초록
신호펩디드 가수분해는 모든 생물에 필수적인 단백질로 N-말단에 신호서열을 가진 단백질들의 신호서열을 잘라내는 효소로 serine과 lysine을 활성부위로 하는 특이한 dyad serine 효소류로 알려져있다. 최근에 신호펩디드 가수분해효소의 특성으로 새로운 항생제 이용 가능성 때문에 그 활성 메카니즘의 연구가 중요시되고 있다. 본 연구는 E. coli 신호펩디드 효소가 serine/lysine/aspartic acid로 하는 전형적인 triad serine 효소류의 메카니즘을 갖는다는 가정하에 Type Ⅰ 신호펩디드류의 하나인 E. coli에서 aspartic acid 99을 alanine으로 치환하여 효소의 enzymatic activity를 조사하였다. 그 결과 D99잔기는 E. coli Type Ⅰ 신호펩디드 효소의 활성메카니즘에 직접적으로 관여한다기보다는 그 효소를 구조적으로 안정화하는데 중요한 역할을 할 가능성이 있는 것으로 암시되어진다.
Type Ⅰ signal peptidase is an integral membrane protein that functions to cleave signal peptides from secreted and membrane proteins. The enzyme serves as a potential target for the development of novel antibacterial agents due to its unique physiological properties. Despite being one of the best characterized enzymes, the catalysis of Type Ⅰ signal peptidase still remains controversy over the catalytic serine/lysine dyad mechanism. It appears that the dyad proteases are generally less efficient than the prototypical serine/histidine/aspartic acid triad found in most enzymes, although Type Ⅰ signal peptidase is an exception to this rule. In this paper, we have proposed that Type Ⅰ signal peptidase may act as the serine/lysine/aspartic acid triad cataltytic mechanism. Therefore, the aspartic acid 99 residue in the E. coli signal peptidase was chosen and mutated to an alanine to see if there is any possible role of the aspartic acid in the catalytic function. Type Ⅰ signal peptidase D99A protein was inactive in vitro assay using the procoat synthesized by in vitro transcription translation. However, the mutant was active using a highly sensitive in vivo assay. Pulse-chase experiments show that the replacement of aspartic acid 99 with alanine results in a very unstable signal peptidase molecule. Therefore, we conclude that it is unlikely that the residue is directly involved in catalysis, but rather plays an important role in stabilizing the protein structure.