A Direct Measure of the Contribution of Solvent Reorganization to the Enthalpy of Binding

Chervenak, Mary C.; Toone, Eric J.

doi:10.1021/ja00102a021

Cited by 242 publications

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“…Less negative ⌬H values associated with water release have been observed previously as well as more negative ⌬H values associated with water uptake (55,56). Water-mediated enthalpy-entropy compensation may be the mechanism by which changes in enthalpy occur (57)(58)(59)(60).…”

Section: Role Of Water In K Cat /K M(dhf)mentioning

confidence: 70%

A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

Chopra

Dooling

Horner

et al. 2008

Journal of Biological Chemistry

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R67 dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate using NADPH as a cofactor. This enzyme is a homotetramer possessing 222 symmetry, and a single active site pore traverses the length of the protein. A promiscuous binding surface can accommodate either DHF or NADPH, thus two nonproductive complexes can form (2NADPH or 2DHF) as well as a productive complex (NADPH⅐DHF). The role of water in binding was monitored using a number of different osmolytes. From isothermal titration calorimetry (ITC) studies, binding of NADPH is accompanied by the net release of 38 water molecules. In contrast, from both steady state kinetics and ITC studies, binding of DHF is accompanied by the net uptake of water. Although different osmolytes have similar effects on NADPH binding, variable results are observed when DHF binding is probed. Sensitivity to water activity can also be probed by an in vivo selection using the antibacterial drug, trimethoprim, where the water content of the media is decreased by increasing concentrations of sorbitol. The ability of wild type and mutant clones of R67 DHFR to allow host Escherichia coli to grow in the presence of trimethoprim plus added sorbitol parallels the catalytic efficiency of the DHFR clones, indicating water content strongly correlates with the in vivo function of R67 DHFR.R67 dihydrofolate reductase, a type II DHFR, 2 catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate. The gene for this enzyme is carried by an R-plasmid, and its presence confers resistance to the antibiotic drug, trimethoprim (TMP). Trimethoprim is a competitive inhibitor of chromosomal DHFR with a picomolar K i (1). Although R67 DHFR is not a good catalyst, it is not inhibited by TMP very well, and thus it allows cell growth in the presence of this antibacterial drug (2, 3). R67 DHFR shares no homology in sequence or structure with chromosomal DHFR and has been proposed to be a good model of a primitive enzyme (4).R67 DHFR is a homotetramer with a single active site pore; the overall structure possesses 222 symmetry as seen in Fig. 1 (5). The symmetry of the active site results in overlapping binding sites for substrate, DHF, and cofactor, NADPH. This can be observed as R67 DHFR binds a total of two ligands as follows: either two NADPH molecules or two folate/DHF molecules or one NADPH plus one folate/DHF molecule (6). The first two complexes are dead-end (binary) complexes, whereas the third is the productive ternary complex. Because of the 222 symmetry, binding to either ligand is unlikely to be optimal.The active site pore of R67 DHFR is unusual in its hourglass shape as well as its large size (2938 Å 3 for apoR67 DHFR with hydrogens added, calculated by CASTp (4, 7)). Because of this large volume, DHF and NADPH cannot occupy all the space in the pore and must use water to mediate some contacts with the protein.How do substrate and cofactor bind to R67 DHFR? From NMR, crystallography, and docking studies, the pteridine ring of DHF/fo...

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Section: Role Of Water In K Cat /K M(dhf)mentioning

confidence: 70%

A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

Chopra

Dooling

Horner

et al. 2008

Journal of Biological Chemistry

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“…The Role of Water in Carbohydrate Binding-Solvent reorganization is thought to account for 25-100% of the observed binding enthalpy (46), which makes the study of water structure in the sugar-binding site potentially interesting. However, in crystal structures, only the solvent molecules for which the position is well determined by the electron density can be evaluated for their importance in solvent reorganization.…”

Section: Man(␣1-3)man(␣1-o)me/man(␣1-6)man(␣1-o)me⅐cona Complexes 29193mentioning

confidence: 99%

The Crystal Structures of Man(α1–3)Man(α1-O)Me and Man(α1–6)Man(α1-O)Me in Complex with Concanavalin A

Bouckaert

Hamelryck

Wyns

et al. 1999

Journal of Biological Chemistry

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“…This is seen for many of the carbohydrate complexes of con A (Chervenak et a i, 1992; Mandai et al, 1994b), the Erythrina corallodendron lectin (Ecor L) (Gupta et a l, 1996; Surolia et a l, 1996), mannose binding protein C (MBP-C) (Quesenberry et a l, 1997) and wheat germ agglutinin (WGA) (Bains et a l, 1992). The data shows that almost all lectin -carbohydrate interactions are entropically unfavourable in water (Chervenak and Toone, 1994). Calorimetric studies have shown that in general, a significant proportion of the enthalpy arises from solvent reorganisation (Chervenak and Toone, 1994) (the transfer of solvent molecules between the bulk of the solvent and the solvation shell of the protein and ligand).…”

Section: Energetics Of Protein -Carbohydrate Interactionsmentioning

confidence: 99%

“…The thermodynamics of con A binding to pyranose sugars has been well documented (Chervenak and Toone, 1994;Chervenak and Toone, 1996;Brewer et al" 1997; Gupta et a l, 1997), and the structural basis of recognition is being investigated (Derewenda et a l, 1989;Harrop et a l, 1996;Naismith and Field, 1996;Moothoo and Naismith, 1998;Bradbrook et al, 1998; Chapters 2, 3 and 4 of this thesis). The binding of con A to furanose sugars has not been as well documented.…”

Section: Introductionmentioning

confidence: 99%

Protein-Carbohydrate Interactions

Abbott¹,

Bueren²

2017

Methods in Molecular Biology

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All rights reserved INFORMATION TO ALL USERSThe quality of this reproduction is d e p e n d e n t upon the quality of the copy subm itted.In the unlikely e v e n t that the a u thor did not send a c o m p le te m anuscript and there are missing pages, these will be noted. Also, if m aterial had to be rem oved, a n o te will ind ica te the deletion. uestProQuest 10171019Published by ProQuest LLO (2017). C opyright of the Dissertation is held by the Author. In submitting this thesis to the University of St. Andrews I understand that I am giving permission for it to be made available for use in accordance with the regulations of the University Library for the time being in force, subject to any copyright vested in the work not being affected thereby. I also understand that the title and abstract will be published, and that a copy of the work may be made and supplied to any bona fide library or research worker. 15Acknowledgements.Firstly, thanks go to my supervisor Jim Naismith for his constant enthusiasm and encouragement over the past three years, and for helping me to achieve my goals. Thanks also go to Steve Homans, JTrevor Rutherford, Charlie Weller and Tony Chiovotti for useful discussions. A special thanks goes to everyone in the Naismith group, especiallyStephen for making the lab a good place to work.Thanks are not enough for my parents, they are truly amazing.Finally, a special thanks goes to Alex, who achieved the impossible and made the sun shine every day in St. Andrews. 16Chapter 1Protein -Carbohydrate Interactions. 17 SummaryCarbohydrates are ubiquitous in biological systems. A sophisticated array of these molecules are found on cell surfaces and they are recognised with varying degrees of specificities by carbohydrate binding proteins such as lectins, anti-carbohydrate antibodies, bacterial periplasmic binding proteins and some enzymes. The interactions between proteins and carbohydrates are attractive therapeutic targets. However, the ubiquity of carbohydrates presents a huge challenge for rational design of therapeutics.The physical basis of protein -carbohydrate interactions is still too poorly understood to permit such design. Improving our understanding needs an atomic level understanding of the recognition of a carbohydrate by a protein. In recent years. X-ray crystallography together with isothermal titration microcalorimetry has begun to provide a powerful insight into the molecular processes governing protein -carbohydrate interactions. This chapter will summarise the current information available from X-ray structures and thermodynamics studies, identifying common features of protein -carbohydrate interactions.Concanavalin A from the Jack bean {Canavalia enisform is\ is the most well studied lectin. It was the first of the lectins to have its three dimensional structure solved by Xray crystallography, the first to have a carbohydrate bound structure solved and the first lectin to be solved to atomic resolution. The thermodynamics of its binding to both natural and modified carbohydrates ...

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A Direct Measure of the Contribution of Solvent Reorganization to the Enthalpy of Binding

Cited by 242 publications

References 0 publications

A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

The Crystal Structures of Man(α1–3)Man(α1-O)Me and Man(α1–6)Man(α1-O)Me in Complex with Concanavalin A

Protein-Carbohydrate Interactions

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