Merli Angelo, Szilágyi Andrea N, Flachner Beáta, Rossi Gian Luigi, Vas Mária
Department of Biochemistry and Molecular Biology, University of Parma, I-43100, Italy.
Biochemistry. 2002 Jan 8;41(1):111-9. doi: 10.1021/bi0115380.
Binding constants for the nucleotide substrates were determined in two different crystalline forms of pig muscle 3-phosphoglycerate kinase (PGK): the binary complex with 3-phosphoglycerate (3-PG) in which the two domains are in an open conformation (Harlos, Vas, and Blake (1992) Proteins, 12, 133-144) and the ternary complex with 3-PG and the Mg salt of the ATP analogue, beta,gamma-methyleneadenosine-5'-triphosphate (AMP-PCP), the structure of which is under resolution. Competitive titrations have been performed in the presence of the chromophoric analogue of ATP, 2'3'-O-(2,4,6-trinitrophenyl)ATP (TNP-ATP), similar to those previously carried out in solution, where a weakening of the binding of the nucleotide substrates in the presence of the other substrate, 3-PG, has been observed (Vas, Merli, and Rossi (1994) Biochem. J. 301, 885-891). Here the K(d) values for MgADP were found to be 0.096 +/- 0.021 and 0.045 +/- 0.016 mM, respectively, for the crystals of the binary and ternary complexes. Both K(d) values are significantly smaller than the one obtained in solution in the presence of 3-PG (0.38 +/- 0.05 mM) and are close to the values determined in solution in the absence of 3-PG (0.06 +/- 0.01 mM). Thus, the "substrate antagonism" observed in solution is not present in either of the investigated crystal forms. Further nucleotide binding studies with the solubilized enzyme have shown that 3-PG has no effect on ADP (Mg(2+)-free) binding (K(d) = 0.34 +/- 0.05 mM), while it weakens MgADP binding. Thus, 3-PG abolishes the strengthening effect of the Mg(2+) ion on the binding of ADP. This phenomenon is apparently due to the interaction between the carboxyl group of 3-PG and the protein, since the carboxyl-lacking analogue glycerol-3-phosphate has no detectable effect on MgADP binding. Comparison of the crystallographic data of different PGK binary (with either 3-PG or MgADP) and ternary (with both 3-PG and MgADP) complexes, having open and closed conformations, respectively, provides a possible structural explanation of the substrate antagonism. We suggest that the specific interaction between the 3-PG carboxylic group and a conserved arginine side chain is changed during domain closure, and, through interdomain communication, this change may be transmitted to the site in which Mg(2+) binds the ADP phosphates. This effect is abolished in the crystals of pig muscle PGK, in which lattice forces stabilize the open domain conformation.
在猪肌肉3 - 磷酸甘油酸激酶（PGK）的两种不同晶体形式中测定了核苷酸底物的结合常数：与3 - 磷酸甘油酸（3 - PG）形成的二元复合物，其中两个结构域处于开放构象（哈罗斯、瓦斯和布莱克（1992年），《蛋白质》，12卷，133 - 144页），以及与3 - PG和ATP类似物β，γ - 亚甲基腺苷 - 5'-三磷酸（AMP - PCP）的镁盐形成的三元复合物，其结构正在解析中。在ATP的发色类似物2'3'-O -（2,4,6 - 三硝基苯基）ATP（TNP - ATP）存在下进行了竞争性滴定，类似于之前在溶液中进行的滴定，在溶液中已观察到在另一种底物3 - PG存在下核苷酸底物的结合减弱（瓦斯、梅利和罗西（1994年），《生物化学杂志》，301卷，885 - 891页）。在此，二元和三元复合物晶体中MgADP的K(d)值分别为0.096±0.021和0.045±0.016 mM。这两个K(d)值均明显小于在3 - PG存在下溶液中获得的值（0.38±0.05 mM），并且接近在无3 - PG的溶液中测定的值（0.06±0.01 mM）。因此，在溶液中观察到的“底物拮抗”在任何一种研究的晶体形式中均不存在。对溶解酶进行的进一步核苷酸结合研究表明，� - PG对ADP（无Mg(2+)）的结合没有影响（K(d)=0.34±0.05 mM），而它会减弱MgADP的结合。因此，3 - PG消除了Mg(2+)离子对ADP结合的增强作用。这种现象显然是由于3 - PG的羧基与蛋白质之间的相互作用，因为缺乏羧基的类似物甘油 - 3 - 磷酸对MgADP的结合没有可检测到的影响。分别具有开放和封闭构象的不同PGK二元（与3 - PG或MgADP）和三元（与3 - PG和MgADP两者）复合物的晶体学数据比较，为底物拮抗提供了一种可能的结构解释。我们认为，在结构域闭合过程中，3 - PG羧基与保守精氨酸侧链之间的特定相互作用发生了变化，并且通过结构域间通讯，这种变化可能传递到Mg(2+)结合ADP磷酸基团的位点。在猪肌肉PGK晶体中这种效应被消除，其中晶格力稳定了开放结构域构象。
