中文摘要 酮醇酸還原異構酶(KARI) 是支鏈胺基酸(BCAA) 生物合成途徑中的第二個酵素， 為雙功能酶可進行兩步驟的催化反應。第一步進行鎂離子專一性的異構反應，隨後為輔 酶NAD(P)H 與鎂離子或其他二價金屬離子(錳離子或鈷離子) 依賴性的還原反應。在 先前研究結果，已得知嗜酸熱硫化葉菌的KARI (Sac-KARI) 具有熱穩定性與輔酶雙專 一性，可以利用NADPH 與NADH 兩種輔酶進行催化反應，且相對偏好於NADPH 。 在本次研究中，我們利用X-ray 蛋白質晶體繞射，解析出Sac-KARI-NADPH 與 Sac-KARI-NADH 兩種複合物晶體結構，解析度分別為2.72 Å 與 1.68 Å 。分析 Sac-KARI 與兩種輔酶複合物結構，發現於活化位β2αB-loop 構形並無明顯改變，但在 Sac-KARI-NADH 結構中發現來自母液的磷酸根佔據了相對於Sac-KARI-NADPH 結 構中NADPH 的2 端磷酸根基團的位置。並於高溫的酵素動力學實驗，比較不同的緩 衝液(HEPES 與磷酸鹽) 對Sac-KARI 相對活性的影響。其結果表示當酵素在使用 NADH 時，在緩衝液含有磷酸根離子環境中，會有相對較高的活性。證實了磷酸根或 是硫酸根離子是可以幫助Sac-KARI 在使用NADH 時的催化活性 。;Abstract Ketol-acid reductoisomerase (KARI) is the second enzyme in branched chain amino acid (BCAA) biosynthetic pathway. The catalytic reaction of this bi-functional enzyme is consisted of two steps, including Mg2+-dependent alkyl migration, followed by the NAD(P)H-dependent reduction reaction. In the previous study, we found that KARI from Sulfolobus acidocaldarius (Sac-KARI) is a thermostable and bi-cofactor-utilizing enzyme. In this study, we determined two crystal structures of Sac-KARI-NADPH (2.72 Å) and Sac-KARI-NADH (1.68 Å) complexes. The crystal structural analysis shows that R49 undergoes the typical π-cation stacking interaction against the adenine ring and forms a salt bridge with the 2´-phosphate of the NADPH. The S53 forms H bonds both with 2´-phosphate and 3´-OH of the NADPH. The R49 and S53 make similar contacts with NADH, however, the phosphate ions mimic the 2´-phosphate of NADPH in the cofactor binding pocket. The enzyme activity assays further confirm that the Sac-KARI has higher activity in the phosphate than that in the HEPES buffer at 50ºC by using NADH as a cofactor. On the other hand, the optimum pH for Sac-KARI activity is in the pH range of 7-7.5 at 50ºC.
