Susan Wilke-Mounts

F1-ATPase, Roles of Three Catalytic Site Residues

Three critical residues, β-Lys-155, β-Asp-242, and β-Glu-181, situated close to the γ-phosphate of MgATP in F1-ATPase catalytic sites, were investigated. The mutations βK155Q, βD242N, and βE181Q were each combined with the βY331W mutation; the fluorescence signal of β-Trp-331 was used to determine MgATP, MgADP, ATP, and ADP binding parameters for the three catalytic sites of the enzyme. The quantitative contribution of side chains to binding energy at all three catalytic sites was calculated. The following conclusions were made. The major functional interaction of β-Lys-155 is with the γ-phosphate of MgATP and is of primary importance at site 1 (the site of highest affinity) and site 2. Release of MgATP during oxidative phosphorylation requires conformational re-positioning of this residue. The major functional interaction of β-Asp-242 is with the magnesium of the magnesium nucleotide at site 1; it has little or no influence at site 2 or 3. In steady-state turnover, the MgATP hydrolysis reaction occurs at site 1. β-Glu-181 contributes little to nucleotide binding; its major catalytic effect derives apparently from a role in reaction chemistry per se This work also emphasizes that nucleotide binding cooperativity shown by the three catalytic sites toward MgATP and MgADP is absolutely dependent on the presence of magnesium.

Conserved Walker A Ser Residues in the Catalytic Sites of P-glycoprotein Are Critical for Catalysis and Involved Primarily at the Transition State Step

P-glycoprotein mutants S430A/T and S1073A/T, affecting conserved Walker A Ser residues, were characterized to elucidate molecular roles of the Ser and functioning of the two P-glycoprotein catalytic sites. Results showed the Ser-OH is critical for MgATPase activity and formation of the normal transition state, although not for initial MgATP binding. Mutation to Ala in either catalytic site abolished MgATPase and transition state formation in both sites, whereas Thr mutants had similar MgATPase to wild-type. Trapping of 1 mol of MgADP/mol of P-glycoprotein by vanadate, shown here with pure protein, yielded full inhibition of ATPase. Thus, congruent with previous work, both sites must be intact and must interact for catalysis. Equivalent mutations (Ala or Thr) in the two catalytic sites had identical effects on a wide range of activities, emphasizing that the two catalytic sites function symmetrically. The role of the Ser-OH is to coordinate Mg2+ in MgATP, but only at the stage of the transition state are its effects tangible. Initial substrate binding is apparently to an “open” catalytic site conformation, where the Ser-OH is dispensable. This changes to a “closed” conformation required to attain the transition state, in which the Ser-OH is a critical ligand. Formation of the latter conformation requires both sites; both sites may provide direct ligands to the transition state.

α-Aspartate 261 Is a Key Residue in Noncatalytic Sites of Escherichia coli F1-ATPase

X-ray structure analysis of the noncatalytic sites of F1-ATPase revealed that residue α-Asp261 lies close to the Mg of bound Mg-5′-adenylyl-β,γ-imidodiphosphate. Here, the mutation αD261N was generated in Escherichia coli and combined with the αR365W mutation, allowing nucleotide binding at F1 noncatalytic sites to be specifically monitored by tryptophan fluorescence spectroscopy. Purified αD261N/αR365W F1-ATPase showed catalytic activity similar to wild-type. An important feature was that, without any resort to nucleotide-depletion procedures, the noncatalytic sites in purified native enzyme were already empty. Binding studies with MgATP, MgADP, and the corresponding free nucleotides led to the following conclusions. Residue α-Asp261 interacts with the Mg of Mg-nucleotide in noncatalytic sites and provides a large component of the binding energy (∼3 kcal/mol). It is the primary determinant of the preference of noncatalytic sites for Mg-nucleotide. The natural ligands at these sites in wild-type enzyme are the Mg-nucleotides and free nucleotides bind poorly. Under conditions where noncatalytic sites were empty, αD261N/αR365W F1 showed significant hydrolysis of MgATP. This establishes unequivocally that occupancy of noncatalytic sites by nucleotide is not required for catalysis.

E. coli F1 -ATPase: Site-directed mutagenesis of the β-subunit

Residues βGlu‐181 and βGlu‐192 of E. coli F1‐ATPase (the DCCD‐reactive residues) were mutated to Gln. Purified βGln‐181 F1 showed 7‐fold impairment of ‘unisite’ Pi formation from ATP and a large decrease in affinity for ATP. Thus the β‐181 carboxyl group in normal F1 significantly contributes to catalytic site properties. Also, positive catalytic site cooperativity was attenuated from 5 × 104‐ to 548‐fold in βGln‐181 F1. In contrast, purified βGln‐192 F1 showed only 6‐fold reduction in ‘multisite’ ATPase activity. Residues βGly‐149 and βGly‐154 were mutated to Ile singly and in combination. These mutations, affecting residues which are strongly conserved in nucleotide‐binding proteins, were chosen to hinder conformational motion in a putative ‘flexible loop’ in β‐subunit. Impairment of purified F1‐ATPase ranged from 5 to 61%, with the double mutant F1 less impaired than either single mutant. F1 preparations containing βIle‐154 showed 2‐fold activation after release from membranes, suggesting association with F0 restrained turnover on F1 in these mutants.

Directed mutagenesis of the dicyclohexylcarbodiimide-reactive carboxyl residues in β-subunit of F1-ATPase of Escherichia coli

Previous studies in which dicyclohexylcarbodiimide (DCCD) was used to inactivate F1-ATPase enzymes have suggested that two glutamate residues in the beta-subunit are essential for catalysis. In the Escherichia coli F1-ATPase, these are residues beta-Glu-181 and beta-Glu-192. Oligonucleotide-directed mutagenesis was used to change these residues to beta-Gln-181 and beta-Gln-192. The beta-Gln-181 mutation produced strong impairment of oxidative phosphorylation in vivo and also of ATPase and ATP-driven proton-pumping activities in membranes assayed in vitro. A low level of each activity was detected and an F1-ATPase appeared to be assembled normally on the membranes. Therefore, it is suggested that the carboxyl side chain at residue beta-181 is important, although not absolutely required, for catalysis in both directions on E. coli F1-ATPase. The beta-Gln-192 mutation produced partial inhibition of oxidative phosphorylation in vivo and membrane ATPase activity was reduced by 78%. These results contrast with the complete or near-complete inactivation seen when E. coli F1-ATPase is reacted with DCCD and imply that DCCD-inactivation is attributable more to the attachment of the bulky DCCD molecule than to the derivatization of the carboxyl side chain of residue beta-Glu-192. M. Ohtsubo and colleagues (Biochem. Biophys. Res. Commun. (1987) 146, 705-710) described mutagenesis of the F1-beta-subunit of thermophilic bacterium PS3. Mutations (Glu----Gln) of the residues homologous to Glu-181 and Glu-192 of E. coli F1-beta-subunit both caused total inhibition of ATPase activity. Therefore, there was a marked difference in results obtained when the same residues were modified in the PS3 and E. coli F1-beta-subunits.

F1-ATPase with cysteine instead of serine at residue 373 of the α subunit

Abstract Escherichia coli strain AN718 contains the αS373F mutation in F 1 F 0 -ATP synthase which blocks ATP synthesis (oxidative phosphorylation) and steady-state F 1 -ATPase activity. The revertant strain AN718SS2 containing the mutation αC373 was isolated and shown to confer a phenotype of higher growth yield than that of the wild type in liquid medium containing limiting glucose, succinate, or LB. Purified F 1 from strain AN718SS2 was found to have 30% of wild-type steady-state ATPase activity and 60% of wild-type oxidative phosphorylation activity. Azide sensitivity of ATPase activity and ADP-induced enhancement of bound aurovertin fluorescence, both of which are lost in αS373F mutant F 1 , were regained in αC373 F 1 . N -Ethylmaleimide (NEM) inactivated αC373 f 1 steady-state ATPase potently but had no effect on unisite ATPase. Complete inactivation of αC373 f 1 steady-state ATPase corresponded to incorporation of one NEM per f 1 (mol/mol), in just one of the three α subunits. NEM-inactivated enzyme showed azide-insensitive residual ATPase activity and loss of ADP-induced enhancement of bound aurovertin fluorescence. The data confirm the view that placement at residue α373 of a bulky amino acid side-chain (phenylalanyl or NEM-derivatized cysteinyl) blocks positive catalytic cooperativity in F 1 . The fact that NEM inhibits steady-state ATPase when only one α subunit of three is reacted suggests a cyclical catalytic mechanism.