蛇肌果糖1,6-二磷酸酯酶与腺苷—磷酸的相互作用和抑制的机制——紫外差光谱的研究

本文证明别构抑制剂AMP和dAMP与蛇肌果糖1,6-二磷酸酯酶结合的差光谱,都有270,280和288nm正峰,此外dAMP尚有275nm正峰。计算得到两者结合常数分别为2.0×10~6M~(-1)和1.2×10~6M~(-1)。两者抑制行为不同和AMP的核糖上的2′-羟基与酶的别构部位相互作用有关。胰蛋白酶的限制性酶解引起该酶活力增加,同时受AMP抑制作用丧失,在差光谱上表现为280和288nm负峰,其特征和酶的脲素差光谱一致。说明高活性酶是处于结构松散状态,受AMP抑制的酶则处于紧凑状态,此二种状态均伴随着酪氨酸残基的微环境的改变。     

蛇肌果糖1,6-二磷酸酯酶的限制性酶解

蛇肌果糖1.6-二磷酸酯酶被枯草杆菌蛋白酶或胰蛋白酶限制性酶解,可分别产生29000和7000或32000和4000的肽段。酶解产物在pH7.5和pH9.2分别有2倍和4—5倍的激活,pH7.5的激活作用,仅可用无机磷测活法观察到。5′AMP对该产物的别构抑制作用部分或全部丧失。本文与文献[1]的结果使我们提出了天然状态的蛇肌酶的催化部位的结构是不完善的看法。被限制性酶解的蛇肌酶受6-磷酸果糖激活,同时又受6-磷酸葡萄糖酸,6-磷酸葡萄糖和还原型辅酶Ⅱ的抑制。因此限制性酶解作用可能在蛇肌中有一定生理功能。     

LIMITED PROTEOLYSIS OF SNAKE MUSCLE FRUCTOSE 1, 6-BISPHOSPHATASE

Limited proteolysis of snake muscle fructose 1,6-bisphosphatase (EC. 3. 1. 3. 11) with subtilisin resulted in a larger fragment of 29,000 daltons and a smaller fragment of 7000 daltons, and limited tryptic digestion for fructose 1, 6-bisphosphatase mainly produced a 32,000 daltons fragment. Both of the modified enzymes have a 4 to 5 fold increase in activity at pH 9.2 as well as a twofold increase in activity at pH 7.5. At 6μM of AMP more than 90% of the activity was inhibited for the native enzyme but not for the modified enzymes. In case of subtilisin, only 50% of activity was inhibited and in case of trypsin, non inhibition was observed. Results indicated the cleavage sites for the two proteases were apparently different.The above results and the fact, as reported previeusly, that snake muscle fructose 1, 6-bisphosphatase modified with 5, 5'-dithiobis(2-nitrobenzoic acid) also caused a twofold increase in its activity lead us to propose that the catalytic site of snake muscle fructose 1, 6-bis-phosp     

INTERACTION BETWEEN ADENOSINE-5′-MONOPHOSPHATE AND FRUCTOSE 1,6-BISPHOSPHATASE AND MECHANISM OF ALLOSTERIC INHIBITION——ULTRAVIOLET SPECTROSCOPIC STUDIES

Ultraviolet difference spectroscopic studies of AMP and dAMP upon binding to snake muscle fructose 1, 6-bisphosphatase showed that both of the ligands interacting with the enzyme generated three positive peaks located at 270 nm, 280 nm and 288 nm. The binding constants calculated from the titration curve of difference spactra by the successive addition of nueleotides were 2.0×10~6M~(-1) and 1.2×10~6M~(-1) respectively. The fact of the same binding constants for the two ligands indicated that the unique inhibition behavior of dAMP differing from AMP was not due to the difference of binding between AMP and dAMP, but due to the absence of 2'-OH group on the ribose of dAMP. In contrast to the difference spectra of AMP binding, the U. V. difference spectra produced by tryptic digestion of fructose 1, 6-bisphosphatase gave two negative bands at 280 nm and 288 nm and had a close resemblance to that of the enzyme in urea. The opposite signs of the two kinds of difference U. V. spectra produced by AMP binding