Dataset Information

Interaction between ?C87 and ?R242 residues participates in energy coupling between catalysis and proton translocation in Escherichia coli ATP synthase.

ABSTRACT: Functioning as a nanomotor, ATP synthase plays a vital role in the cellular energy metabolism. Interactions at the rotor and stator interface are critical to the energy transmission in ATP synthase. From mutational studies, we found that the ?C87K mutation impairs energy coupling between proton translocation and nucleotide synthesis/hydrolysis. An additional glutamine mutation at ?R242 (?R242Q) can restore efficient energy coupling to the ?C87K mutant. Arrhenius plots and molecular dynamics simulations suggest that an extra hydrogen bond could form between the side chains of ?C87K and ?TPE381 in the ?C87K mutant, thus impeding the free rotation of the rotor complex. In the enzyme with ?C87K/?R242Q double mutations, the polar moiety of ?R242Q side chain can form a hydrogen bond with ?C87K, so that the amine group in the side chain of ?C87K will not hydrogen-bond with ?E381. As a conclusion, the intra-subunit interaction between positions ?C87 and ?R242 modulates the energy transmission in ATP synthase. This study should provide more information of residue interactions at the rotor and stator interface in order to further elucidate the energetic mechanism of ATP synthase.

SUBMITTER: Li Y

PROVIDER: S-EPMC6668360 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Interaction between γC87 and γR242 residues participates in energy coupling between catalysis and proton translocation in Escherichia coli ATP synthase.

Li Yunxiang Y Ma Xinyou X Weber Joachim J

Biochimica et biophysica acta. Bioenergetics 20190625 8

Functioning as a nanomotor, ATP synthase plays a vital role in the cellular energy metabolism. Interactions at the rotor and stator interface are critical to the energy transmission in ATP synthase. From mutational studies, we found that the γC87K mutation impairs energy coupling between proton translocation and nucleotide synthesis/hydrolysis. An additional glutamine mutation at γR242 (γR242Q) can restore efficient energy coupling to the γC87K mutant. Arrhenius plots and molecular dynamics simu ...[more]

PMID: 31251901

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Project description:Residue-to-alanine mutations and a two-amino acid deletion have been made in the highly conserved catalytic loop (residues 100-109) of Salmonella typhimurium OMP synthase (orotate phosphoribosyltransferase, EC 2.4.2.10). As described previously, the K103A mutant enzyme exhibited a 10(4)-fold decrease in k(cat)/K(M) for PRPP; the K100A enzyme suffered a 50-fold decrease. Alanine mutations at His105 and Glu107 produced 40- and 7-fold decreases in k(cat)/K(M), respectively, and E101A, D104A, and G106A were slightly faster than the wild-type (WT) in terms of k(cat), with minor effects on k(cat)/K(M). Equilibrium binding of OMP or PRPP in binary complexes was affected little by loop mutation, suggesting that the energetics of ground-state binding have little contribution from the catalytic loop, or that a favorable binding energy is offset by costs of loop reorganization. Pre-steady-state kinetics for mutants showed that K103A and E107A had lost the burst of product formation in each direction that indicated rapid on-enzyme chemistry for WT, but that the burst was retained by H105A. ?102?106, a loop-shortened enzyme with Ala102 and Gly106 deleted, showed a 10(4)-fold reduction of k(cat) but almost unaltered K(D) values for all four substrate molecules. The 20% (i.e., 1.20) intrinsic [1'-(3)H]OMP kinetic isotope effect (KIE) for WT is masked because of high forward and reverse commitment factors. K103A failed to express intrinsic KIEs fully (1.095 ± 0.013). In contrast, H105A, which has a smaller catalytic lesion, gave a [1'-(3)H]OMP KIE of 1.21 ± 0.0005, and E107A (1.179 ± 0.0049) also gave high values. These results are interpreted in the context of the X-ray structure of the complete substrate complex for the enzyme [Grubmeyer, C., Hansen, M. R., Fedorov, A. A., and Almo, S. C. (2012) Biochemistry 51 (preceding paper in this issue, DOI 10.1021/bi300083p )]. The full expression of KIEs by H105A and E107A may result from a less secure closure of the catalytic loop. The lower level of expression of the KIE by K103A suggests that in these mutant proteins the major barrier to catalysis is successful closure of the catalytic loop, which when closed, produces rapid and reversible catalysis.

Dataset Information

Interaction between ?C87 and ?R242 residues participates in energy coupling between catalysis and proton translocation in Escherichia coli ATP synthase.

Publications

Interaction between γC87 and γR242 residues participates in energy coupling between catalysis and proton translocation in Escherichia coli ATP synthase.

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