Avidin is a slow-binding inhibitor of pyruvate carboxylase

Duggleby, Ronald G.; Attwood, Paul V.; Wallace, John C.; Keech, D.B.

doi:10.1021/bi00257a018

Cited by 79 publications

(76 citation statements)

References 23 publications

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“…The stoichiometry of binding of avidin to pyruvate carboxylase, as determined from the end-point of the titration, was found to be close to 1, as reported previously [8]. This means that 1 mol biotin contained in the enzyme is bound by 1 mol biotin-binding sites in the avidin.…”

Section: Dye-displacement Titrutionsupporting

confidence: 86%

“…while ['4C]biotin was obtained from Amersham Australia. All other materials were high-purity preparations as described earlier [8]. Chicken liver pyruvate carboxylase was purified as described by Goss et al [I].…”

Section: Materials a N D Methodsmentioning

confidence: 99%

“…In addition, the structure of avidin has been examined using both chemical and electron microscopic techniques [6,7] and the arrangement of the biotin-binding sites on the tetramer has been elucidated. In a previous paper on the kinetics of the inhibition of pyruvate carboxylase by avidin, Duggleby et al [8] [2] have observed the formation of clusters and chains between the avidin molecules and pyruvate carboxylase tetramers at these low concentrations of the proteins. In addition they observed single avidin molecules attached at the periphery of the enzyme tetramers.…”

mentioning

confidence: 99%

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Localisation of the Active Site of Pyruvate Carboxylase by Electron Microscopic Examination of Avidin‐Enzyme Complexes

Johannssen

Attwood

Wallace

et al. 1983

European Journal of Biochemistry

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Negatively stained enzyme-avidin complexes, seen at different stages of the titration of the biotin-binding sites on avidin with chicken liver pyruvate carboxylase, have been studied using electron microscopy. Formation of linear, unbranched polymers of the enzyme-avidin complex is seen to occur when the ratio of avidin to enzyme is between 2 : 1 and 1 : 2 ; beyond these limits only single enzyme tetramers are visible. The single avidin molecules observed seem to have a cuboid structure. The orientation and dimensions of the enzyme tetramers within the polymers indicate that the tetrahedron-like structure, observed in the single molecules, has been preserved. From the structure of the polymers and the observation of single enzyme-avidin complexes, we deduce that the biotin groups on the enzvme are located on the external faces of each subunit close to, and probably within 3 nm of, the

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Section: Dye-displacement Titrutionsupporting

confidence: 86%

Section: Materials a N D Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Localisation of the Active Site of Pyruvate Carboxylase by Electron Microscopic Examination of Avidin‐Enzyme Complexes

Johannssen

Attwood

Wallace

et al. 1983

European Journal of Biochemistry

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“…23,24 An example of this type of inhibition is seen with 1 (SAHA) 11,23 and, presumably, other hydroxamic acid-bearing HDAC inhibitors. However, if the rate of substrate conversion decreases over time (before the substrate has been significantly depleted), then the inhibitor can be described as having a slow-binding mechanism of action.…”

mentioning

confidence: 99%

“…However, if the rate of substrate conversion decreases over time (before the substrate has been significantly depleted), then the inhibitor can be described as having a slow-binding mechanism of action. 23,24 From the data plotted in Figure 3a−c, it appears that 26 binds HDACs 1−3 with a fast-on/fast-off mechanism, thus rendering less likely the involvement of protein conformational changes or an allosteric mode of inhibition. Moreover, to establish whether substrate and inhibitor 26 binding to the enzyme were mutually exclusive events, we measured product formation under conditions of varying substrate and inhibitor concentration for HDACs 1 and 3, followed by a Lineweaver− Burk analysis ( Figure S2 in the Supporting Information).…”

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confidence: 99%

Discovery of HDAC Inhibitors That Lack an Active Site Zn²⁺-Binding Functional Group

Vickers

Olsen

Leman

et al. 2012

ACS Med. Chem. Lett.

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Natural and synthetic histone deacetylase (HDAC) inhibitors generally derive their strong binding affinity and high potency from a key functional group that binds to the Zn 2+ ion within the enzyme active site. However, this feature is also thought to carry the potential liability of undesirable off-target interactions with other metalloenzymes. As a step toward mitigating this issue, here, we describe the design, synthesis, and structure−activity characterizations of cyclic α 3 β-tetrapeptide HDAC inhibitors that lack the presumed indispensable Zn 2+ -binding group. The lead compounds (e.g., 15 and 26) display good potency against class 1 HDACs and are active in tissue culture against various human cancer cell lines. Importantly, enzymological analysis of 26 indicates that the cyclic α 3 β-tetrapeptide is a fast-on/ off competitive inhibitor of HDACs 1−3 with K i values of 49, 33, and 37 nM, respectively. Our proof of principle study supports the idea that novel classes of HDAC inhibitors, which interact at the activesite opening, but not with the active site Zn 2+ , can have potential in drug design.

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Slow Binding Inhibitors

2013

Evaluation of Enzyme Inhibitors in Drug Discovery

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Avidin is a slow-binding inhibitor of pyruvate carboxylase

Cited by 79 publications

References 23 publications

Localisation of the Active Site of Pyruvate Carboxylase by Electron Microscopic Examination of Avidin‐Enzyme Complexes

Localisation of the Active Site of Pyruvate Carboxylase by Electron Microscopic Examination of Avidin‐Enzyme Complexes

Discovery of HDAC Inhibitors That Lack an Active Site Zn²⁺-Binding Functional Group

Slow Binding Inhibitors

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Avidin is a slow-binding inhibitor of pyruvate carboxylase

Cited by 79 publications

References 23 publications

Localisation of the Active Site of Pyruvate Carboxylase by Electron Microscopic Examination of Avidin‐Enzyme Complexes

Localisation of the Active Site of Pyruvate Carboxylase by Electron Microscopic Examination of Avidin‐Enzyme Complexes

Discovery of HDAC Inhibitors That Lack an Active Site Zn2+-Binding Functional Group

Slow Binding Inhibitors

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Discovery of HDAC Inhibitors That Lack an Active Site Zn²⁺-Binding Functional Group