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. 2025 Nov 7;5(11):5568–5577. doi: 10.1021/jacsau.5c01069

Commonality of Mechanism in Glycoside Hydrolases, Nucleoside Hydrolases, and Phosphorylases: Importance of Side-Chain Conformation Preorganization

Po-Sen Tseng †,‡,§, Jonathan C K Quirke †,‡,§, W Jonathan Lin †,‡,§, David Crich †,‡,§,*
PMCID: PMC12648331  PMID: 41311960

Abstract

A survey of the Protein Data Bank reveals that the arabinofuranosidase class of enzymes broadly restrict their substrate side chains to the gauche,gauche (gg) conformation that provides maximum electrostatic stabilization to oxocarbenium ion-like transition states and so employ the strategy reported previously for the majority of glycoside hydrolases, transglycosidases, and glycosyltransferases acting on pyranosyl substrates. The fructofuranosidases, ribonucleosidases, ribonucleoside phosphorylases, and nucleoside 2′-deoxyribosyltransferases, whose gg conformation is sterically hindered, restrict their substrate side chains to the next most positive charge-stabilizing gauche,trans (gt) conformation. These conclusions are supported by extensive literature studies on the mechanisms of C–N bond cleavage by members of the nucleosidase and nucleoside phosphorylase families and are discussed in terms of Warshel’s concept of the electrostatic origin of the catalytic power of enzymes and the role of preorganized active sites.

Keywords: glycoside hydrolases, nucleoside hydrolases, nucleoside phosphorylases, electrostatic transition-state stabilization, substrate preorganization

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Introduction

Glycoside hydrolases (GHs) and glycosyltransferases (GTs), enzymes that catalyze the hydrolysis or formation of glycosidic bonds with exceptional efficiency, with limited exceptions, − function by stabilizing transition states with significant degrees of oxocarbenium ion character irrespective of their classifications as inverting or retaining (double inverting) systems. − Optimal chemical glycosylation reactions are similarly best understood as proceeding via SN2-like mechanisms with exploded transition states with considerable oxocarbenium ion character. − In chemical glycosylation or glycoside hydrolysis at the anomeric center of pyranoses, it has been established that the side-chain conformation influences both reactivity and stereoselectivity, ,− with the gauche,gauche (gg) conformation providing maximal electrostatic stabilization to nascent positive charge at the oxocarbenium ion-like transition state. This influence of the side-chain conformation on reactivity at the anomeric center is analogous to the manner by which axial and pseudoaxial C–O bonds on pyranosyl rings enhance reactivity compared to the corresponding electron-withdrawing equatorial or pseudoequatorial bonds (Figure ). − Mining of the Protein Data Bank (PDB) revealed that GHs proceeding via classical mechanisms, Leloir GTs, and transglycosidases use hydrogen bonding to restrict the side chains of their pyranosyl substrates to approximate gg conformations and, we have argued, in doing so provide additional electrostatic stabilization to oxocarbenium ion-like transition states. , Exceptions to this phenomenon mostly result from the enforcement of nonstandard conformations of the pyranoside ring on binding to the enzyme. Such nonstandard ring conformations either energetically disfavor the gg side-chain conformation or provide additional electrostatic stabilization from pseudoaxial C–O bonds, akin to the synthetic chemists’ concept of superarming, , so minimizing the need for side-chain conformational control. , Glycosidase inhibitors with appropriately restricted side-chain conformations, whether synthetic or natural, can show improved inhibitory properties over their unrestricted counterparts. ,,

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Gauche,gauche (gg), gauche,trans (gt), and trans,gauche (tg) conformations of sugar side chains illustrated for six-membered cyclic oxocarbenium ions and parallels of the gg and tg conformations with pseudoaxial and pseudoequatorial C–O bonds at the 4-position of galactosyl and glucosyl oxocarbenium ion-like transition states, respectively.

The concept of the control of substrate conformation by enzymes as a component of catalysis has a long history. − It has also been strongly disputed by Warshel, who has argued that enzymes gain little by restraining the reactive fragments of their substrates and proposed that “the polar preorganization of enzyme active sites is the most important factor in enzyme catalysis” in large part by overcoming the reorganization energy of water molecules needed to electrostatically stabilize transition states. −

In this paper, we extend our PDB-driven analysis of side-chain conformational restriction by GHs and related enzymes to encompass GHs acting on furanosides, nucleoside hydrolases (NHs), nucleoside phosphorylases (NPs), nucleoside 2′-deoxyribosyltransferases (NDTs), 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidases (MTANs), and 5′-methylthioadenosine phosphorylases (MTAPs) (Figure ).

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Transformations catalyzed by the l-arabinofuranosidases (AFs), d-fructofuranosidases (FFs), d-nucleosidases (NHs), and d-nucleoside phosphorylases (NPs).

We draw parallels with previous mechanistic work on the importance of the substrate side-chain hydroxy group in NHs and NPs, culminating in the development of the immucillins, , to underline the importance of side-chain conformational control by GHs, GTs, and related enzymes. Finally, we argue that the restriction of side-chain conformation, and so substrate preorganization, by GHs, GTs, and related enzymes bridges the gap between classical physical organic-based substrate preorganization, with its emphasis on confining the reactive termini of the substrate and reducing the entropic penalty, − and the electrostatic hypothesis propounded by Warshel and others in that the enzymes in question restrict the side-chain conformation to provide additional electrostatic stabilization to the transition state. − ,

Results

The side-chain conformational equilibria of pentofuranosides have been investigated by Serianni and co-workers by classical NMR methods and by DFT calculations, leading to the approximate populations for the arabino and xylofuranosides set out in Figure . , Serianni and co-workers also noted the coupling of ring and side-chain conformations in these systems, as was confirmed in a molecular dynamics (MD) study by Wang and Woods. In contrast, a subsequent MD study by Nester and Plazinski found little correlation between ring and side-chain conformations in the furanosides with ≤0.7 kcal mol–1 difference in the ribo and arabinofuranosides. Using a combination of VT-NMR and DFT methods, Lowary and co-workers determined the impact of side-chain conformation on anomeric reactivity in a series of 5-O-benzoyl-α- and β-d-lyxofuranosides and α- and β-furanosyl triflates finding a consistent tg > gt > gg stability order. The restriction of side-chain conformation in furanosides, whether by inclusion in a bicyclic system or simply by the introduction of an additional C–C bond, as in the hexofuranosides, has been demonstrated to impact anomeric reactivity. − Overall, while the conformational equilibria and reactivity patterns do not follow exactly those found in the pyranosides, the same gross trends are found in the furanosides.

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Side-chain conformation distributions of furanosides in solution.

Mining of the PDB using the Carbohydrate Active Enzymes database (CAZy, http://www.cazy.org) for structures of GHs binding furanosides and their analogs in their active (−1) site with resolution ≤2.5 Å, as described for the pyranosides, , with data curation using the Privateer software to minimize errors, resulted in a data set (Tables and ) comprised of α- and β-l-arabinofuranosidases, β-d-fructofuranosidases, β-d-transfructofuranosidases, and one α-d-arabinofuranosidase/fructofuranosidase (see Supporting Information for full details). Clearly, the arabinofuranosidases (AFs), whether α- or β-, restrict the side chains of their substrates to the gg conformation, while the β-d-fructofuranosidases (FFs) enforce the gt conformation.

1. Side-Chain and Ring Conformations of Furanoside Ligands Bound to l-Arabinose Processing Enzymes.

enzyme function gg gt tg ring conformation total H-bonding to side chain
α-l-arabinofuranosidase 19 0 0 E 4/E 2 19 16
β-l-arabinofuranosidase 10 0 0 E 4/3 T 2 10 9
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2. Side-Chain and Ring Conformations of Furanoside Ligands Bound to d-Fructose Processing Enzymes.

  side-chain conformation anomeric side-chain conformation      
enzyme function gg gt tg gg gt tg ring conformation total H-bonding to side chain
β-d-fructofuranosidase 0 28 0 1 26 1 E 3 28 27
β-d-transfructofuranosidase 1 11 0 4 8 0 E 3/4 T 3 13 7
α-d-arabinofuranosidase/α-d-fructofuranosidase 2 0 0 0 0 1 3 T 4/E 5 2 2
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a

One structure has an ambiguous side-chain conformation.

b

The arabinofuranosyl substrate has the 3 T 4 ring conformation, while the fructofuranosyl substrate has the E 5 ring conformation.

We next examined the NHs and NPs, whose mechanisms have been reviewed and also involve exploded oxocarbenium-ion-like transition states, − restricting ourselves to the enzymes operating on nucleosides as substrates. By searching the Enzyme Commission (EC) numbers in the PDB and using the same criteria as described for the GHs above, we obtained the data set summarized in Table for NHs (EC 3.2.2.1, EC 3.2.2.3, EC 3.2.2.8, and EC 3.2.2.22), NPs (EC 2.4.2.1, EC 2.4.2.2, EC 2.4.2.3, and EC 2.4.2.4), NDTs (EC 2.4.2.6), MTANs (EC 3.2.2.9 and 3.2.2.16), and MTAPs (EC 2.4.2.28).

3. Side-Chain and Ring Conformations of Ligands in Complexes with d-Nucleoside Processing Enzymes.

enzyme function gg gt tg ring conformation total H-bonding to side chain
d-nucleoside hydrolase (NH) 0 19 0 4 E 19 19
d-nucleoside phosphorylase (NP) 5 83 1 4 E 89 81
d-nucleoside 2′-deoxyribosyl-transferase (NDT) 0 6 0 E O 6 6
d-5′-methylthioadenosine/d-S-adenosylhomocysteine nucleosidase (MTAN) 1 3 41 2 T 3/4 E 45 4
d-5′-methylthioadenosine phosphorylase (MTAP) 0 21 0 4 E/O E 21 1
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For the NHs, NPs, NDTs, and MTAPs, there is a very strong preference for binding the substrates with side-chain conformations that approximate the gt conformation, while for the MTANs, substrate binding with the tg conformation of the side chain is strongly preferred.

Discussion

Side-Chain Conformation

The contrast between the AFs (Table ), which enforce the gg conformation of their side chains, and the FFs (Table ), NHs, NPs, NDTs, and MTAPs (Table ), which in contrast predominantly restrict the side chains of their substrates to the gt conformation, is striking. Yet a further contrast is apparent with the MTANs (Table ), which have evolved to bind their substrates with the tg conformation of the side chains. We first address the AFs, FFs, NHs, NPs, and NDTs, all of which bear a hydroxy group in their substrate side chains, before returning to the MTANs and MTAPs with their sulfur-based substituents in the side chain of their substrates.

For the α-l-AFs when the bound ligand is l-arabinofuranose-derived or lacks an anomeric substituents, as in l-1,4-iminoarabinitol, it is very predominantly bound in the E 4 conformation (Figure ) with a single exception having the 3 E conformation. In contrast, when the anomeric substituent of the ligand is on the β-face, the E 2 conformation is preferred, with the exception of a covalently bound adduct arising from invertive ring opening by an active-site glutamate residue of a carbasugar-derived α-cyclic sulfate that carries a residual sulfate on the α-face. All ligands bound in complex with an α-l-AF retain the gg conformation of the side chain, whether the anomeric configuration is α- or β-, and the implication is that the α-l-AFs bind their substrates as an E 4 envelope with a pseudoequatorial side chain and restrict it to the gg conformation. After pseudorotation to the proximal 3 E envelope, still with a pseudoequatorial side chain restricted by hydrogen bonding to the gg conformation, invertive displacement at the anomeric center, whether by water or an active site residue, takes place through a 3 T 2-like transition state ultimately providing a β-configured product in the E 2 conformation. Finally, this initial product conformer may undergo pseudorotation to the closely related 1 E conformation, with the gg conformation of the side chain retained throughout the entire process (Figure ). A single example of a difructose dianhydride I synthase/hydrolase exhibiting α-d-AF activity in complex with β-d-arabinofuranose (PDB 7V1W) has the ligand bound in an approximate 3 T 4 conformation, midway between the E 4 and 3 E envelopes, with its side chain restricted by hydrogen bonding to the gg conformation.

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Pseudorotational wheels of α-d-arabinofuranoside (A) and α-l-arabinofuranoside (B) rings. Only the envelope conformations are shown. P, pseudorotational phase angle.

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Apparent pseudorotational itinerary followed by substrates during hydrolysis by α-l-arabinofuranosidases. Hydroxy groups at the 2- and 3-positions are omitted for clarity.

For the β-l-AFs, arabinose-derived ligands with the β-anomeric configuration are bound mainly in either the 3 E or 3 T 2 conformations, always with the side chain held in an approximate gg conformation by hydrogen bonding. On the other hand, β-l-AF structures with α-configured l-arabinofuranose derivatives, located in the form of covalent adducts with the protein, are found as either E 2 or E 4 envelopes, still with the side chain maintained in the gg conformation. This pattern holds even in recent examples that employ active-site cysteine residues as nucleophiles. ,

Arabinofuranose and fructofuranose share the relative arabino-configuration of their nonanomeric positions (Figure ), yet the β-d-FFs bind their fructose-derived ligands very predominantly in the E 3 conformation (equivalent to the E 2 conformation in the d-arabinofuranosides) and enforce the gt conformation of their side chains, both of which differ from the ligand conformation preferences of the AFs discussed above.

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Structures of β-d-arabinofuranose and β-d-fructofuranose.

The difference in ring conformation between arabinofuranosides and fructofuranosides arises from the presence of the additional hydroxymethyl side chain at the anomeric center in the latter, hereinafter called the anomeric side chain, and the need to accommodate it in a pseudoequatorial position. Like the side chain itself, the anomeric side chain of the fructofuranosides can adopt three staggered conformations, which by analogy we dub the gg, gt, and tg conformations. Of the twenty-eight FF–ligand complexes located, all from GH32, twenty-seven adopt approximate E 3 conformations, while the remaining one takes up the adjacent 4 T 3 ring conformation. All of these twenty-eight structures enforce the gt conformation of the side chain and twenty-six of them similarly enforce approximate gt conformations on the anomeric side chain. There is therefore a strong preference among the β-d-FFs to bind either β-d-fructofuranose itself or β-d-fructofuranosides with an E 3-like ring pucker with both side chains in the gt conformation (Figure ). With the ring in the E 3 envelope conformation, the anomeric substituent is pseudoaxial and would be subject to a steric clash with the 6-OH group were the side chain restricted to the gg conformation. We suggest that the preference for the gt conformation of the anomeric side chain arises because, in addition to providing modest stabilization to nascent positive charge at the transition state for hydrolysis, it benefits from stabilizing gauche interactions of the C1–O1 bond with both the C2–OR and C2–O5 bonds and has no steric gauche interactions with a C–C bond. In contrast, the gg and tg conformations both have only one stabilizing gauche interaction and suffer from one steric gauche interaction (Figure ). It is apparent therefore that the FFs have evolved to reach a compromise holding both side chains of their substrates in the moderately activating gt conformation.

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Predominant E 3, gt, and gt conformations of β-d-fructofuranos­(id)­es in the active site of GH-32 β-d-fructofuranosidases and the staggered conformations about the C1–C2 bond.

Four trans-FFs from GH32 closely follow the FFs from the same family binding β-d-fructofuranose in either the E 3 or the adjacent 4 T 3 ring conformations with the gt conformation of the side chain and (with the exception of one gg example) the gt conformation of the anomeric side chain. Similarly, five trans-FFs from GH68 bind β-d-fructofuranose in the E 3 conformation with the gt conformation of the side chain, and three of the five bind the anomeric side chain in the gt conformation, while the other two enforce the gg conformation of the anomeric side chain. Four structures meeting our selection criteria are available for the GH91 FF-transferases (PDB 9J4I, 5ZKU, 5ZLA, and 8HUI), − which cleave difructofuranose from fructans initially providing various difructose anhydrides. The data from these structures, however, are ambiguous, perhaps because the imposed conformations reflect the need to position the nonreducing terminal residue of the substrates so as to act as a nucleophile in the formation of the anhydride products and are not analyzed further. Finally, a trans-FF from Bifidobacterium dentium belonging to the newly assigned GH class 172 additionally displays α-d-AF activity and α-d-FF activity. It binds β-d-fructofuranose in the E 5 with the gg conformation of its side chain (PDB 7V1X) and β-d-arabinofuranose in the adjacent (taking account of the numbering shift) 3 T 4 conformation with the gg-oriented side chain (PDB 7V1W) and so more closely resembles the AFs in other GH classes than the more common GH32 class of FFs and the GH91 trans-FFs. The anomeric side chain of the β-d-fructofuranose ligand in complex with the B. dentium trans-FF is hydrogen bonded in the deactivating tg conformation, thereby highlighting the difference with the FFs from GH32 and the trans-FFs from GH classes 68 and 91.

The NHs and the NPs from both the NP-I and NP-II families, with their distinctly different folds, almost exclusively bind their ribo-configured substrates and substrate analogs with the gt conformation of the side chain and show a very significant preference for the 4 E ring conformation, with occasional exceptions having the E 3, E O, or 2 T 3 ring conformations. All ring conformations are therefore in the southwest quadrant of the pseudorotational wheel for d-furanosides and importantly place the side chain in a pseudoaxial orientation (Figure ). The situation for the NHs and NPs therefore closely parallels that of the FFs, with the furanoside ring bound in a conformation that excludes the gg side-chain conformation because of the steric clash it would engender with the leaving group at the anomeric position, leading to the imposition of the second most activating gt side-chain conformation. Finally, although the number of structures is limited (Table ), NDTs from both classes I and II, which catalyze the transfer of the 2′-deoxyribosyl moiety from a nucleoside to an acceptor nucleobase, , bind substrate analogs and the nucleoside products with a preference for the E O ring conformation and the gt side-chain conformation. −

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Predominant bound ring and side-chain conformations of the ribonucleoside hydrolase and ribonucleoside phosphorylase substrates.

Turning to the MTANs and MTAPs in which the C5′–OH bond of the substrates is replaced by a longer and less electron-withdrawing C5′–SR bond, the data reveal that MTAN substrates and their analogs are mostly bound in the 2 T 3 and 4 E ring conformations like the NHs and NPs, but with their side chains almost exclusively restricted to the tg conformation (Figure ). The MTAPs on the other hand bind their substrates and substrate analogs with predominantly the 4 E and O E ring conformations and the gt side chain conformation. The more diffuse electron density around the 5′-sulfur atom of the MTAN and MTAP substrates and their longer 5′-C–S bonds combine to reduce any electrostatic transition-state stabilization afforded by side chains in the gg or gt conformations. At the same time, the lower electronegativity of sulfur compared to oxygen renders the tg side chain conformation less destabilizing toward nascent positive charge at the transition state, such that overall there is little to be gained for the MTANs and MTAPs by restricting the side chains of their substrates to the gg or gt conformations. Presumably, the MTANs have evolved to bind their substrates in the tg conformation to remove the steric bulk of the 5′-residue from the vicinity of the reaction center particularly when functioning as S-adenosylhomocysteine nucleosidases, while the MTAPs have retained the preference for the gt conformation enjoyed by the NPs in general.

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Predominant bound ring and tg side-chain conformations of the 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase substrates.

Overall, examination of the crystallographic record provides strong evidence that AFs, FFs, NHs, NPs, and NDTs restrict the conformation of their substrates by hydrogen bonding to provide electrostatic stabilization by the 5′-hydroxy group of their substrates to nascent positive charge at the anomeric center and in doing so enhance catalysis. When the substrate is bound in a ring conformation that readily accommodates it as in the AFs, the more stabilizing gg conformation of the side chain is preferred, as is found in the vast majority of the α- and β-glucopyranosidases and, with certain well-defined exceptions, the α- and β-mannopyranosidases. When the substrate is bound in a ring conformation in which the energy of the gg side-chain conformation would be increased by a steric clash with the leaving group (FFs, NHs, and NPs), the side chain is instead preferentially bound in the second most activating gt conformation. This compromise is akin to that found in the α-galactopyranosidases where the gg side-chain conformation of the substrate is higher in energy due to dipolar repulsion with the C4–O4 bond, resulting in the evolution of the preference for binding in the second most activating gt conformation. We consider the MTANs, with their preference for binding their substrates with the tg side-chain conformation because of the relative absence of electrostatic stabilization available from the side-chain substituent, as exceptions that reinforce the overall rule.

Correlation with the Literature on the Role of the Substrate Side Chain in the Action of NHs and NPs

Our initial search of the PDB leading to the recognition that pyranosidases restrict the side-chain conformation of their substrates to enhance catalysis was driven by our insight into the role of the side chain in chemical glycosylation reactions. The results presented above suggest that nature widely applies the same phenomenon to furanosidases, nucleosidases, and nucleoside phosphorylases. The case for side-chain conformational control by NHs and NPs is strongly supported by a considerable body of biochemical and physical organic studies on individual enzymes over many years. Thus, Schramm and co-workers, studying the trypanosome Crithidia fasciculata NH, found (i) that 5′-deoxyinosine is not a substrate and (ii) a secondary 3H kinetic isotope effect at the 5′-position of the substrate inosine leading them to implicate a “transition-state function for the 5′-hydroxyl” and to initially propose hyperconjugative stabilization of the developing charge at C4′ by a C5′–H5′ bond from a gt conformation of the side chain. − In a subsequent computational analysis, this transition-state hypothesis was revised to place the side chain in an approximate gg conformation in such a manner that “more electrostatic binding energy could be developed by placing a negatively charged group or suitable dipole over the ribose ring”, leading the authors to state that “structural features of the transition state, sometimes remote from the site of bond breaking and forming, are likely to be used to distribute or localize charge development to optimize structures which can be stabilized at enzymatic transition states”. , A subsequent X-ray structure of this NH in complex with the inhibitor p-aminophenyliminoribitol generally confirmed this hypothesis and revealed a O5′–C5′–C4′–N4′ torsion angle of 336°, i.e., midway between the gt conformation and one in which O4′ and O5′ are eclipsed with stabilization of the positive charge by a “neighboring group” (Figure A; PDB 2MAS). Steyaert and co-workers studied the Trypanosoma vivax NH and similarly found the 5′-deoxy analog of the natural substrate adenosine to be a very poor substrate and calculated that the 5′-hydroxy group contributes 5.4 kcal mol–1 to the catalytic efficiency of hydrolysis of adenosine. Working with a D10A mutant, lacking a catalytic aspartate residue, they were able to determine structures of the complex with the natural substrate inosine, representing a snapshot of pretransition-state species, and of that with the inhibitor 3-deazainosine: in both structures (PDB 1KIC and 1KIE), the side chain was held in an approximate gt conformation. The complex of the natural T. vivax NH with the transition-state analog inhibitor immucillin H also retains the approximate gt conformation of the inhibitor side chain, although with some deformation toward eclipsing with the C4′–O4′ bond (Figure B; PDB 2FF2), albeit alternative rationalizations were proposed for the role of the 5′-hydroxy group on the basis of computational work. The importance of the substrate 5′-hydroxy group to hydrolysis has also been demonstrated for the Trypanosoma brucei NH, although in this case, its contribution was considered to arise more from formation of the Michaelis complex than to catalysis. In contrast to the NHs, with their significant secondary 3H KIEs and requirements for the presence of the 5′-hydroxy group, the MTANs show only small secondary KIEs at the 5′-position, attributed to hyperconjugation with sulfur, − and are able to process their 5′-unsubstituted congeners.

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Active site–ligand interactions from (A) PDB 2MAS and (B) PDB 2FF2 showing protein–ligand H-bonding holding the ligand side chain in the gt conformation.

The human purine NP (PNP) binds inosine derivatives carrying modifications at the 5′-position, such as alkylthio, halo, and deoxy derivatives, but they are very poor substrates. , Secondary 3H KIE studies on the hydrolysis of inosine by human PNP in the absence of phosphate revealed the involvement of the 5′-position in the transition state, prompting the authors to propose an analogous transition state to that for the NHs with the side chain in an approximate gt conformation. These KIEs, together with a series of crystallographic snapshots of PNPs with the inhibitor immucillin H and inorganic phosphate (Figure A; PDB 1B8O) and the product α-d-ribofuranosyl phosphate (Figure B; PDB 1A9T), led to the proposition of an early oxocarbenium ion-like transition state. In this transition state, the side chain takes up a conformation midway between gt and gg in which the C5′–O5′ bond eclipses the C4′–O4′ bond so as, in combination with the electron density on the α-face provided by the phosphate nucleophile, to “favor the release of electrons from O4′, cleavage of the C1′–N9 bond, and formation of the ribooxocarbenium ion transition state (electron push)”. Multiple subsequent structures of PNPs with substrates or inhibitors support this analysis, ultimately leading to an understanding of the mechanism in which the 5′-hydroxy group is hydrogen bonded to His257 at the level of the Michaelis complex with moderate distortion toward the transition state. Finally, at the transition state, the 5′-hydroxy group approaches an eclipsed conformation with O4′ and provides “neighboring group participation” at the transition state. Transition path sampling simulations were used to probe the approach of O5′ to O4′ and showed it to reach a minimum of approximately 2.85 Å at the transition state. The extensive studies of Schramm and co-workers toward the characterization of the PNP mechanism have been reviewed. ,

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Active site–ligand interactions from (A) PDB 1B8O and (B) PDB 1A9T.

Schramm’s concept of a push from the electron density on O5′ when eclipsing the C4′–O4′ bond that facilitates departure of the base from C1′ in the NHs and NPs and our concept of side-chain preorganization into the arming gg conformation, and when that is sterically inaccessible the gt conformation, are evidently two faces of the same coin. Any subtle differences between the two descriptions are attributable to conformations imposed by the enzymes on the ring and possibly to the extent of charge delocalization onto the ring oxygen at the transition state. Setting aside these minor variations on the theme, it is difficult to escape the conclusion that the majority of GHs, NHs, and NPs and, as is clear from our database searches, transglycosidases and Leloir GTs have evolved to bind their substrates so that their side chains are conformationally preorganized to provide additional electrostatic stabilization to oxocarbenium ion-like transition states.

Provision of Electrostatic Stabilization to Transition States by Substrate Preorganization

We now argue that side-chain preorganization by this broad group of glycoside and nucleoside processing enzymes bridges the gap between Warshel, who has strongly disputed the concept that enzymes employ the conformation restriction of their substrates to enhance catalysis, and multiple authors who have strongly advocated for such restriction often on the basis of physical organic studies of model systems. − We argue that the restriction of substrate side-chain conformation by GHs and related enzymes to provide electrostatic stabilization to at least partially positively charged transition states is consistent with Warshel’s concept that, in addition to providing correctly positioned catalytic residues, enzymes achieve catalysis largely by preorganizing the dipoles of active-site polar groups to maximize electrostatic stabilization of transition states. ,, Thus, adopting Warshel’s analysis, in the uncatalyzed reaction in aqueous solution as the substrate passes from the reactant state to the transition state, it suffers a change in charge distribution, which causes the dipoles of the surrounding water molecules to orient themselves so as to stabilize the charges on the transition state, for which there is an energy penalty, known as the reorganization energy. In the enzymic reaction, on the other hand, active-site polar residues are already correctly aligned and do not have to undergo significant reorganization to stabilize charges in the transition state. As the degree of stabilization of the charge on the transition state by reorganized water and active-site residues is comparable, a significant contribution to catalysis is the removal of reorganization energy for the reaction in water. It is apparent that active-site residues in GHs and related enzymes are preorganized to restrict the substrate side chain to the gg conformation where it is poised to provide electrostatic stabilization to the transition state. When the gg conformation is sterically disfavored because of the conformation imposed on the ring at the transition state, the enzyme is preorganized to select the second most stabilizing gt conformation. Roughly speaking, the penalty paid for restricting the side-chain conformation will be comparable to the reorganization energy of a single appropriately placed water molecule in the aqueous reaction or to the retention and conformational restriction of a single water molecule in the active-site in place of the side-chain hydroxy group. Of note in this regard is the minimal effect that removal of the hydroxymethyl side chain from the substrate has on the hydrolysis of β-d-galactopyranosyl substrates by the Escherichia coli (lacZ) β-galactosidase, which was later rationalized in terms of possible binding of a water molecule in the place of the hydroxymethyl side chain.

Regarding the magnitude of the transition-state stabilization afforded by the conformationally restricted side-chain hydroxy group, Steyaert and co-workers estimate that relative to the 5′-deoxy analog, the 5′-hydroxy group contributes 5.4 kcal mol–1 to the catalytic efficiency of adenosine hydrolysis by the T. vivax NH, which likely defines an upper limit. This is because deoxygenation or deoxyhalogenation of the side chain leaves a methyl or halomethyl residue that is likely too large to allow a water molecule to fill the cavity. Consequently, the reduction in activity due to the loss of electrostatic transition-state stabilization on deletion of the side-chain hydroxy group will be accentuated by the reduction in affinity arising from the absence of hydrogen bonding to the active-site residue(s) responsible for the restriction of the side-chain conformation in the native substrate. The beneficial effect of the substrate side-chain hydroxy group in various pyranosidases that are similarly found to process 6-deoxy analogs more slowly than the actual substrates − has been attributed to hydrogen bonding with the active-site, but as crystallographic studies have shown hydrogen bonding with restriction of conformation of the side chain typically begins at the level of the enzyme substrate complex and is retained through the transition state to the enzyme product complex. This suggests that the hydrogen bonding to the side chain per se contributes mainly to K M. Contributions to k cat on the other hand will arise from the electrostatic interaction of the side-chain hydroxy group with the partially positively charged transition state and any minor conformational adjustments of the side-chain conformation required to optimize it, as found by Schramm in his studies on mechanism of nucleoside phosphorylation by human PNP.

Finally, we note that the concept of substrate preorganization to provide electrostatic stabilization to the transition state is not restricted to the substrate side chain. Thus, the GH47 α-mannopyranosidases benefit from chelation of Ca2+ to bind their substrates in a 3 S 1 twist boat conformation on the way to the proximal 3 H 4 half chair transition state for hydrolysis in which O3 and O4 are pseudoaxial and provide maximal electrostatic stabilization. Yet other glycosidases appear to use steric hindrance to force their substrates into conformations enriched in pseudoaxial hydroxy groups poised to provide electrostatic transition-state stabilization.

Conclusion

Mining of the PDB reveals that the arabinofuranosidases enforce the gg conformation on the side chains of their substrates, while the fructofuranosidases, ribonucleoside hydrolases, phosphorylases, and transferases, for which the gg conformation is sterically hindered, very largely restrict the side-chain conformations of their substrates to the gt conformation. Hydrolases that catalyze reactions via partially positively charged furanosyl oxocarbenium ion-like transition states therefore follow the pattern described earlier for glycoside hydrolases, transglycosidases, and Leloir glycosyltransferases acting on pyranosyl substrates by restricting side-chain conformation to provide additional electrostatic stabilization to the transition state. These conclusions are supported by extensive literature studies on the importance of the substrate 5′-hydroxyl group in C–N bond cleavage reactions by nucleoside hydrolases and phosphorylases and are consistent with the electrostatic basis for enzyme catalysis propounded by Warshel and others. ,,

Supplementary Material

au5c01069_si_001.pdf (242.4KB, pdf)

Acknowledgments

D.C. thanks the NIH (GM62160) for partial support of this work. W.J.L. was partly supported by NIGMS training grant T32 107004.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.5c01069.

  • Additional information on Privateer analysis and tables of raw crystal structure data (PDF)

The authors declare no competing financial interest.

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