Substrate recognition by the hetero-octameric ATP phosphoribosyltransferase from Lactococcus lactis
Substrate recognition by the hetero-octameric ATP phosphoribosyltransferase from Lactococcus lactis
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DOI：
10.1021/bi061802v
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发表时间：
2006-12-19
期刊：
影响因子：
2.9
通讯作者：
Francklyn, Christopher S.
中科院分区：
文献类型：
--
作者：
Champagne, Karen S.;Piscitelli, Elise;Francklyn, Christopher S.
ATP磷酸核糖转移酶（ATP-PRT）家族在组氨酸生物合成的第一反应中连接ATP和5-磷酸核糖-1焦磷酸（PRPP）。它们由在所有三个界中发现的同六聚体形式和主要限于细菌的异八聚体形式组成。异八聚体ATP-PRT由四个与周质结合蛋白相关的HisG（S）催化亚基和四个类似于组氨酰-tRNA合成酶的HisZ调节亚基组成。为了澄清两个家庭的ATP-PRT之间的关系和磷酸核糖基转移酶一般，我们确定了稳态动力学的杂八聚体形式和其特征在于通过诱变的活性位点。PRPP底物的Km PRPP（18.4 +/- 3.5 AM）和k（cat）（2.7 +/- 0.3 s（-1））值与六聚体ATP-PRT相似，但ATP的Km（2.7 +/- 0.3 mM）高4倍，表明能量电荷的调节更严格。组氨酸和AMP分别被确定为非竞争性（Ki = 81.1 μ M）和竞争性（Ki = 1.44 μ M）抑制剂，其值接近其细胞内浓度。旨在研究识别PRPP的侧链的诱变实验显示，5 '-磷酸接触（T159 A和T162 A）具有k（cat）/K-m的最大降低（分别为25倍和155倍），而较小的降低在与焦磷酸部分交叉亚基接触（K50 A和K8 A）或与2'-OH基团接触的突变体中观察到。尽管它们的四级结构明显不同，但六聚体和杂八聚体ATRP-PRT表现出相似的功能参数，并采用使人想起更广泛的PRT超家族的机制策略。
Two families of ATP phosphoribosyl transferases (ATP-PRT) join ATP and 5-phosphoribosyl-1 pyrophosphate ( PRPP) in the first reaction of histidine biosynthesis. These consist of a homohexameric form found in all three kingdoms and a hetero-octameric form largely restricted to bacteria. Heterooctameric ATP-PRTs consist of four HisG(S) catalytic subunits related to periplasmic binding proteins and four HisZ regulatory subunits that resemble histidyl-tRNA synthetases. To clarify the relationship between the two families of ATP-PRTs and among phosphoribosyltransferases in general, we determined the steady state kinetics for the hetero-octameric form and characterized the active site by mutagenesis. The Km PRPP (18.4 +/- 3.5 AM) and k(cat) (2.7 +/- 0.3 s(-1)) values for the PRPP substrate are similar to those of hexameric ATP-PRTs, but the Km for ATP (2.7 +/- 0.3 mM) is 4-fold higher, suggestive of tighter regulation by energy charge. Histidine and AMP were determined to be noncompetitive (K-i = 81.1 mu M) and competitive (K-i = 1.44 mu M) inhibitors, respectively, with values that approximate their intracellular concentrations. Mutagenesis experiments aimed at investigating the side chains recognizing PRPP showed that 5'- phosphate contacts (T159A and T162A) had the largest (25- and 155-fold, respectively) decreases in k(cat)/K-m, while smaller decreases were seen with mutants making cross subunit contacts (K50A and K8A) to the pyrophosphate moiety or contacts to the 2'-OH group. Despite their markedly different quaternary structures, hexameric and hetero-octameric ATRP-PRTs exhibit similar functional parameters and employ mechanistic strategies reminiscent of the broader PRT superfamily.