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首页> 外文期刊>Biochemistry >The characteristics of the (alpha(VC)-C-371)(3)(beta(RC)-C-337)(3 gamma) double mutant subcomplex of the TF1-ATPase indicate that the catalytic site at the alpha(TP)-beta(TP) interface with bound MgADP in crystal structures of MF1 represents a cataly
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The characteristics of the (alpha(VC)-C-371)(3)(beta(RC)-C-337)(3 gamma) double mutant subcomplex of the TF1-ATPase indicate that the catalytic site at the alpha(TP)-beta(TP) interface with bound MgADP in crystal structures of MF1 represents a cataly

机译：（α（vc）-c-371）（3）（β（β（β）（β（rc）-c-337）（3γ）双突变体亚复合物的TF1-ATP酶的特征表明α（TP）处的催化位点 -beta（TP）与MF1晶体结构中的带有结合MGADP的界面表示催化

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In the MF1 crystal structure with the MgADP-fluoroaluminate complex bound to two catalytic sites [Menz, R. I., Walker, J. E., and Leslie, A. G. W. (2001) Cell 106, 331-341], the guanidinium of betaR(337) is within 2.9 Angstrom of the alpha-oxygen of alphaS(370) and 3.7 Angstrom of a methyl group of alphaV(371) at the alphaE-beta(HC) interface. To examine the functional role of this contact, the (alphaV(371)C)(3)(betaR(337)C)(3)gamma subcomplex of the TF1-ATPase was prepared and characterized. Steady state ATPase activity of the reduced double-mutant is 30% of that of the wild type. Inactivation of the double mutant containing empty catalytic sites or MgADP bound to one catalytic site with CuCl2 cross-linked two alpha-beta pairs, whereas a single alpha-beta pair cross-linked when at least two catalytic sites contained MgADP. The reduced double mutant hydrolyzed substoichiometric ATP 100-fold more rapidly than the enzyme containing two cross-linked alpha-beta pairs. Addition of AlCl3 and NaF to the reduced double mutant after incubation with stoichiometric MgADP or 200 muM MgADP irreversibly inactivated the steady state ATPase activity with rate constants of 1.5 x 10(-2) and 4.1 x 10(-2) min(-1), respectively. In contrast, addition of AlCl3 and NaF to the cross-linked enzyme after incubation with stoichiometric or 200 muM MgADP irreversibly inactivated ATPase activity with a common rate constant of similar to10(-4) min(-1). Correlation of these results with crystal structures of MF1 suggests that the catalytic site at the alpha(TP)-beta(TP) interface is loaded first upon addition of nucleotides to nucleoticle-depleted F-1-ATPases and that the catalytic site at the alpha(TP)-beta(TP) interface with bound MgADP in crystal structures represents a catalytic site containing inhibitory MgADP.
机译：在MF1晶体结构与MGADP-氟铝酸盐复合物结合到两个催化位点[MENZ，RI，WALKER，JE和LESLIE，AGW（2001）电池106,331-341]，ketar（337）的胍（337）在2.9内在α-β（HC）界面处的α-α（370）和3.7埃α的α-氧气（370）和3.7埃的血管。为了检查该接触的功能作用，制备和表征TF1-ATP酶的（α）（371）C）（3）（β（337）c）（3）γpromplect。减少双突变体的稳态ATP酶活性是野生型的30％。将含有空催化位点的双突变体的灭活与CuCl2交联的两个α-β对结合到一个催化位点，而当至少两个催化位点含有MGADP时，单个α-β对交联。减少的双突变体水解的替代计量ATP比含有两个交联α-β对的酶更快地迅速。与化学计量MgADP或200毫米MGADP孵育后，将AlCl3和NaF加入到还原的双突变体中，不可逆地灭活稳态ATP酶活性，速率常数为1.5×10（-2）和4.1×10（-2）min（-1） ， 分别。相反，在与化学计量或200毫米MGADP温育后，将AlCl3和NaF加入交联酶，不可逆地灭活的ATP酶活性，其具有相似于10（-4）min（-1）的共同速率常数。这些结果与MF1的晶体结构的相关性表明，在向核苷酸耗尽的核苷酸加入核苷酸时首先加载α（TP）-β（TP）界面处的催化位点，并且α催化位点（TP）-beta（TP）晶体结构中的MGADP界面表示含有抑制作用型MgAdp的催化位点。

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