Liquid-Vapor Interface of Aqueous Ethylene Glycol Solutions: A Molecular Dynamics Study.

While the organic constituent in an aqueous binary solution enriches its liquid-vapor (l-v) interface, the extent of enrichment can depend nonlinearly on its mole fraction. A microscopic quantification and rationalization of this behavior are crucial to understand the dependence of properties such as surface tension and evaporation rate of the solution on its composition. Extensive all-atom molecular dynamics simulations of aqueous ethylene glycol (EG) solutions show that the composition of the solution at the l-v interface deviates the most from that in the bulk solution at an EG mole fraction of 0.3. The population of EG molecules with their central C-C dihedral in the gauche conformation was found to be higher at the l-v interface than that in the bulk solution to facilitate the orientation of its hydrophobic methyl groups toward the vapor phase. Free energy calculations reveal that in dilute EG solutions, an EG molecule is most stable at the l-v interface. The behavior of vapor pressure in aqueous EG solutions is ideal and follows Raoult's law, while in contrast, the aqueous solution of dimethyl sulfoxide does not. A rationale for the same is provided through the orientational distribution of interfacial water molecules in the respective solutions.

Revisiting the Burden Borne by Fumarase: Enzymatic Hydration of an Olefin.

Fumarate hydratase (FH) is a remarkable catalyst that decreases the free energy of the catalyzed reaction by 30 kcal mol-1, much larger than most exceptional enzymes with extraordinary catalytic rates. Two classes of FH are observed in nature: class-I and class-II, which have different folds, yet catalyze the same reversible hydration/dehydration reaction of the dicarboxylic acids fumarate/malate, with equal efficiencies. Using class-I FH from the hyperthermophilic archaeon Methanocaldococcus jannaschii (Mj) as a model along with comparative analysis with the only other available class-I FH structure from Leishmania major (Lm), we provide insights into the molecular mechanism of catalysis in this class of enzymes. The structure of MjFH apo-protein has been determined, revealing that large intersubunit rearrangements occur across apo- and holo-protein forms, with a largely preorganized active site for substrate binding. Site-directed mutagenesis of active site residues, kinetic analysis, and computational studies, including density functional theory (DFT) and natural population analysis, together show that residues interacting with the carboxylate group of the substrate play a pivotal role in catalysis. Our study establishes that an electrostatic network at the active site of class-I FH polarizes the substrate fumarate through interactions with its carboxylate groups, thereby permitting an easier addition of a water molecule across the olefinic bond. We propose a mechanism of catalysis in FH that occurs through transition-state stabilization involving the distortion of the electronic structure of the substrate olefinic bond mediated by the charge polarization of the bound substrate at the enzyme active site.

Lipase A from Bacillus subtilis: Substrate Binding, Conformational Dynamics, and Signatures of a Lid.

Protein-ligand binding studies are crucial for understanding the molecular basis of biological processes and for further advancing industrial biocatalysis and drug discovery. Using computational modeling and molecular dynamics simulations, we investigated the binding of a butyrate ester substrate to the lipase A (LipA) enzyme of Bacillus subtilis. Besides obtaining a close agreement of the binding free energy with the experimental value, the study reveals a remarkable reorganization of the catalytic triad upon substrate binding, leading to increased essential hydrogen bond populations. The investigation shows the distortion of the oxyanion hole in both the substrate-bound and unbound states of LipA and highlights the strengthening of the same in the tetrahedral intermediate complex. Principal component analysis of the unbound ensemble reveals the dominant motion in LipA to be the movement of Loop-1 (Tyr129-Arg142) between two states that cover and uncover the active site, mirroring that of a lid prevalent in several lipases. This lid-like motion of Loop-1 is also supported by its tendency to spontaneously open up at an oil-water interface. Overall, this study provides valuable insights into the impact of substrate binding on the structure, flexibility, and conformational dynamics of the LipA enzyme.

Supramolecular Polymerization of a Pyrene-Substituted Diamide and Its Ensemble of Kinetically Trapped Configurations.

The prevalence of kinetically accessible states in supramolecular polymerization pathways has been exploited to control the growth of the polymer and thereby to obtain niche morphologies. Yet, these pathways themselves are not easily amenable for experimental delineation but could potentially be understood through molecular dynamics (MD) simulations. Herein, we report an extensive investigation of the self-assembly of pyrene-substituted diamide (PDA) monomers in solution, conducted using atomistic MD simulations and advanced sampling methods. We characterize such kinetic and thermodynamic states as well as the transition pathways and free energy barriers between them. PDA forms a dimeric segment with the N- to C-termini vectors of the diamide moieties arranged either in parallel or anti-parallel fashion. This characteristic, combined with the molecule's torsional flexibility and pyrene-solvent interactions, presents an ensemble of molecular configurations contributing to the kinetic state in the polymerization pathway. While this ensemble primarily comprises short oligomers containing a mix of anti-parallel and parallel dimeric segments, the thermodynamic state of the assembly is a right-handed polymer featuring parallel ones only. Our work thus offers an approach by which the landscape of any specific supramolecular polymerization can be deconstructed.

Gate Opening without Volume Change Triggers Cooperative Gas Interactions, Underpins an Isotherm Step in Metal-Organic Frameworks.

Three halogenated metal-organic frameworks (MOFs) reported recently exhibited a second step in their CO2 gas adsorption isotherms. The emergence of halogen-bonding interactions beyond a threshold gas pressure between the framework halogen and the CO2 guest was conjectured to be the underlying reason for the additional step in the isotherm. Our investigation employing periodic density functional theory calculations did not show significant interactions between the halogen and CO2 molecules. Further, using a combination of DFT-based ab initio molecular dynamics and grand canonical Monte Carlo simulations, we find that the increased separation of framework nitrate pairs facing each other across the pore channel enables the accommodation of an additional CO2 molecule which is further stabilized by cooperative interactions─an observation that facilely explains the second isotherm step. The increased separation between the nitrate groups can occur without any lattice expansion, consistent with experiments. The results point to a structural feature to achieve this isotherm step in MOFs that neither possess large pores nor exhibit large-scale structural changes such as breathing.

Molecular simulations explain the exceptional thermal stability, solvent tolerance and solubility of protein-polymer surfactant bioconjugates in ionic liquids.

Proteins complexed electrostatically with polymer surfactants constitute a viscous liquid by themselves, called the solvent-free protein liquid (SFPL). A solution of SFPL in a room temperature ionic liquid (PS-IL) offers the protein hyperthermal stability, higher solubility and greater IL tolerance. A generic understanding of these protein-polymer systems is obtained herein through extensive atomistic molecular dynamics simulations of three different enzymes (lipase A, lysozyme and myoglobin) under various conditions. Along with increased intra-protein hydrogen bonding, the surfactant coating around the proteins imparts greater thermal stability, and also aids in screening protein-IL interactions, endowing them IL tolerance. The reduced surface polarity of the protein-polymer bioconjugate and hydrogen bonding between the ethylene glycol groups of the surfactant and the IL cation contribute to the facile solvation of the protein in its PS-IL form. The results presented here rationalize several experimental observations and will aid in the improved design of such hybrid materials for sustainable catalysis.

Liquid ethylene glycol: prediction of physical properties, conformer population and interfacial enrichment with a refined non-polarizable force field.

Periodic density functional theory based molecular dynamics simulations confirm the fraction of molecules in neat liquid ethylene glycol with their central OCCO dihedral in the trans conformation to be 21% at ambient conditions, while the rest are gauche conformers. Using this result as a benchmark, two non-polarizable force fields are developed herein to reproduce the conformer populations in the liquid, an important aspect inadequately addressed in several generic force fields. The mean dipole moment of a molecule in the liquid is estimated to be about 40% enhanced over its value in the gas phase, a feature discerned via AIMD simulations and fairly reproduced by our force fields. They are also shown to quantitatively predict all the physical properties of the liquid. Molecules present at the liquid-vapor interface of ethylene glycol are oriented with their methylene groups pointing towards the vapor phase, a requirement that enriches the interface with gauche conformers, in line with polarized sum frequency generation spectroscopy results.

Ileal Perforation and Enteric Fever: Implications for Burden of Disease Estimation.

BACKGROUND: Ileal perforation occurs in about 1% of enteric fevers as a complication, with a case fatality risk (CFR) of 20%-30% in the early 1990s that decreased to 15.4% in 2011 in South East Asia. We report nontraumatic ileal perforations and its associated CFR from a 2-year prospective enteric fever surveillance across India. METHODS: The Surveillance for Enteric Fever in India (SEFI) project established a multitiered surveillance system for enteric fever between December 2017 and March 2020. Nontraumatic ileal perforations were surveilled at 8 tertiary care and 6 secondary care hospitals and classified according to etiology. RESULTS: Of the 158 nontraumatic ileal perforation cases identified,126 were consented and enrolled. Enteric fever (34.7%), tuberculosis (19.0%), malignancy (5.8%), and perforation of Meckel diverticulum (4.9%) were the common etiology. In those with enteric fever ileal perforation, the CFR was 7.1%. CONCLUSIONS: Enteric fever remains the most common cause of nontraumatic ileal perforation in India, followed by tuberculosis. Better modalities of establishing etiology are required to classify the illness, and frame management guidelines and preventive measures. CFR data are critical for comprehensive disease burden estimation and policymaking.

Structural basis for the hyperthermostability of an archaeal enzyme induced by succinimide formation.

Stability of proteins from hyperthermophiles (organisms existing under boiling water conditions) enabled by a reduction of conformational flexibility is realized through various mechanisms. A succinimide (SNN) arising from the post-translational cyclization of the side chains of aspartyl/asparaginyl residues with the backbone amide -NH of the succeeding residue would restrain the torsion angle Ψ and can serve as a new route for hyperthermostability. However, such a succinimide is typically prone to hydrolysis, transforming to either an aspartyl or ß-isoaspartyl residue. Here, we present the crystal structure of Methanocaldococcus jannaschii glutamine amidotransferase and, using enhanced sampling molecular dynamics simulations, address the mechanism of its increased thermostability, up to 100°C, imparted by an unexpectedly stable succinimidyl residue at position 109. The stability of SNN109 to hydrolysis is seen to arise from its electrostatic shielding by the side-chain carboxylate group of its succeeding residue Asp110, as well as through n → π∗ interactions between SNN109 and its preceding residue Glu108, both of which prevent water access to SNN. The stable succinimidyl residue induces the formation of an α-turn structure involving 13-atom hydrogen bonding, which locks the local conformation, reducing protein flexibility. The destabilization of the protein upon replacement of SNN with a Φ-restricted prolyl residue highlights the specificity of the succinimidyl residue in imparting hyperthermostability to the enzyme. The conservation of the succinimide-forming tripeptide sequence (E(N/D)(E/D)) in several archaeal GATases strongly suggests an adaptation of this otherwise detrimental post-translational modification as a harbinger of thermostability.

Fluorocarbon-Functionalized Superhydrophobic Metal-Organic Framework: Enhanced CO2 Uptake via Photoinduced Postsynthetic Modification.

The design and synthesis of porous materials for selective capture of CO2 in the presence of water vapor is of paramount importance in the context of practical separation of CO2 from the flue gas stream. Here, we report the synthesis and structural characterization of a photoresponsive fluorinated MOF {[Cd(bpee)(hfbba)]·EtOH}n (1) constructed by using 4,4'-(hexafluoroisopropylidene)bis(benzoic acid) (hfbba), Cd(NO3)2, and 1,2-bis(4-pyridyl)ethylene (bpee) as building units. Due to the presence of the fluoroalkyl -CF3 functionality, compound 1 exhibits superhydrophobicity, which is validated by both water vapor adsorption and contact angle measurements (152°). The parallel arrangement of the bpee linkers makes compound 1 a photoresponsive material that transforms to {[Cd2(rctt-tpcb)(hfbba)2]·2EtOH}n (rctt-tpcb = regio cis,trans,trans-tetrakis(4-pyridyl)cyclobutane; 1IR) after a [2 + 2] cycloaddition reaction. The photomodified framework 1IR exhibits increased uptake of CO2 in comparison to 1 under ambient conditions due to alteration of the pore surface that leads to additional weak electron donor-acceptor interactions with the -CF3 groups, as examined through periodic density functional theory calculations. The enhanced uptake is also aided by an expansion of the pore window, which contributes to increasing the rotational entropy of CO2, as demonstrated through force field based free energy calculations.

Dipolar relaxation in thin films of supramolecular stacks of benzenecarboxamides and insights to enhance their ferroelectric characteristics.

The relationship between molecular structure and ferroelectric behaviour of thin films is explored in an all-organic supramolecular polymer material based on benzenecarboxamides, using atomistic molecular dynamics simulations. While increasing the number of amide groups around the phenyl core increases the dipole density of a molecule, increasing the length of the corresponding alkyl groups decreases the same. The interplay between these two contributions displays a rich behaviour on key material characteristics, in particular, the polarisation retention time. The latter is shown to be inversely proportional to the alkyl chain length, a consequence of weaker interactions between macrodipoles of stacks. Polarisation retention time was observed to be the highest in a molecule with five amide groups around the aromatic phenyl core which is explained as due to the large barrier for amide group rotation, which is one of the crucial channels for dipolar relaxation. Simulations also demonstrate that the barrier, however, does not affect the switchability of polarization, upon field reversal.

An atomistic view of solvent-free protein liquids: the case of Lipase A.

Solvent-free enzymes hold the promise of being able to deliver higher activity at elevated temperatures by virtue of them being not limited by the boiling point of the solvent. They have been realized in the liquid phase through a polymer surfactant coating on the protein surface. However, a clear understanding of intermolecular interactions, structure, dynamics, and the behaviour of the minuscule amount of water present in the solvent-free protein liquid is essential to enhance the activity of these biofluids. Using atomistic molecular dynamics simulations, we demonstrate that the scaled spatial correlations between proteins in the hybrid liquid phase of Lipase A enzymes are comparable to the inter-particle correlations in a noble gas fluid. The hydrophilic region of the surfactants forms a coronal layer around each enzyme which percolates throughout the liquid, while the hydrophobic parts are present as disjointed clusters. Inter-surfactant interactions, determined to be attractive and in the range of -200 to -300 kcal mol-1, stabilize the liquid state. While the protein retains its native state conformational dynamics in the solvent-free form, the fluxionality of its side chains is much reduced; at 333 K, the latter is found to be equivalent to that of the enzyme in an aqueous solution at 249 K. Despite the sluggishness of the solvent-free enzyme, some water molecules exhibit high mobility and transit between enzymes primarily via the interspersed hydrophilic regions. These microscopic insights offer ideas to improve substrate diffusion in the liquid to enable the enhancement of catalytic activity.

A Dynamic Chemical Clip in Supramolecular Framework for Sorting Alkylaromatic Isomers using Thermodynamic and Kinetic Preferences.

Adsorptive chemical separation is at the forefront of future technologies, for use in chemical and petrochemical industries. In this process, a porous adsorbent selectively allows a single component from a mixture of three or more chemical components to be adsorbed or permeate. To separate the unsorted chemicals, a different adsorbent is needed. A unique adsorbent which can recognize and separate each of the chemicals from a mixture of three or more components is the necessity for the next generation porous materials. In this regard, we demonstrate a "dynamic chemical clip" in a supramolecular framework capable of thermodynamic and kinetics-based chemical separation. The dynamic space, featuring a strong preference for aromatic guests through π-π and C-H⋅⋅⋅π interactions and adaptability, can recognize the individual chemical isomers from mixtures and separate those based on thermodynamic and kinetic factors. The liquid-phase selectivity and separation of the aromatic isomers are possible by the adaptability of the "chemical clip" and here we elucidate the prime factors in a combinatorial approach involving crystallographic evidence and detailed computational studies.

How a purine salvage enzyme singles out the right base.

Two phosphoribosyltransferases in the purine salvage pathway exhibit exquisite substrate specificity despite the chemical similarity of their distinct substrates, but the basis for this discrimination was not fully understood. Ozeir et al. now employ a complementary biochemical, structural, and computational approach to deduce the chemical constraints governing binding and propose a distinct mechanism for catalysis in one of these enzymes, adenine phosphoribosyltransferase. These insights, built on data from an unexpected finding, finally provide direct answers to key questions regarding these enzymes and substrate recognition more generally.

Low-Symmetry Self-Assembled Coordination Complexes with Exclusive Diastereoselectivity: Experimental and Computational Studies.

A pyridine/aniline appended unsymmetrical bidentate ligand N-(4-(4-aminobenzyl)phenyl)nicotinamide, investigated in this work has two well-separated coordination sites. Combination of the ligand with cis-protected palladium(II) (i.e., PdL') and palladium(II) in separate reactions produced the corresponding Pd2L'2Lun2 and extremely rare Pd2Lun4 type self-assembled binuclear complexes, respectively. Notably, both varieties of complexes are prepared from a common ligand system. Two diastereomers (i.e., (2,0) and (1,1)-forms) are possible for Pd2L'2Lun2 type complex, whereas four diastereomers (i.e., (4,0), (3,1), trans(2,2), and cis(2,2)-forms) can be imagined for the Pd2Lun4 type complex. However, exclusive diastereoselectivity was observed, and the complexes formed belong to (1,1)-Pd2L'2Lun2 and cis(2,2)-Pd2Lun4 forms. The diastereomers are predicted from NMR study in solution and DFT calculations in gas-phase and implicit-solvent media; however, single-crystal structures of both the complexes provided unambiguous support. The rare Pd2Lun4 type complex is studied in further detail. Parameters like counteranion, concentration, temperature, and stoichiometry of metal to ligand did not influence the diastereoselectivity in complex formation. DFT calculations show the cis(2,2) form to be the most stable, followed by the (3,1) isomer. The lowest conformational strain in the bound ligand strands in the cis(2,2)-arrangement along with optimal intermolecular interactions makes it the energetically most stable of all the isomers. Molecular dynamics (MD) simulations were carried out to visualize the self-assembly process toward the formation of Pd2Lun4 type complex and the free energy difference between the cis(2,2) and (3,1) isomers. Snapshots of MD simulation elucidate the step-by-step progress of complexation leading to the cis(2,2)-isomer.

Role of W181 in modulating kinetic properties of Plasmodium falciparum hypoxanthine guanine xanthine phosphoribosyltransferase.

Hypoxanthine-guanine-xanthine phosphoribosyltransference (HGXPRT), a key enzyme in the purine salvage pathway of the malarial parasite, Plasmodium falciparum (Pf), catalyses the conversion of hypoxanthine, guanine, and xanthine to their corresponding mononucleotides; IMP, GMP, and XMP, respectively. Out of the five active site loops (I, II, III, III', and IV) in PfHGXPRT, loop III' facilitates the closure of the hood over the core domain which is the penultimate step during enzymatic catalysis. PfHGXPRT mutants were constructed wherein Trp 181 in loop III' was substituted with Ser, Thr, Tyr, and Phe. The mutants (W181S, W181Y and W181F), when examined for xanthine phosphoribosylation activity, showed an increase in Km for PRPP by 2.1-3.4 fold under unactivated condition and a decrease in catalytic efficiency by more than 5-fold under activated condition as compared to that of the wild-type enzyme. The W181T mutant showed 10-fold reduced xanthine phosphoribosylation activity. Furthermore, molecular dynamics simulations of WT and in silico W181S/Y/F/T PfHGXPRT mutants bound to IMP.PPi.Mg2+ have been carried out to address the effect of the mutation of W181 on the overall dynamics of the systems and identify local changes in loop III'. Dynamic cross-correlation analyses show a communication between loop III' and the substrate binding site. Differential cross-correlation maps indicate altered communication among different regions in the mutants. Changes in the local contacts and hydrogen bonding between residue 181 with the nearby residues cause altered substrate affinity and catalytic efficiency of the mutant enzymes. Proteins 2016; 84:1658-1669. © 2016 Wiley Periodicals, Inc.

Host-Guest [2+2] Cycloaddition Reaction: Postsynthetic Modulation of CO2 Selectivity and Magnetic Properties in a Bimodal Metal-Organic Framework.

Simultaneous tuning of permanent porosity and modulation of magnetic properties by postsynthetic modification (PSM) with light in a metal-organic framework is unprecedented. With the aim of achieving such a photoresponsive porous magnetic material, a 3D photoresponsive biporous framework, MOF1, which has 2D channels occupied by the guest 1,2-bis(4-pyridyl)ethylene (bpee), H2 O, and EtOH molecules, has been synthesized. The guest bpee in 1 is aligned parallel to pillared bpee with a distance of 3.9 Šbetween the ethylenic groups; this allows photoinduced PSM of the pore surface through a [2+2] cycloaddition reaction to yield MOF2. Such photoinduced PSM of the framework structure introduces enhanced CO2 selectivity over that of N2 . The higher selectivity in MOF2 than that of MOF1 is studied through theoretical calculations. Moreover, MOF2 unveils reversible changes in Tc with response to dehydration-rehydration. This result demonstrates that photoinduced PSM is a powerful tool for fabricating novel functional materials.

Dynamic Entangled Porous Framework for Hydrocarbon (C2-C3) Storage, CO2 Capture, and Separation.

Storage and separation of small (C1-C3) hydrocarbons are of great significance as these are alternative energy resources and also can be used as raw materials for many industrially important materials. Selective capture of greenhouse gas, CO2 from CH4 is important to improve the quality of natural gas. Among the available porous materials, MOFs with permanent porosity are the most suitable to serve these purposes. Herein, a two-fold entangled dynamic framework {[Zn2 (bdc)2 (bpNDI)]⋅4DMF}n with pore surface carved with polar functional groups and aromatic π clouds is exploited for selective capture of CO2 , C2, and C3 hydrocarbons at ambient condition. The framework shows stepwise CO2 and C2 H2 uptake at 195 K but type I profiles are observed at 298 K. The IAST selectivity of CO2 over CH4 is the highest (598 at 298 K) among the MOFs without open metal sites reported till date. It also shows high selectivity for C2 H2 , C2 H4 , C2 H6 , and C3 H8 over CH4 at 298 K. DFT calculations reveal that aromatic π surface and the polar imide (RNC=O) functional groups are the primary adsorption sites for adsorption. Furthermore, breakthrough column experiments showed CO2 /CH4 C2 H6 /CH4 and CO2 /N2 separation capability at ambient condition.

Crystal Dynamics in Multi-stimuli-Responsive Entangled Metal-Organic Frameworks.

An understanding of solid-state crystal dynamics or flexibility in metal-organic frameworks (MOFs) showing multiple structural changes is highly demanding for the design of materials with potential applications in sensing and recognition. However, entangled MOFs showing such flexible behavior pose a great challenge in terms of extracting information on their dynamics because of their poor single-crystallinity. In this article, detailed experimental studies on a twofold entangled MOF (f-MOF-1) are reported, which unveil its structural response toward external stimuli such as temperature, pressure, and guest molecules. The crystallographic study shows multiple structural changes in f-MOF-1, by which the 3 D net deforms and slides upon guest removal. Two distinct desolvated phases, that is, f-MOF-1 a and f-MOF-1 b, could be isolated; the former is a metastable one and transformable to the latter phase upon heating. The two phases show different gated CO2 adsorption profiles. DFT-based calculations provide an insight into the selective and gated adsorption behavior with CO2 of f-MOF-1 b. The gate-opening threshold pressure of CO2 adsorption can be tuned strategically by changing the chemical functionality of the linker from ethanylene (-CH2 -CH2 -) in f-MOF-1 to an azo (-N=N-) functionality in an analogous MOF, f-MOF-2. The modulation of functionality has an indirect influence on the gate-opening pressure owing to the difference in inter-net interaction. The framework of f-MOF-1 is highly responsive toward CO2 gas molecules, and these results are supported by DFT calculations.

Product Release Pathways in Human and Plasmodium falciparum Phosphoribosyltransferase.

Atomistic molecular dynamics (MD) simulations coupled with the metadynamics technique were carried out to delineate the product (PPi.2Mg and IMP) release mechanisms from the active site of both human (Hs) and Plasmodium falciparum (Pf) hypoxanthine-guanine-(xanthine) phosphoribosyltransferase (HG(X)PRT). An early movement of PPi.2Mg from its binding site has been observed. The swinging motion of the Asp side chain (D134/D145) in the binding pocket facilitates the detachment of IMP, which triggers the opening of flexible loop II, the gateway to the bulk solvent. In PfHGXPRT, PPi.2Mg and IMP are seen to be released via the same path in all of the biased MD simulations. In HsHGPRT too, the product molecules follow similar routes from the active site; however, an alternate but minor escape route for PPi.2Mg has been observed in the human enzyme. Tyr 104 and Phe 186 in HsHGPRT and Tyr 116 and Phe 197 in PfHGXPRT are the key residues that mediate the release of IMP, whereas the motion of PPi.2Mg away from the reaction center is guided by the negatively charged Asp and Glu and a few positively charged residues (Lys and Arg) that line the product release channels. Mutations of a few key residues present in loop II of Trypanosoma cruzi (Tc) HGPRT have been shown to reduce the catalytic efficiency of the enzyme. Herein, in silico mutation of corresponding residues in loop II of HsHGPRT and PfHGXPRT resulted in partial opening of the flexible loop (loop II), thus exposing the active site to bulk water, which offers a rationale for the reduced catalytic activity of these two mutant enzymes. Investigations of the product release from these HsHGPRT and PfHGXPRT mutants delineate the role of these important residues in the enzymatic turnover.