Glucose Binding Isotope Effects in the Ternary Complex of Brain Hexokinase Demonstrate Partial Relief of Ground-State Destabilization

Lewis, Brett E.; Schramm, Vern L.

doi:10.1021/ja029852k

Cited by 20 publications

(19 citation statements)

References 13 publications

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“…Kinetic isotope effects near the site of bond loss (the C-N bond connecting ribose and hypoxanthine) compared to the remote 5′-3 H V/K KIE for nucleoside hydrolase from Crithidia fasciculata [17]. Binding isotope effects for individually labeled 3 H-labeled glucose molecules binding to the catalytic domain of human brain hexokinase [20,21]. The binary complex consists of glucose and hexokinase.…”

Section: Discussionmentioning

confidence: 99%

“…The extent and magnitude of BIEs was tested systematically using binary and ternary complexes of human brain hexokinase [20,21]. Tritium was incorporated at every carbon-H bond to give a family of [ [20].…”

Section: Hexokinasementioning

confidence: 99%

“…BIEs with the ternary complex of hexokinase•glucose•β-γ-CH 2 -ATP gave substantial changes at C6-H 2 , C5-H and C1-H ( Figure 4). Using the crystal structure of human hexokinase, the changed BIEs for the ternary complex were interpreted in terms of conformational changes of Glu742 positioned near C1 and Asn683 located under the glucosyl ring near C5 ( Figure 5) [21]. Computational modeling with 2-propanol was used to interpret isotope effects at each position.…”

Section: Hexokinasementioning

confidence: 99%

“…Computational modeling with 2-propanol was used to interpret isotope effects at each position. Significant BIEs occur with subtle bond length, angle and sp 3 geometric distortions that are too small to be observed by crystallography, but when combined with crystallography, interpretation of the BIE effect changes is augmented [21]. The isotope effects on the α-, βanomeric equilibrium of glucose were also considered and were shown to have an insignificant effect on the BIE shown in Figure 4…”

Section: Hexokinasementioning

confidence: 99%

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Binding isotope effects: boon and bane

Schramm

2007

Current Opinion in Chemical Biology

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Kinetic isotope effects are increasingly applied to investigate enzyme reactions and have been used to understand transition state structure, reaction mechanisms, quantum mechanical hydride ion tunneling and to design transition state analogue inhibitors. Binding isotope effects are an inherent part of most isotope effect measurements but are usually assumed to be negligible. More detailed studies have established surprisingly large binding isotope effects with lactate dehydrogenase, hexokinase, thymidine phosphorylase, and purine nucleoside phosphorylase. Binding reactants into catalytic sites immobilizes conformationally flexible groups, polarizes bonds, and distorts bond angle geometry, all of which generate binding isotope effects. Binding isotope effects are easily measured and provide high-resolution and detailed information on the atomic changes resulting from ligand-macromolecular interactions. Although binding isotope effects complicate kinetic isotope effect analysis, they also provide a powerful tool for finding atomic distortion in molecular interactions.

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Section: Discussionmentioning

confidence: 99%

Section: Hexokinasementioning

confidence: 99%

Section: Hexokinasementioning

confidence: 99%

Section: Hexokinasementioning

confidence: 99%

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Binding isotope effects: boon and bane

Schramm

2007

Current Opinion in Chemical Biology

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“…All BIE measurements were performed using a rapid equilibrium dialysis device (Thermo Fisher Scientific) in an analogous approach to the ultrafiltration method 15 previously used by our lab to measure BIEs for purine nucleoside phosphorylase, 16 uridine phosphorylase, 17 thymidine phosphorylase, 18 and hexokinase. 19,20 …”

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confidence: 99%

Binding Isotope Effects for para-Aminobenzoic Acid with Dihydropteroate Synthase from Staphylococcus aureus and Plasmodium falciparum

et al. 2015

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Dihydropteroate synthase is a key enzyme in folate biosynthesis and is the target of the sulfonamide class of antimicrobials. Equilibrium binding isotope effects and density functional theory calculations indicate that the substrate binding sites for para-aminobenzoic acid on the dihydropteroate synthase enzymes from Staphylococcus aureus and Plasmodium falciparum present distinct chemical environments. Specifically, we show that para-aminobenzoic acid occupies a more sterically constrained vibrational environment when bound to dihydropteroate synthase from P. falciparum relative to that of S. aureus. Deletion of a nonhomologous, parasitespecific insert from the plasmodial dihydropteroate synthase abrogated the binding of paraaminobenzoic acid. The loop specific to P. falciparum is important for effective substrate binding and therefore plays a role in modulating the chemical environment at the substrate binding site. Graphical abstract *Corresponding Author. Phone: 718-430-2813. vern.schramm@einstein.yu.edu. † These authors contributed equally to this work. ASSOCIATED CONTENT Supporting InformationThe Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.5b00490. Supporting Information Figure S1 and Supporting Information Tables S1-S3. Complete experimental procedures for expression and purification of wild-type SaDHPS, wild-type PfHPPK-DHPS, and Δ628-668PfHPPK-DHPS; measurement of equilibrium BIEs; measurement of V/K KIEs; and all DFT calculations (PDF) NotesThe authors declare no competing financial interest. Figure 1a). The sulfonamide class of antimicrobials, which function as pABA analogs, are competitive inhibitors of DHPS and act to disrupt folate biosynthesis. 1 Though the efficacy of sulfonamides has been hampered by the development of resistance, 2,3 therapies combining sulfonamides with the dihydrofolate reductase (DHFR) inhibitors pyrimethamine or trimethoprim are still widely used in the prophylaxis and treatment of malaria. 4 In addition, recent studies suggest therapies targeting DHPS may be effective alternatives for the treatment of bacterial infections caused by methicillin-resistant Staphylococcus aureus (MRSA). 5,6 As in most bacteria, DHPS in S. aureus (SaDHPS) is expressed as a monofunctional enzyme. 7 By contrast, DHPS in many lower eukaryotes is often expressed as a domain of a multifunctional polypeptide. [8][9][10][11] For example, DHPS from the malarial parasite Plasmodium falciparum (PfDHPS) is expressed as a bifunctional protein with 7,8-dihydro-6-hydroxymethylpterin pyrophosphokinase (PfHPPK), the enzyme preceding DHPS in folate biosynthesis. 12,13 Due to the lack of crystallographic data for PfHPPK-DHPS, there is currently limited information on the association of the PfHPPK and PfDHPS domains and how this association may impact protein-substrate interactions relative to monofunctional DHPS variants. In the current study, we use binding isotope effects (BIEs) and computational modeling to investigate differences in t...

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General Synthesis of Ordered Crystallized Metal Oxide Nanoarrays Replicated by Microwave‐Digested Mesoporous Silica

et al. 2003

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sulated between any two points on the exposed area of two adjacent grids. Therefore, the transferred PPy lines are electrically separated and so might find application in plastic electronics. [19] In summary, we have demonstrated for the first time to our knowledge that electropolymerization on semiconductor substrates patterned with self-assembled films results in patternamplified conducting polymer microstructures. It is a facile and versatile way to directly form positively patterned threedimensional conducting polymer structures, which we believe will find applications in related high-technology fields, such as nano-/microscale electromechanical systems and biosensors. ExperimentalPatterning was realized making use of the well-established lCP method, using a PDMS stamp to transfer OTS onto freshly cleaned substrate [8]. The substrates used include three types of silicon wafers (n-type (111), 0.015 X cm; n-type (100), 4 X cm; p-type (111), 15 X cm); and ITO (4±7 X cm). Electrodeposition was preformed on a Chi660a (Shanghai Chenhua Apparatus Co. Ltd) electrochemical workstation. A three-electrode system was used, with the patterned substrate as the working electrode, Pt wire as the counter electrode, and Ag/Ag + (10 mM AgNO 3 in acetonitrile) as the reference electrode. The plating solution of acetonitrile contains 0.2 M pyrrole and 0.1 M 1-hexyl-3-methylimidazolium tetrafluoroborate (a gift from Dr. Wang and Dr. Ye of our laboratory) as the supporting electrolyte. To transfer the PPy microstructure, the PPy pattern on the substrate was coated with Sylgard 184 (Dow Corning) and cured at 100 C for 2 h. Then the silicone elastomer was peeled off from the substrate.Resistance measurements were performed making use of a two-probe method. The adhesion strength of the PPy to the silicone elastomer was determined by a peel-off test using a domestic adhesive tape. AFM measurements were taken using an SPM-9500 apparatus (Shimazu). Optical microscopic images were collected on a charge-coupled device (CCD) camera (USA) equipped to a microhardness tester (USA). A variety of synthetic pathways has been proposed for the development of nanostructures because of their numerous potential applications. Received[1] The use of soft templates [2±4] (chelating agents, surfactants, DNA, etc.) and hard templates [5±11] (anionic alumina, carbon nanotubes, and mesoporous materials) has sparked wonderful contributions. Though various metal and semiconductor nanostructures have successfully been exploited, [2±4] uniform mesostructured crystallized metal oxide patterns are rarely reported, [10n,o,12] probably a result of the difficulty of choosing the proper synthesis precursor and the auxiliary reagents. In this respect, a general synthetic strategy for mesostructured metal oxides guided by ªhost± guest chemistryº is much desired. For the preparation of ordered nanostructure arrays, a hard template has some advantages when compared with a soft template, especially in its specific topological stability, veracity, predictability, and...

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Glucose Binding Isotope Effects in the Ternary Complex of Brain Hexokinase Demonstrate Partial Relief of Ground-State Destabilization

Cited by 20 publications

References 13 publications

Binding isotope effects: boon and bane

Binding isotope effects: boon and bane

Binding Isotope Effects for para-Aminobenzoic Acid with Dihydropteroate Synthase from Staphylococcus aureus and Plasmodium falciparum

General Synthesis of Ordered Crystallized Metal Oxide Nanoarrays Replicated by Microwave‐Digested Mesoporous Silica

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