Catalytic Contributions from Remote Regions of Enzyme Structure

Lee, Jeeyeon; Goodey, Nina M.

doi:10.1021/cr100042n

Cited by 98 publications

(96 citation statements)

References 216 publications

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“…Structure-function studies of enzymes have moved beyond an exclusive focus on active site protein side chains, with the growing recognition that distal residues can have a significant impact on catalytic bond cleavage events (1,2). Topological features within the protein matrix, such as cavities and channels, can also play key roles in the flux of biomolecules.…”

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confidence: 99%

Control of the Position of Oxygen Delivery in Soybean Lipoxygenase-1 by Amino Acid Side Chains within a Gas Migration Channel

Collazo

Klinman

2016

Journal of Biological Chemistry

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Understanding gas migration pathways is critical to unraveling structure-function relationships in enzymes that process gaseous substrates such as O 2 , H 2 , and N 2 . This work investigates the role of a defined pathway for O 2 in regulating the peroxidation of linoleic acid by soybean lipoxygenase 1. Computational and mutagenesis studies provide strong support for a dominant delivery channel that shuttles molecular oxygen to a specific region of the active site, thereby ensuring the regio-and stereospecificity of product. Analysis of reaction kinetics and product distribution in channel mutants also reveals a plasticity to the gas migration pathway. The findings show that a single site mutation (I553W) limits oxygen accessibility to the active site, greatly increasing the fraction of substrate that reacts with oxygen free in solution. They also show how a neighboring site mutation (L496W) can result in a redirection of oxygen toward an alternate position of the substrate, changing the regio-and stereospecificity of peroxidation. The present data indicate that modest changes in a protein scaffold may modulate the access of small gaseous molecules to enzyme-bound substrates.Structure-function studies of enzymes have moved beyond an exclusive focus on active site protein side chains, with the growing recognition that distal residues can have a significant impact on catalytic bond cleavage events (1, 2). Topological features within the protein matrix, such as cavities and channels, can also play key roles in the flux of biomolecules. For proteins with gaseous substrates, e.g. O 2 , H 2 , N 2 , and NH 3 , specific channels have been proposed either to transport reactive intermediates between active sites within a multifunctional enzyme (3) or to guide these small molecules from the solvent to the active site. The likely importance of the latter is relevant to a wide range of enzymes that includes oxidases, monooxygenases, nitrogenases, and hydrogenases (4 -17). The involvement of functionally evolved gas channels represents a significant departure from earlier held views in which random, transient fluctuations within a protein were proposed to facilitate the delivery of gaseous substrates to their site of binding and/or reactivity.Studies of lipoxygenases have aided in this shift in perception, given their requirement to capture O 2 via highly regio-and stereospecific pathways. As illustrated in Fig. 1 for the reaction catalyzed by soybean lipoxygenase-1 (SLO-1), 3 the delocalized nature of the free radical intermediate generated from the preferred substrate linoleic acid (LA) predicts that four unique products will be produced in equal amounts in solution. By contrast, native SLO-1 produces the 13S-hydroperoxide product (13S-HPOD) in greater than 90% yield for reaction of WT enzyme under optimal conditions. The precise mechanism by which SLO-1, as well as other lipoxygenases, maintain the regio-and stereospecificity of product peroxidation has engendered a variety of proposals that include: (i) an alteration in s...

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confidence: 99%

Control of the Position of Oxygen Delivery in Soybean Lipoxygenase-1 by Amino Acid Side Chains within a Gas Migration Channel

Collazo

Klinman

2016

Journal of Biological Chemistry

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“…First, it is clear that monomeric proteins can show functionally important allosteric effectsfor example, the regulatory proteins CheY and NtrC (Cui and Karplus 2008;McDonald et al 2012;Villali et al 2014;Volkman et al 2001). The transmission of the effects of distal mutations is analogous in many ways to the transmission of the effects of ligand binding, and is of course seen in many monomeric proteins (e.g., Clarkson et al 2006;Lee and Goodey 2011), while it has been studied in particular detail in dihydrofolate reductase (Boehr et al 2013;Wong et al 2005). Secondly, such effects are clearly not restricted to small molecule ligands-allosteric effects involving the binding of other macromolecules or covalent modification (such as phosphorylation) are of fundamental importance in the regulation of signal transduction and of metabolism.…”

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The role of protein dynamics in allosteric effects—introduction

Roberts

2015

Biophys Rev

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“…Distal residues can have a variety of effects on the structure [Chalissery et al, 2007;Chaptal et al, 2007], substrate binding affinity [Heitman et al, 2006;Carpten et al, 2007;Chalissery et al, 2007;Mirza et al, 2007], stability [Hong et al, 2007], and function [El Omari et al, 2006;Dupre et al, 2007;Klyuyeva et al, 2008] of a wide variety of different enzymes [Lee et al, 2011]. The semantics of defining the ''shell'' to which a specific amino acid residue belongs can be challenging, in particular in enzymes with large substrates, such as DNA polymerases.…”

Section: Discussionmentioning

confidence: 99%

“…AminoacyltRNA synthetases (aaRSs) are examples of enzymes that have very large nucleic acid substrates and whose activity is affected by mutations of distant residues [Lue et al, 2007]. The aaRSs must bind to and activate their cognate amino acids and tRNAs, and then transfer the activated aminoacyl adenylate to the tRNA [Arnez et al, 1997;Lee et al, 2011]. There is a need for communication between distinct domains in aaRSs for efficient and accurate aminoacylation [Alexander et al, 2001].…”

Section: Discussionmentioning

confidence: 99%

Effects of non‐catalytic, distal amino acid residues on activity of E. coli DinB (DNA polymerase IV)

Walsh

Parasuram

Rajput

et al. 2012

Environ and Mol Mutagen

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DinB is one of two Y family polymerases in E. coli and is involved in copying damaged DNA. DinB is specialized to bypass deoxyguanosine adducts that occur at the N 2 position, with its cognate lesion being the furfuryl adduct. Active site residues have been identified that make contact with the substrate and carry out deoxynucleotide triphosphate (dNTP) addition to the growing DNA strand. In DNA polymerases, these include negatively charged aspartate and glutamate residues (D8, D103, and E104 in E. coli DNA polymerase IV DinB). These residues position the essential magnesium ions correctly to facilitate nucleophilic attack by the primer hydroxyl group on the a-phosphate group of the incoming dNTP. To study the contribution of DinB residues to lesion bypass, the computational methods THEMATICS and POOL were employed. These methods correctly predict the known active site residues, as well as other residues known to be important for activity. In addition, these methods predict other residues involved in substrate binding as well as more remote residues. DinB variants with mutations at the predicted positions were constructed and assayed for bypass of the N 2 -furfuryl-dG lesion. We find a wide range of effects of predicted residues, including some mutations that abolish damage bypass. Moreover, most of the DinB variants constructed are unable to carry out the extension step of lesion bypass. The use of computational prediction methods represents another tool that will lead to a more complete understanding of translesion DNA synthesis. Environ. Mol. Mutagen. 53:766-776, 2012. V V C 2012 Wiley Periodicals, Inc.

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Catalytic Contributions from Remote Regions of Enzyme Structure

Abstract: Introduction 7595 2. What Properties of Protein Structure and Function Are Affected by Distal Mutations? 7596 3. Methods for Studying the Effect of Distal Mutations 7596 3.1. Functional Methods 7596 3.1.1. Amino Acid Mutagenesis and Kinetics 7596 3.

Cited by 98 publications

References 216 publications

Control of the Position of Oxygen Delivery in Soybean Lipoxygenase-1 by Amino Acid Side Chains within a Gas Migration Channel

Control of the Position of Oxygen Delivery in Soybean Lipoxygenase-1 by Amino Acid Side Chains within a Gas Migration Channel

The role of protein dynamics in allosteric effects—introduction

Effects of non‐catalytic, distal amino acid residues on activity of E. coli DinB (DNA polymerase IV)

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