The Structural Mechanism for Half-the-Sites Reactivity in an Enzyme, Thymidylate Synthase, Involves a Relay of Changes between Subunits

Anderson, Amy C.; O'Neil, R.H.; DeLano, Warren L.; Stroud, Robert M.

doi:10.1021/bi991610i

Cited by 82 publications

(95 citation statements)

References 28 publications

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“…During PDDF titration, Trp fluorescence at 332 nm was quenched related to the binding of PDDF, but an emission at 379 nm appeared, suggesting a fluorescence resonance energy transfer (FRET) between the WT TS and PDDF. A similar red shift in maximum emission during binding was reported in the literature for TS from Lactobacillus casei (Sharma & Kisliuk, 1975) and Pneumocystis carinii (Anderson, O'Neil, DeLano & Stroud, 1999). Trp fluorescence experiments indicate that the K48Q mutant has a lower affinity for both antifolates, consistent with the ITC data.…”

Section: Folate Binding For the K48q Mutantsupporting

confidence: 88%

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Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: Calorimetric and crystallographic analysis of the K48Q mutant

Arvizu‐Flores

Sugich-Miranda

Arreola

et al. 2008

The International Journal of Biochemistry & Cell Biology

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Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) using methylene tetrahydrofolate (CH 2 THF) as cofactor, the glutamate tail of which forms a water-mediated hydrogen-bond with an invariant lysine residue of this enzyme. To understand the role of this interaction, we studied the K48Q mutant of Escherichia coli TS using structural and biophysical methods. The k cat of the K48Q mutant was 430 fold lower than wild-type TS in activity, while the the K m for the (R)-stereoisomer of CH 2 THF was 300 µM, about 30 fold larger than K m from the wild-type TS. Affinity constants were determined using isothermal titration calorimetry, which showed that binding was reduced by one order of magnitude for folate-like TS inhibitors, such as propargyl-dideaza folate (PDDF) or compounds that distort the TS active site like BW1843U89 (U89). The crystal structure of the K48Q-dUMP complex revealed that dUMP binding is not impaired in the mutamt, and that U89 in a ternary complex of K48Q-nucleotide-U89 was bound in the active site with subtle differences relative to comparable wild type complexes. PDDF failed to form ternary complexes with K48Q and dUMP. Thermodynamic data correlated with the structural determinations, since PDDF binding was dominated by enthalpic effects while U89 had an important entropic component. In conclusion, K48 is critical for catalysis since it leads to a productive CH 2 THF binding, while mutation at this residue does not affect much the binding of inhibitors that do not make contact with this group.

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Section: Folate Binding For the K48q Mutantsupporting

confidence: 88%

“…Trp fluorescence was used to observe the antifolate binding regarding to the Trp residues at the active site (W80 and W83) (Anderson, O'Neil, DeLano & Stroud, 1999;Felder, Dunlap, Dix & Spencer, 2002;Lovelace, Gibson & Lebioda, 2007;Sharma & Kisliuk, 1975). Samples were prepared by dialysis as described above for ITC.…”

Section: Tryptophan Fluorescencementioning

confidence: 99%

Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: Calorimetric and crystallographic analysis of the K48Q mutant

Arvizu‐Flores

Sugich-Miranda

Arreola

et al. 2008

The International Journal of Biochemistry & Cell Biology

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“…The utility of negative cooperativity in the nitrogenase mechanism is not obvious, just as, to our knowledge, there are no cases of substantiated advantages of this phenomenon in other enzymes (32)(33)(34)37). That said, it can be imagined that when the events on one side are communicated to the other side, this may facilitate conformational changes that contribute to unidirectional electron flow from the Fe protein to the FeMo-co, or that favor the dissociation of the discharged Fe protein.…”

Section: Discussionmentioning

confidence: 97%

Negative cooperativity in the nitrogenase Fe protein electron delivery cycle

Danyal

Shaw

Page

et al. 2016

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

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Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen (N2) to two ammonia (NH3) molecules through the participation of its two protein components, the MoFe and Fe proteins. Electron transfer (ET) from the Fe protein to the catalytic MoFe protein involves a series of synchronized events requiring the transient association of one Fe protein with each αβ half of the α2β2 MoFe protein. This process is referred to as the Fe protein cycle and includes binding of two ATP to an Fe protein, association of an Fe protein with the MoFe protein, ET from the Fe protein to the MoFe protein, hydrolysis of the two ATP to two ADP and two Pi for each ET, Pi release, and dissociation of oxidized Fe protein-(ADP)2 from the MoFe protein. Because the MoFe protein tetramer has two separate αβ active units, it participates in two distinct Fe protein cycles. Quantitative kinetic measurements of ET, ATP hydrolysis, and Pi release during the presteady-state phase of electron delivery demonstrate that the two halves of the ternary complex between the MoFe protein and two reduced Fe protein-(ATP)2 do not undergo the Fe protein cycle independently. Instead, the data are globally fit with a two-branch negative-cooperativity kinetic model in which ET in one-half of the complex partially suppresses this process in the other. A possible mechanism for communication between the two halves of the nitrogenase complex is suggested by normal-mode calculations showing correlated and anticorrelated motions between the two halves.

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“…Although limited, crystallographic studies of such oligomeric systems have indicated that binding of the first ligand may distort the shape of the unliganded subunit, thereby rendering the binding of the second ligand more unfavorable 18,39 . Nevertheless, other crystallographic studies have failed to reveal any obvious structural effect on the unfilled subunit in the intermediate complex that could account for the observed negative cooperativity 18,40,41 , raising the possibility that other mechanisms may act to increase the energy of the second binding step.…”

Section: Discussionmentioning

confidence: 99%

Dynamically driven protein allostery

Popovych

Sun

Ebright

et al. 2006

Nat Struct Mol Biol

585

628

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Allosteric interactions are typically considered to proceed through a series of discrete changes in bonding interactions that alter the protein conformation. Here we show that allostery can be mediated exclusively by transmitted changes in protein motions. We have characterized the negatively cooperative binding of cAMP to the dimeric catabolite activator protein (CAP) at discrete conformational states. Binding of the first cAMP to one subunit of CAP dimer has no effect on the conformation of the other subunit. The dynamics of the system, however, are modulated in a distinct way by the sequential ligand binding process, with the first cAMP partially enhancing and the second cAMP completely quenching protein motions. As a result, a pronounced conformational entropic penalty is incurred by the second cAMP binding that is entirely responsible for the observed cooperativity. The results provide strong support of the existence of purely dynamics-driven allostery.Allosteric regulation is widely used in biological systems as a very effective mechanism to control protein activity [1][2][3][4] . Although a variety of allosteric systems have been studied for many decades, the mechanisms that underlie the communication of distant sites and energetically couple them remain largely elusive. According to the classical "mechanical" view allosteric interactions are mediated by a series of discrete conformational changes that alter protein structure [5][6][7] . Allosteric regulation within or across protein subunits requires that spatially separated sites be energetically coupled. Because allostery is fundamentally thermodynamic in nature, long-range communication may be mediated not only from a change in the mean conformation (enthalpic contribution) but also from a change in the dynamic fluctuations about the mean position (entropic contribution) 8,9 . The current view of allostery tends to explain the phenomenon only in pure structural terms, neglecting the important role of protein motions. To the other extreme, allosteric processes could, in theory, proceed solely through alteration in protein dynamics in the absence of conformational changes 10 . While there is increasing evidence suggesting a role of conformational dynamics in cooperative ligand binding processes 1,[11][12][13][14][15][16][17] , it has not been known whether dynamic phenomena can dominate allosteric mechanisms in proteins. Here, we present direct experimental evidence that allostery can be driven solely by changes in protein dynamics.Typically, studies of systems exhibiting homotropic cooperativity have focused on comparison of unliganded beginning and ligand-bound end states. However, the key conformational state with the potential to provide detailed insight into the mechanisms that underpin the allosteric process is the intermediate state. These states cannot be isolated in positively cooperative proteins because the singly liganded intermediates are poorly populated. In contrast, negatively *To whom correspondence should be addressed: babis@an...

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The Structural Mechanism for Half-the-Sites Reactivity in an Enzyme, Thymidylate Synthase, Involves a Relay of Changes between Subunits

Cited by 82 publications

References 28 publications

Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: Calorimetric and crystallographic analysis of the K48Q mutant

Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: Calorimetric and crystallographic analysis of the K48Q mutant

Negative cooperativity in the nitrogenase Fe protein electron delivery cycle

Dynamically driven protein allostery

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