GAMMA-GLUTAMYL-TRANSPEPTIDASE OF ESCHERICHIA-COLI K-12
EFFECT OF SITE-DIRECTED MUTATIONS ON PROCESSING AND ACTIVITY OF GAMMA-GLUTAMYL-TRANSPEPTIDASE OF ESCHERICHIA-COLI K-12
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DOI：
10.1093/oxfordjournals.jbchem.a124894
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发表时间：
1995-07-01
影响因子：
2.7
通讯作者：
KUMAGAI, H
中科院分区：
文献类型：
--
作者：
HASHIMOTO, W;SUZUKI, H;KUMAGAI, H
大肠杆菌K-12的γ-谷氨酰转肽酶[EC 2.3.2.2]被认为是通过翻译后加工从单个前体多肽合成为异二聚体形式。γ-谷氨酰转肽酶高产菌株E. coliK-12中的γ-谷氨酰转肽酶，周质部分仅含有成熟的γ-谷氨酰转肽酶，膜部分仅含有γ-谷氨酰转肽酶的前体，胞质部分未检测到γ-谷氨酰转肽酶的前体，通过定点突变，将加工成大小亚基的切割位点的氨基酸残基替换。通过Western blot分析，检测了6个突变体的加工表型，并测量其γ-谷氨酰转肽酶活性。小亚基N端氨基酸残基突变（Thr-391、Thr-392和His-393）阻止了酶的成熟，未成熟突变体不显示酶活性，大亚基（Gln-390）C端残基的突变对加工和酶活性的影响较小，这些结果表明，小亚基N端的苏氨酰-苏氨酰-组氨酸残基序列对E.大肠杆菌K-12 γ-谷氨酰转肽酶的表达，这一加工过程是表达大肠杆菌K-12 γ-谷氨酰转肽酶活性所必需的。coli K-12。
gamma-Glutamyltranspeptidase [EC 2.3.2.2] of Escherichia coli K-12 is thought to be synthesized from a single precursor polypeptide into a heterodimeric form through post-translational processing. Cells of a gamma-glutamyltranspeptidase-overproducing transformant of E. coli K-12 were fractionated and the localization of the enzyme was examined by Western blot analysis, The periplasmic fraction only contained the mature form of gamma-glutamyltranspeptidase, membrane fraction only contained the precursor of gamma-glutamyltranspeptidase, and no precursor of gamma-glutamyltranspeptidase was detected in the cytoplasmic fraction, Amino acid residues at the cleavage site for processing into the large and small subunits were substituted by site-directed mutagenesis, The processing phenotypes of six mutants were examined by Western blot analysis, and their gamma-glutamyltranspeptidase activities were measured. Mutations at the N-terminal amino acid residues of the small subunit (Thr-391, Thr-392, and His-393) prevented the maturation of the enzyme and the immature mutants exhibited no enzymatic activity, A mutation at the C-terminal residue of the large subunit (Gln-390) had less effect on the processing and enzymatic activity, These results suggest that the sequence of threonyl-threonyl-histidinyl residues at the N-terminal of the small subunit is very important for the processing of E. coli K-12 gamma-glutamyltranspeptidase and this processing is essential to the expression of gamma-glutamyltranspeptidase activity of E. coli K-12.