5'-腺苷磷酰硫酸激酶及其R68K突变体对5'-腺苷一磷酸3'羟基的磷酸化

Phosphorylation of 3'-hydroxyl Group of 5'-adenosine Monophosphate by Adenosine 5'-phosphosulfate Kinase and Its R68K Mutant

硫作为生命活动的必需元素,主要以-2价和+6价发挥生物学功能。硫的同化代谢包括胞内活化、转移以及还原等反应。其活化是同化代谢的关键反应,包括ATP硫酸化酶(ATP sulfurylase,ATPS)催化硫酸盐与ATP反应生成腺苷-5'-磷酰硫酸(adenosine 5'-phosphosulfate,APS)和焦磷酸(pyrophosphate,PPi)以及腺苷-5'-磷酰硫酸激酶(adenosine 5'-phosphosulfate kinase,APSK)催化APS 3'羟基磷酸化生成3'-磷酸腺苷-5'-磷酰硫酸(3'-phosphoadenosine 5'-phosphosulfate,PAPS),APSK催化APS磷酸机理已经较为清楚。利用APSK对AMP的磷酸化进行了初步分析,发现AMP可作为APSK的底物,反应生成3,5'二磷酸腺苷(3'-phosphoadenosine 5'-phosphate,PAP);对APSK的三维结构进行分析发现,R68同时和APS的磷酸根和硫酸根形成氢键,稳定APS的结合,而K侧链基团比R短2.4魡,R68K突变将导致K不能和距离较远的硫酸根离子相互作用,从而减弱APS的亲和力,而增加与磷酸根离子的相互作用,可能提高AMP的亲和力。研究结果表明,R68K突变体的最适底物变为AMP,KmAMP是对照的0.2倍,而催化效率是对照的5倍。以R68K为偶联酶成功测定了具有较低KmPAP的酵母3,5二磷酸核苷酸酶(3',5'-bisphosphate nucleotidase,YND)动力学常数,为分析测定AMP底物的酶活提供了工具。

Sulfur, an essential element for all organisms, plays its biological roles mainly with valence status of -2 and ＋6. Intracellular sulfate activating, first step for the assimilation of sulfate, includes biosynthesis of adenosine 5＇-phosphosulfate （AlaS） catalyzed by ATP sulfurylase （ATlaS） with side product pyrophosphate （PPi） and the subsequent phosphorylation of APS by adenosine 5＇-phosphosulfate kinase（APSK） to form 3＇-phosphoadenosine 5＇-phosphosulfate （PAPS）, the universal sulfuryl group donor. Although the mechanism of APS phosphorylation by APSK had been reported in detail, it remains to be elucidated that if the APS analogue - AMP could be phosphorylated by APSK. Preliminary studies showed that AMP could be phosphorylated by APSK with product 3＇-phosphoadenosine 5＇-phosphate （PAP）. Structural analysis indicated that R68 would form hydrogen bonds with oxygen on phosphate and sulfate to stabilize the binding of APS. Shortening R group by R68K mutation would weaken the interaction to sulfate, strengthen interaction to phosphoate, and possibly discriminate APS from AMP. Our results clearly demonstrated that optimized substrate for APSK turned to AMP from APS, with 5 times enhancement of catalytic efficiency and binding affinity. R68K was also further successfully used as coupling enzymes to keep PAP concentration constant and eliminate product AMP in solution to facilitate the initial rate measurements for PAP hydrolysis activity catalyzed by yeast 3＇,5＇-bisphosphate nucleotidase （YND）. R68K was proved to be an alternating coupling enzyme to facilitate characterization of AMP producing enzymes.