The Characteristics of the (alphaV~(371)C)_3(betaR~(227)C)_3gamma Double Mutant Subcomplex of the TF_1-ATPase Indicate that the Catalytic Site at the alpha_(TP)-beta_(TP) Interface with Bound MgADP in Crystal Structures of MF_1 Represents a Catalytic

摘要

In the MF_1 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 A of the a-oxygen of alphaS~(370) and 3.7 A of a methyl group of alphaV~(371) at the alpha_E-beta_(HC) interface.To examine the functional role of this contact,the (alphaV~(371)C)_3(betaR~(337)C)_3gamma subcomplex of the TF_1-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 CuCl_2 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 A1Cl_3 and NaF to the reduced double mutant after incubation with stoichiometric MgADP or 200muM 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 A1Cl_3 and NaF to the cross-linked enzyme after incubation with stoichiometric or 200muM MgADP irreversibly inactivated ATPase activity with a common rate constant of approx10~(-4) min~(-1).Correlation of these results with crystal structures of MF_1 suggests that the catalytic site at the alpha_(TP)-beta_(TP) interface is loaded first upon addition of nucleotides to nucleotide-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.