Substrate Binding Causes Movement in the ATP Binding Domain of <i>Escherichia coli</i> Adenylate Kinase

Bilderback, Tim R.; Fulmer, Tim; Mantulin, William W.; Gläser, Michael

doi:10.1021/bi951833i

Cited by 31 publications

(31 citation statements)

References 24 publications

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“…2b, demonstrates that the lid domain of AK is in dynamical equilibrium between the open and closed conformational modes with each mode containing an ensemble of substates. Our results are in sharp contrast to the common view that AK's lid should remain open in the absence of substrates (13,(15)(16)(17), whereas binding would cause a global conformational change resulting in lid closure. That the substrate-free enzyme should remain in the open form has also been assumed (a reasonable supposition given the existing ensemble-averaged results) in the analysis of NMR relaxationdispersion experiments performed on AK (19).…”

Section: Resultscontrasting

confidence: 99%

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Illuminating the mechanistic roles of enzyme conformational dynamics

Hanson

Duderstadt

Watkins

et al. 2007

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

280

430

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Many enzymes mold their structures to enclose substrates in their active sites such that conformational remodeling may be required during each catalytic cycle. In adenylate kinase (AK), this involves a large-amplitude rearrangement of the enzyme's lid domain. Using our method of high-resolution single-molecule FRET, we directly followed AK's domain movements on its catalytic time scale. To quantitatively measure the enzyme's entire conformational distribution, we have applied maximum entropy-based methods to remove photon-counting noise from single-molecule data. This analysis shows unambiguously that AK is capable of dynamically sampling two distinct states, which correlate well with those observed by x-ray crystallography. Unexpectedly, the equilibrium favors the closed, active-site-forming configurations even in the absence of substrates. Our experiments further showed that interaction with substrates, rather than locking the enzyme into a compact state, restricts the spatial extent of conformational fluctuations and shifts the enzyme's conformational equilibrium toward the closed form by increasing the closing rate of the lid. Integrating these microscopic dynamics into macroscopic kinetics allows us to model lid opening-coupled product release as the enzyme's rate-limiting step.conformational equilibrium ͉ rate-limiting step ͉ single-molecule FRET ͉ adenylate kinase P roteins such as enzymes are flexible with a range of motions spanning from picoseconds for localized vibrations to seconds for concerted global conformational rearrangements (1). Despite their randomly fluctuating environment, in which stochastic collisions with solvent molecules drive changes in tertiary structure, enzymes have evolved to catalyze reactions efficiently and specifically. Indeed, conformational transitions have been postulated to play a central role in enzyme functions in a wide variety of ways, including direct contribution to catalysis (2), allosteric regulation (3), and large-scale conformational changes in response to ligand binding (4). Most of our current understanding of structural motions in solution comes from NMR experiments (5) as well as from molecular dynamics simulations (6), approaches that are best suited to study dynamics in the picoto millisecond time scales. Because catalysis in enzymes frequently occurs in the submillisecond to minute time regime, our current understanding of the relationship between enzyme function and conformational dynamics comes from NMR experiments involving relatively localized motions of active site forming loops on the submillisecond time scale (7-10). However, many enzymes contain active sites located in between domains in which large-amplitude, low-frequency domain motions are required to complete their Michaelis-Menten enzyme-substrate complexes. Even simple questions regarding these transitions remain generally unanswered: What is the number and range of conformational states accessible to enzymes during their catalytic cycle? How does the enzyme's conformation respond to interac...

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Section: Resultscontrasting

confidence: 99%

“…AK was chosen for this study because many aspects of this enzyme have been studied both experimentally (11)(12)(13)(14)(15)(16)(17)(18)(19) and theoretically (20)(21)(22)(23)(24). It is an ubiquitous enzyme that helps maintain the energy balance in cells by catalyzing the reversible reaction, Mg 2ϩ ⅐ATP ϩ AMP º Mg 2ϩ ⅐ADP ϩ ADP.…”

mentioning

confidence: 99%

Illuminating the mechanistic roles of enzyme conformational dynamics

Hanson

Duderstadt

Watkins

et al. 2007

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

280

430

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“…Although the reactivity of rabbit muscle AK is slightly inhibited at higher AMP concentrations (29, 32), E. coli AK exhibits its maximum turnover rate around 0.2 mM AMP followed by a steep drop, which plateaus at still higher AMP concentrations (33)(34)(35). This observation has been traditionally attributed to greater substrate inhibition by AMP in E. coli AK compared with the rabbit isoform; yet, the issue of whether the reaction involves competitive or non-competitive inhibition by AMP at the ATP binding site remains unresolved (15,33,(35)(36)(37).Here, we report a comprehensive kinetic study of the forward reaction of AK, exploring concentrations of nucleotides and Mg 2ϩ that are comparable to those inside E. coli cells, [Mg 2ϩ ] ϳ 1-2 mM (38) and [ATP] up to 3 mM (39). We discovered a previously unreported phenomenon: an increase in the forward reaction rate of AK with increasing Mg 2ϩ concentrations, where the stoichiometry of Mg 2ϩ to the enzyme is greater than one.…”

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confidence: 99%

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confidence: 99%

Direct Mg2+ Binding Activates Adenylate Kinase from Escherichia coli

Tan

Hanson

Yang

2009

Journal of Biological Chemistry

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We report evidence that adenylate kinase (AK) from Escherichia coli can be activated by the direct binding of a magnesium ion to the enzyme, in addition to ATP-complexed Mg 2؉ . By systematically varying the concentrations of AMP, ATP, and magnesium in kinetic experiments, we found that the apparent substrate inhibition of AK, formerly attributed to AMP, was suppressed at low magnesium concentrations and enhanced at high magnesium concentrations. This previously unreported magnesium dependence can be accounted for by a modified random bi-bi model in which Mg 2؉ can bind to AK directly prior to AMP binding. A new kinetic model is proposed to replace the conventional random bi-bi mechanism with substrate inhibition and is able to describe the kinetic data over a physiologically relevant range of magnesium concentrations. According to this model, the magnesium-activated AK exhibits a 23-؎ 3-fold increase in its forward reaction rate compared with the unactivated form. The findings imply that Mg 2؉ could be an important affecter in the energy signaling network in cells. Adenylate kinase (AK)2 is a ϳ24-kDa enzyme involved in cellular metabolism that catalyzes the reversible phosphoryl transfer reaction (1) as in Reaction 1.It is recognized to play an important role in cellular energetic signaling networks (2, 3). A deficiency in human AK function may lead to such illness as hemolytic anemia (4 -8) and coronary artery disease (9); the latter is thought to be caused by a disruption of the AMP signaling network of AK (10). The ubiquity of AK makes it an ideal candidate for investigating evolutionary divergence and natural adaptation at a molecular level (11,12). Indeed, extensive structure-function studies have been carried out for AK (reviewed in Ref. 13). Both structural and biophysical studies have suggested that large-amplitude conformational changes in AK are important for catalysis (14 -19). More recently, the functional roles of conformational dynamics have been investigated using NMR (20 -22), computer simulations (23-27), and single-molecule spectroscopy (28). Given the critical role of AK in regulating cellular energy networks and its use as a model system for understanding the functional roles of conformational changes in enzymes, it is imperative that the enzymatic mechanism of AK be thoroughly characterized and understood.The enzymatic reaction of adenylate kinase has been shown to follow a random bi-bi mechanism using isotope exchange experiments (29). Isoforms of adenylate kinases characterized from a wide range of species have a high degree of sequence, structure, and functional conservation. Although all AKs appear to follow the same random bi-bi mechanistic framework (15, 29 -33), a detailed kinetic analysis reveals interesting variations among different isoforms. For example, one of the most puzzling discrepancies is the change in turnover rates with increasing AMP concentration between rabbit muscle AK and Escherichia coli AK. Although the reactivity of rabbit muscle AK is slightly inhibited at high...

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“…The enzyme contains two distinct nucleotide binding sites: the MgATP site, which binds MgATP and MgADP, and the AMP site, which is specific for AMP and uncomplexed ADP. The substrate-induced conformation changes in AK have been the subject of a number of investigations (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Based on the comparison of AK crystal structures representing the enzyme in different ligand forms, apo-form (from pig muscle), enzyme-AMP binary complex (from beef heart mitochondrial matrix), and enzyme-AP 5 A complex (from Escherichia coli), Schulz and co-workers (15,18) suggested that AK undergoes large structural changes upon substrates binding.…”

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Evidence for at Least Two Native Forms of Rabbit Muscle Adenylate Kinase in Equilibrium in Aqueous Solution

Zhang

Sheng

Niu

et al. 1998

Journal of Biological Chemistry

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The time course of 8-anilino-1-naphthalenesulfonic acid (ANS) binding to adenylate kinase (AK) is a biphasic process. The burst phase ends in the dead-time of the stopped-flow apparatus (about 15 ms), whereas the slow phase completes in about 10 min. A Job's plot tests of the binding stoichiometry demonstrates that there is one ANS binding site on AK, but only about 70% of the enzyme can rapidly bind with ANS, indicating that the conformation of native AK molecules is not homogeneous. Further kinetic analysis shows that the effects of ANS and substrates concentration on the burst and slow phase fluorescence building agree well with the multiple native forms mechanism. One form (denoted N 1 ) binds with ANS, whereas the other (denoted N 2 ) does not. ANS binding to N 1 results in a burst phase fluorescence increase, followed by the interconversion of N 2 to N 1 , to give the slow phase ANS binding. Under urea denaturation conditions, N 2 is easily perturbed by urea and unfolds completely at low denaturant concentrations, whereas N 1 is relatively resistant to denaturation and unfolds at higher denaturant concentrations. The existence of multiple native forms in solution may shed some light on the interpretation of the enzyme catalytic mechanism.It has been accepted that a globular protein in its native state adopts a single, well defined conformation. However, this concept has been challenged by several reports that some proteins may exist in more than one distinct folded form in equilibrium. Evidence for distinguishing multiple native forms of staphylococcal nuclease has come from electrophoretic and NMR studies (1-6). For calbindin D 9K , evidence of multiple forms came not only from NMR studies, but also from x-ray crystal structure (7,8).Adenylate kinase (AK 1 ; EC 2.7.4.3) catalyzes the phosphoryl transfer reaction: MgATP ϩ AMP i MgADP ϩ ADP (9, 10). The enzyme contains two distinct nucleotide binding sites: the MgATP site, which binds MgATP and MgADP, and the AMP site, which is specific for AMP and uncomplexed ADP. The substrate-induced conformation changes in AK have been the subject of a number of investigations (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Based on the comparison of AK crystal structures representing the enzyme in different ligand forms, apo-form (from pig muscle), enzyme-AMP binary complex (from beef heart mitochondrial matrix), and enzyme-AP 5 A complex (from Escherichia coli), Schulz and co-workers (15, 18) suggested that AK undergoes large structural changes upon substrates binding. These conformational changes can be subdivided into two steps; the first change, corresponding to binding to AMP, only involves the displacement of the small ␣-helical domain (residues 30 -59 in E. coli AK), whereas the second change, occurring with additional binding of substrate ATP, mainly involves the displacement of the LID domain (residues 122-159 in E. coli AK). However, it is not clear whether the enzyme achieves its catalytic conformation only upon substrate binding or whether the above confo...

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Substrate Binding Causes Movement in the ATP Binding Domain of Escherichia coli Adenylate Kinase

Cited by 31 publications

References 24 publications

Illuminating the mechanistic roles of enzyme conformational dynamics

Illuminating the mechanistic roles of enzyme conformational dynamics

Direct Mg2+ Binding Activates Adenylate Kinase from Escherichia coli

Evidence for at Least Two Native Forms of Rabbit Muscle Adenylate Kinase in Equilibrium in Aqueous Solution

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