Abstract
大腸桿菌 ( Escherichia coli ) 的甲硫安酸安基生太脢 ( mehionine peptidase, 簡稱 MAP ) 是一個酵素專一性高, 需要二價鈷離子活化的金 屬生太脢 ( metallopeptidase ), MAP在細胞中的功能是切除新合成蛋白 質的 N- 端甲硫安酸 ( N-terminal methionine, 簡稱 N-Met ),由 X- ray 結晶圖知道在 MAP 的活性基有兩個二價鈷離子與 Asp97, Asp108, Glu204, Glu235 and His171 形成配位鍵結, 但其它參與酵 素活性的安基酸殘基仍然不清楚, 因此我們想以化學修飾法更進一步研 究 MAP 的活性基, 首先利用聚合脢鏈反應 ( polymerase chain reaction, 簡稱 PCR ) 由大腸桿菌的基因組 ( genomic ) DNA 擴大出 E. coli map 基因, 將它插入 pBS(+) 載體, 然後送進大腸桿菌中大量表 現經由 DEAE-Sepharose, Superose 12 gel filtration, Mono Q管柱 層析, 得到均質的酵素在本實驗中碘化乙稀安 ( iodoacetamide,簡稱 IAM ) 被使用來修飾 MAP 的半胱安酸 ( cysteine ) 由實驗結果知道 IAM 對 MAP 的抑制是 time-dependent manner, 同時是不可逆反應, 在 25 C, pH 7.5 的條件下, 反應速率常數 K1 = 13 + - 5 1/min /M, 反應 級數 n 值在低濃度 (1 - 4 mM) 及高濃度 ( 4 - 6 mM ) 的 IAM 下分 別為 0.807 + - 0.007 及 2.05, 表示 MAP 活性受 IAM 抑制, 在低濃 度可能是因為一個半胱安酸被修飾所造成的; 而在高濃度則是兩個 double-reciprocal plot 顯示, IAM 對 MAP 的抑制是競爭性型式 ( competitive mode ), Circular dichroism ( CD ) 光譜圖顯示在低濃 度 ( 1 mM ) 的修飾不影響蛋白質的二級結構, 但高濃度 ( 5 mM ) 則 會, 綜合以上的結果, 我們認為應該至少有一個半胱安酸可能位於 MAP 的活性位置 E.coli methionine aminopeptidase ( MAP ) is a cobalt dependent enzyme which removes N-terminal methionone from nascently synthesized peptides. Study of X-ray structure has shown that active site of MAP binds two cobalt ion liganded by the site chains of Asp97, Asp108, Glu204, Glu235 and His 171. However, the nature of the other amino acids involved in the enzymatic activity of MAP remains unknown. In this report. the E.coli map gene was amplified from E.coli genomic DNA by polymerse chain reaction (PCR), inserted into pBS(+), and overexpressed. By three purification steps including DEAE-Sepharose, Superose 12 gel filtration, and Mono Q column chromatography, the MAP was purified to homogeneity. Modification of MAP by iodoacetamide (IAM) shows that the enzyme can be irreversibely inactivated by IAM in a time-dependent manner. Reaction order (n) of 0.807 + - 0.007 and 2.05 was determined by low (1-4 mM) and high (4-6 mM) concentration of IAM respectively. This indicated that the inactivation of MAP was due to modification of one cysteine in or near active site at low concentrations of inhibition, but two in high concentrations. The double -reciprocal plot showed that the mode of inhibition is competitive mode. The effect of IAM on the secondary structure of MAP was determined by circular dichroism which showed that no appreciable conformation change at the level of 1 mM but not for the 5 mM IAM. These results sugest that at least one cysteine reidue may be involved in the enzymatic activity of MAP.