大腸桿菌Thioredoxin蛋白□抑制劑之反影動力論

Abstract

大腸桿菌thioredoxin為一分子量約11.7 kDa之多功能蛋白。在其多項功能中，Lunn等人在1986年即已提出大腸桿菌thioredoxin具有胰蛋白□抑制劑之活性。本實驗首先純化野生株（SK3981）及C32S, G、G33D, K, V、P34G, Y、與G33V/P34Y等九種thioredoxin表現菌株所生產之thioredoxin，再進行胰蛋白□抑制劑反應動力論之研究。大部分的thioredoxin在NaCl鹽梯度達0.3-0.4 M即可沖提出來，僅C32G與G33K需達0.5 M才可收集得到。而在胰蛋白□濃度為8.61×10-7 M時，以Na-benzoyl-L-arginine-p-nitroanilide hydrochloride (L-BAPA)為反應基質所測得之Km=（7.16±0.07）×10-4 M, kcat=316 min-1；而以Na-benzoyl-L-arginine ethyl ester hydrochloride (BAEE)測得之Km=（6.37±0.007）×10-6 M, kcat =91 min-1，由此可知，BAEE對胰蛋白□的親合力較強，而L-BAPA之最大反應速率則較大。接著再利用L-BAPA進行胰蛋白□抑制劑反應動力論，結果發現thioredoxin作為胰蛋白□抑制劑時所呈現的抑制型態為混合型抑制作用（mixed inhibition）。野生株（SK3981）之Ki＝(77.1±1.5) mM，Cys-32之定點突變株方面改為R基為-H之Gly可使其與胰蛋白□之親合力提高兩倍，與野生株相近的R基為-CH2-OH之Ser相較之下則具有更強的抑制效果；而在Gly-33處之定點突變株則是由親水性之R基改為厭水性之Val則Ki與野生株相近甚至較高，同時改為帶正電之Lys與帶負電的Asp其Ki約下降四倍，顯示在此位置若改為帶電荷之R基的胺基酸有助於其抑制作用；Pro-34處則由厭水性之胺基酸改為親水性之Gly與Tyr，二者之Ki值均下降了，尤以P34G之抑制常數為最低。最後一雙定點突變株G33V/P34Y 其Ki值與P34Y相近卻較和野生株相近的G33V低了約兩倍，更加證明了P34Y有助於抑制作用而G33V則無很大的影響。

E. Coli thioredoxin is a low molecular weight (11.7 kDa) protein found in all cells. One of its function is thioredoxin may serve as trypsin inhibitior. In this study, we purified thioredoxins produced from wild type (SK3981) and several mutants (C32G, S; G33D, K, V; P34G, Y; G33V/P34Y), then analyzed the kinetics of thioredoxin as trypsin inhibitor. We found that most all of the thioredoxins can be eluted when the NaCl concentration gradient was up to 0.3-0.4 M, but C32G and G33K were both eluted until the NaCl concentration was about 0.5 M. The protealytic activity of trypsin was Km = (7.16±0.07)×10-4 M; kcat=316 min-1 for Na-benzoyl-L-arginine- p-nitroanilide hydrochloride (L-BAPA), and Km=（6.37±0.007）×10-6 M, kcat =91 min-1 for Na-benzoyl-L-arginine ethyl ester hydrochloride (BAEE). The binding affinity between trypsin and substrates is BAEE＞L-BAPA, but the maximum reaction velocity is L-BAPA＞BAEE. Finally we used L-BAPA to assay the kinetics of trypsin inhibition. The model of inhibition is mixed inhibition，and the Ki of wild type is (77.1±1.5) mM. At the position Cys-32, the inhibition ability of C32G was two fold higher than wild type and C32S. The Ki of charged G33D and G33K is four-fold lower than wild type and G33V. Inhibition ability of P34G is four-fold higher than the wild type. Ki of the mutant G33V/P34Y is about two-fold lower than wild type and G33V, and clos to P34Y.