STRUCTURAL SIMILARITY OF D-AMINOPEPTIDASE TO CARBOXYPEPTIDASE-DD AND BETA-LACTAMASES
STRUCTURAL SIMILARITY OF D-AMINOPEPTIDASE TO CARBOXYPEPTIDASE-DD AND BETA-LACTAMASES
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DOI：
10.1021/bi00123a016
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发表时间：
1992-03-03
期刊：
影响因子：
2.9
通讯作者：
KONDO, K
中科院分区：
文献类型：
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作者：
ASANO, Y;KATO, Y;KONDO, K
d -氨基肽酶（dap）基因已从人类Ochrobactrum anthropi SCRC C1-38中分离出来[Asano， Y., Nakazawa， A., Kato， Y., & Kondo， K.（1989）。化学，264,14233-14239]及其核苷酸序列测定。将基因置于pUC19的lac启动子下游，构建了表达质粒pC138DP （4.5 kb）。大肠埃希菌JM109/pC138DP无细胞提取物中酶的含量提高到28.8万单位/L，是人类O. SCRC C1-38的约3600倍。该酶约占总可提取细胞蛋白的30%。该基因由1560个核苷酸组成的开放阅读框组成，该阅读框指定M(r) 57 257蛋白。该酶的氨基酸序列表明，它与羧肽酶DD、β -内酰胺酶和青霉素结合蛋白有关。通过位点特异性诱变产生了该酶的7个突变体，以探索Ser61-X(aa)-X(aa)-Lys64序列周围的残基的作用，其中X（aa）是任何氨基酸，因为相同的序列也出现在青霉素识别肽水解酶中，活性位点上有丝氨酸。在大肠杆菌中表达的突变酶进行了纯化，并进行了动力学表征。替换Ser61和Lys64位点产生的突变体显示V（max）值显著降低，而K(m)值大部分保持不变。靠近可能的活性中心Ser61的Cys60对Ser和Gly的变化导致酶对PCMB的敏感性降低，其V(max)/K(m)值几乎不变。这种酶似乎是丝氨酸肽酶而不是硫醇酶。PCMB对野生型酶的抑制作用可能是由cys60与PCMB之间形成巯基键引起的。考虑到d -氨基肽酶、羧肽酶DD（一种青霉素结合蛋白）和β -内酰胺酶在识别含有d -氨基酸的肽方面具有共同的特征，并且前两者与含有d -丙烯酰- d -丙氨酸基团的底物催化转肽化反应，我们提出该酶是“青霉素识别酶”的新成员。我们发现，β -内酰胺类化合物，如6-APA、7-ACA、青霉素和氨苄西林，虽然它们不是该酶的底物，但实际上抑制了该酶。讨论了d -氨基肽酶和其他丝氨酸水解酶、β -内酰胺酶和羧基肽酶催化的反应与初级结构的关系。据我们所知，这种酶是第一个在活性位点有丝氨酸的氨基肽酶的例子。
The gene for D-aminopeptidase (dap) has been isolated from the bacterium Ochrobactrum anthropi SCRC C1-38 [Asano, Y., Nakazawa, A., Kato, Y., & Kondo, K. (1989) J. Biol. Chem. 264, 14233-14239] and its nucleotide sequence determined. An expression plasmid pC138DP (4.5 kb) was constructed by placing the gene downstream of the lac promoter of pUC19. The amount of the enzyme in the cell-free extract of Escherichia coli JM109/pC138DP was elevated to 288 000 units/L of culture, which is about 3600-fold over that of O. anthropi SCRC C1-38. The enzyme comprised about 30% of the total extractable cellular protein. The gene consisted of an open reading frame of 1560 nucleotides which specifies a protein of M(r) 57 257. The deduced amino acid sequence of the enzyme showed that it is related to carboxypeptidase DD, beta-lactamases, and penicillin-binding proteins. Seven mutants of the enzyme were generated by site-specific mutagenesis to explore the roles of the residues of interest, around the sequence Ser61-X(aa)-X(aa)-Lys64, where X(aa) is any amino acid, since the identical sequences also appear in the penicillin-recognizing peptide hydrolases with Ser at the active sites. The mutant enzymes expressed in E. coli were purified to homogeneity and kinetically characterized. Replacements of the site at Ser61 and Lys64 yielded mutants showing significantly reduced V(max) values, while most of the K(m) values remained unchanged. Changes at Cys60, which is adjacent to the likely active center Ser61, to Ser and Gly resulted in the production of enzyme less sensitive to PCMB, with almost unaltered V(max)/K(m), values. The enzyme appears to be a serine peptidase rather than a thiol one. The inhibition by PCMB in the wild-type enzyme may have been caused by a formation of a mercaptide bond between Cys 60 and PCMB. Considering that D-aminopeptidase, carboxypeptidase DD (a penicillin-binding protein), and beta-lactamase have a common feature in recognizing peptides containing D-amino acid and that the former two catalyze transpeptidation reactions with substrates containing D-alanyl-D-alanine moieties, we propose that the enzyme is a new member of the "penicillin-recognizing enzymes". We showed that the enzyme is actually inhibited by beta-lactam compounds, such as 6-APA, 7-ACA, benzylpenicillin, and ampicillin, although they are not the substrate for the enzyme. The relationship between the primary structures and the reactions catalyzed by D-aminopeptidase and other serine hydrolases beta-lactamases and carboxypeptidase DD is discussed. To our knowledge, this enzyme is the first example of an aminopeptidase with Ser at the active site.