Lipchock James, Loria J Patrick
Department of Chemistry, Yale University, New Haven, CT 06520, USA.
J Biomol NMR. 2009 Sep;45(1-2):73-84. doi: 10.1007/s10858-009-9337-8. Epub 2009 Jun 30.
IGPS is a 51 kDa heterodimeric enzyme comprised of two proteins, HisH and HisF, that catalyze the hydrolysis of glutamine to produce NH(3) in the HisH active site and the cyclization of ammonia with N'-[(5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) in HisF to produce imidazole glycerol phosphate (IGP) and 5-aminoimidazole-4-carboxamide ribotide (AICAR). Binding of PRFAR and IGP stimulates glutaminase activity in the HisH enzyme over 5,000 and 100-fold, respectively, despite the active sites being >25 A apart. The details of this long-range protein communication process were investigated by solution NMR spectroscopy and CPMG relaxation dispersion experiments. Formation of the heterodimer enzyme results in a reduction in millisecond motions in HisF that extend throughout the protein. Binding of lGP results in an increase in protein-wide millisecond dynamics evidenced as severe NMR line broadening and elevated R (ex) values. Together, these data demonstrate a grouping of flexible residues that link the HisF active site with the protein interface to which HisH binds and provide a model for the path of communication between the IGPS active sites.
IGPS是一种51 kDa的异源二聚体酶，由HisH和HisF两种蛋白质组成，在HisH活性位点催化谷氨酰胺水解生成NH(3)，在HisF中催化氨与N'-[(5'-磷酸核糖基)甲脒基]-5-氨基咪唑-4-甲酰胺-核糖核苷酸(PRFAR)环化生成咪唑甘油磷酸(IGP)和5-氨基咪唑-4-甲酰胺核糖核苷酸(AICAR)。尽管活性位点相距超过25 Å，但PRFAR和IGP的结合分别使HisH酶中的谷氨酰胺酶活性提高了5000倍和100倍以上。通过溶液核磁共振光谱和CPMG弛豫分散实验研究了这种长程蛋白质通讯过程的细节。异源二聚体酶的形成导致HisF中毫秒级运动的减少，这种运动贯穿整个蛋白质。IGP的结合导致全蛋白毫秒级动力学增加，表现为严重的核磁共振谱线展宽和升高的R(ex)值。这些数据共同证明了一组柔性残基的存在，它们将HisF活性位点与HisH结合的蛋白质界面连接起来，并为IGPS活性位点之间的通讯路径提供了一个模型。
