Flip‐Flop Mechanisms in Enzymology

Lazdunski, Michel; Petitclerc, Claude; Chappelet, D.; Lazdunski, Claude

doi:10.1111/j.1432-1033.1971.tb01370.x

Cited by 173 publications

(73 citation statements)

References 64 publications

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“…It was noted that, although glutathione was bound at both active sites of the dimer, the conjugate of glutathione and 4-hydroxynonenal was only found in one subunit. The "active" status was proposed to switch between the two subunits by means of a relay of interacting residues in a flip-flop mechanism (42). Residues implicated in this relay were Arg-15, which is essential for the activity of mGST A4-4 with 4-hydroxynonenal (24, 43), and Arg-69, which is part of the dimer interface (7).…”

Section: Discussionmentioning

confidence: 99%

“…[45][46][47][48][49][50]). It appears that true half-of-the-sites reactivity (42,51) is an unusual phenomenon, with the corollary that enzymes displaying both half-of-the-sites and all-ofthe-sites reactivity are rarer still. Often the half-of-the-sites reactivity noted in previous reports was associated with a change in the quaternary structure of the enzyme (such as a shift in a tetramer-dimer equilibrium) or binding of an enzyme inhibitor or activator (52)(53)(54)(55)(56)(57).…”

Section: Discussionmentioning

confidence: 99%

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Human Glutathione Transferase A1-1 Demonstrates Both Half-of-the-sites and All-of-the-sites Reactivity

Lien

Gustafsson

Andersson

et al. 2001

Journal of Biological Chemistry

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A study of the kinetics of a heterodimeric variant of glutathione transferase (GST) A1-1 has led to the conclusion that, although the wild-type enzyme displays all-of-the-sites reactivity in nucleophilic aromatic substitution reactions, it demonstrates half-of-the-sites reactivity in addition reactions. The heterodimer, designed to be essentially catalytically inactive in one subunit due to a single point mutation (D101K), and the two parental homodimers were analyzed with seven different substrates, exemplifying three types of reactions catalyzed by glutathione transferases (nucleophilic aromatic substitution, addition, and double-bond isomerization reactions). Stopped-flow kinetic results suggested that the wild-type GST A1-1 behaved with halfof-the-sites reactivity in a nucleophilic aromatic substitution reaction, but steady-state kinetic analyses of the GST A1-D101K heterodimer revealed that this was presumably due to changes to the extinction coefficient of the enzyme-bound product. In contrast, steady-state kinetic analysis of the heterodimer with three different substrates of addition reactions provided evidence that the wild-type enzyme displayed half-of-the-sites reactivity in association with these reactions. The half-of-thesites reactivity was shown not to be dependent on substrate size, the level of saturation of the enzyme with glutathione, or relative catalytic rate.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Human Glutathione Transferase A1-1 Demonstrates Both Half-of-the-sites and All-of-the-sites Reactivity

Lien

Gustafsson

Andersson

et al. 2001

Journal of Biological Chemistry

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“…Several investigators have suggested (25,26) that subunit interactions within the LADH dimer play an important role in ordering the course of reaction. From the present structure it is evident that any kinetic model based on direct interaction between the active sites is excluded.…”

Section: Functional Significance Of the Structurementioning

confidence: 99%

Structure of Liver Alcohol Dehydrogenase at 2.9-Å Resolution

Brändén¹,

Eklund²,

Nordström³

et al. 1973

Proc. Natl. Acad. Sci. U.S.A.

176

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The conformation of the polypeptide chain in horse liver alcohol dehydrogenase (EC 1.1.1.1), as well as the binding sites for some inhibitor molecules, have been determined from x-ray crystallographic data to a resolution of 2.9 A. The apoenzyme of LADH crystallizes in space-group C2221 with one subunit per asymmetric unit and cell dimensions a = 56.0 A, b = 75.2 A, and c = 181.6 A (10). The crystallographic 2-fold axis relating the two subunits of the apoenzyme molecule is not present in crystals of complexes between apoenzyme and coenzyme, which suggests that the coenzyme may induce a structural asymmetry in the chemically identical subunits. An electron density map to 5-A resolution of the apoenzyme molecule has been described (11).Here we report the structure of this molecule as deduced from an electron-density distribution at a resolution of 2.9 A. Most important is the analysis of the binding sites for some inhibitor molecules, which permits us to identify functional attributes of the enzyme structure; MATERIALS AND METHODSThe enzyme was isolated from fresh horse livers (Akeson, A. & Lundqvist, G., to be published). Preparation of crystals and heavy-atom derivatives suitable for x-ray studies has been described (10). Methods of isomorphous replacement similar to those used for other protein-structure determinations were applied to obtain the electron density map. The crystallographic data were measured at +40 on a computercontrolled Philips-Stoe four-circle diffractometer equipped with a 32 K disc storage. Data were collected to a resolution of 2.9 X from crystals of the native protein, three heavy-metal derivatives [K2Pt(CN)4, KAu(CN)2, and K2Pt(CN)4 + KAu(CN)2], and one inhibitor complex [adenosine diphosphate ribose (ADP-ribose) ]. Intensities within 4.5 A were also measured on two other inhibitor complexes: 8-Br-ADPribose and 1,10-phenanthroline. A skeletal model of the main chain was built with the Kendrew-type models, with an optical comparator (12). CONFORMATION OF THE SUBUNITIn this section we describe briefly the conformation of the polypeptide chain and some details of the subunit interaction and binding of inhibitor molecules as deduced from our electron-density maps. In the next section we will discuss some implications of this structure.The two highest features in our 2.9-A electron-density map were roughly spherical in shape and were interpreted as the two zinc atoms of the subunit. From our 5-A work we already knew the position of one of these zinc atoms and the subunit

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“…Analysis of transport data for some constructs revealed Hill coefficients with non-Michaelis-Menten behavior (Table 1). This could be due to one or more factors including a flip-flop model where a dimeric transporter has reciprocal conformations, a ping-pong mechanism, or random substrate binding if more than one zinc atom is transported per transport cycle (35,36). It was also determined that zinc transport was roughly linear over the time of the assay (60 min) upon the addition of 21.75 M 65 ZnCl 2 (Fig.…”

Section: Hzip4 Computational Modeling and Functional Studiesmentioning

confidence: 99%

Computation and Functional Studies Provide a Model for the Structure of the Zinc Transporter hZIP4

Antala

Овчинников

Kamisetty

et al. 2015

Journal of Biological Chemistry

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Background: ZIP transporters increase the cytosolic concentration of first row transition metals. Results: We have developed a structural model of hZIP4 by combining protein prediction methods with in situ experiments. Conclusion: Analysis of our experiments provides insight into the permeation pathway of hZIP4. Significance: Comparison of this model to membrane transporter crystal structures provides a structural linkage to MFS proteins.

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Flip‐Flop Mechanisms in Enzymology

Cited by 173 publications

References 64 publications

Human Glutathione Transferase A1-1 Demonstrates Both Half-of-the-sites and All-of-the-sites Reactivity

Human Glutathione Transferase A1-1 Demonstrates Both Half-of-the-sites and All-of-the-sites Reactivity

Structure of Liver Alcohol Dehydrogenase at 2.9-Å Resolution

Computation and Functional Studies Provide a Model for the Structure of the Zinc Transporter hZIP4

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