Cacciapuoti G, Servillo L, Moretti M A, Porcelli M
Dipartimento di Biochimica e Biofisica "F Cedrangolo," Seconda Universita degli Studi di Napoli, Via Costantinopoli 16, 80138, Naples, Italy.
Extremophiles. 2001 Oct;5(5):295-302. doi: 10.1007/s007920100205.
The effect of phosphate, its analogues, and other substrates on structural features of recombinant 5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) was investigated. Phosphate was found to exert a significant stabilizing effect on the protein against the inactivation caused by temperature, sodium dodecyl sulfate (SDS), urea, and proteolytic enzymes. In the presence of 100 mM phosphate: (i) the apparent transition temperature (Tm) of recombinant SsMTAP increased from 111 degrees to 118 degrees C; and (ii) the enzyme still retained 40% and 30% activity, respectively, after 30 min of incubation at 90 degrees C with 2% SDS or 8 M urea. The structure modification of SsMTAP by phosphate binding was probed by limited proteolysis with subtilisin and proteinase K and analysis of polypeptide fragments by SDS-PAGE. The binding of the phosphate substrate protected SsMTAP against protease inactivation, as proven by the disappearance of a previously accessible proteolytic cleavage site that was localized in the N-terminal region of the enzyme. The conformational changes of SsMTAP induced by phosphate and ribose-1-phosphate were analyzed by fluorescence spectroscopy, and modifications of the protein intrinsic fluorophore exposure, as a consequence of substrate binding, were evidenced.
研究了磷酸盐、其类似物及其他底物对来自嗜热栖热菌的重组5'-甲硫基腺苷磷酸化酶（SsMTAP）结构特征的影响。发现磷酸盐对该蛋白质具有显著的稳定作用，可抵抗温度、十二烷基硫酸钠（SDS）、尿素和蛋白水解酶导致的失活。在存在100 mM磷酸盐的情况下：（i）重组SsMTAP的表观转变温度（Tm）从111℃升高至118℃；（ii）在90℃下与2% SDS或8 M尿素孵育30分钟后，该酶仍分别保留40%和30%的活性。通过枯草杆菌蛋白酶和蛋白酶K的有限蛋白水解以及SDS-PAGE对多肽片段的分析，探究了磷酸盐结合对SsMTAP结构修饰 的影响。磷酸盐底物的结合保护SsMTAP免受蛋白酶失活，这一点通过位于该酶N端区域的一个先前可及的蛋白水解切割位点的消失得到证实。通过荧光光谱分析了磷酸盐和核糖-1-磷酸诱导的SsMTAP的构象变化，并证明了由于底物结合导致的蛋白质固有荧光团暴露的修饰。
