Active Site Moieties Research Articles

Toward quantification of active site density and size-dependent ORR activity of ZIF-derived carbons in alkaline electrolyte

ZIF-derived carbons (ZDCs) represent a novel category of non-precious metal catalysts (NPMCs) for the oxygen reduction reaction (ORR). Nevertheless, establishing the correlation between their structure and performance is challenging due to their diverse structural properties and ill-defined active site moieties. Here we investigate the effect of ZIF-67 derived carbon’s particle size and corresponding distribution of assorted nitrogen functionalities on the ORR. Our findings reveal a substantial increase in ORR activity with decreasing ZDC particle size, while the exact influence of nitrogen functionalities remains uncertain. Most importantly, the present work unveils a correlation between the ORR activity and the site density (SD) of Co-Nx within the ZDC materials, which can be attributed to the concurrently increasing SD and accessibility of Co-Nx on the ZDC particles with small sizes. The present work provides novel insights into the optimization of ZDCs as high-performance NPMCs for ORR in alkaline electrolytes.

Spectroscopic Studies on the Conformational Stability of Hemocyanin of Pila virens (Lamarck, 1822) in the Presence of Temperature and Detergents

Gastropods are endowed with a soluble copper containing, blue coloured respiratory protein known as hemocyanin. In the present study, for the first time, hemocyanin from Pila virens, a fresh water gastropod endemic to Indian sub-continent was studied for its conformational stability in the presence of temperature and various detergents by different spectroscopic techniques viz. UV–Vis, fluorescence and circular dichroism spectroscopy. Results from UV–Vis spectroscopy revealed that, absorbance corresponding to active site moieties were decreased with increase in temperature from 30 to 70 °C. Whereas ‘thermal reversibility’ studies showed the increase in absorbance of active site with decrease in temperature from 70 to 20 °C (is an indicative of the characteristic feature of hemocyanins—that oxygen is reversibly bound to the copper in the active site). Results from CD spectroscopy revealed that the temperature-induced conformational changes on the secondary structure of hemocyanin was reversible up to 70 °C. Beyond 80 °C, the protein was completely denatured and assumed an increased random coil conformation. Hemocyanin in the presence of high concentration of detergents lost its structure as evident from UV–Vis fluorescence (evident from the red shifts in the emission maxima accompanied by quenching in emission intensities) and CD spectroscopy. Stability of active site moieties in the presence of different detergents was also studied by UV–Vis spectroscopy. The biophysical techniques gave insights into the varied effects of each of the different denaturants on the conformational stability of hemocyanin from Pila virens.

Rational Generation of Fe−Nx Active Sites in Fe−N−C Electrocatalysts Facilitated by Fe−N Coordinated Precursors for the Oxygen Reduction Reaction

AbstractFe−N−C catalysts synthesized by pyrolysis of Fe and N precursors have been intensively studied due to their remarkable activities for the electrochemical oxygen reduction reaction (ORR). Although Fe−N4 coordinated structures have been suggested as active sites by recent spectroscopic studies, the influence of precursor coordination on the generation of the active sites during high‐temperature pyrolysis is not well understood. In this work, phenanthroline isomers were used as systematic model precursors to reveal the correlation between precursor coordination and active site formation in Fe−N−C catalysts. Coordination between Fe and each phenanthroline isomer was effectively controlled by the molecular structure: monodentate (1,7‐ and 4,7‐phenanthroline) and bidentate coordination (1,10‐phenanthroline). Through X‐ray absorption spectroscopy and X‐ray photoelectron spectroscopy study, large difference in atomic distribution of both Fe and N was revealed; the preferential formation of Fe‐Nx active sites was featured only in Fe(1,10‐phenanthroline)/KB with homogeneously distributed Fe and highly retained pyridinic N. With Fe‐Nx active site moieties, Fe(1,10‐phenanthroline)/KB exhibited superior ORR activity and stability in alkaline half‐cell and full‐cell tests. These results highlight the importance of the use of precursors with multiple coordination (i. e. bidentate) for the effective derivation of Fe‐Nx active sites for highly active and stable ORR electrocatalysts.

On the shoulders of giants.

On the shoulders of giants.

Characteristics and applications of a recombinant alkaline serine protease from a novel bacterium Bacillus lehensis

A highly alkaline protease (BLAP) from a novel psychrotolerant and alkaliphilic bacterium, Bacillus lehensis was cloned and expressed in Escherichia coli. BLAP belongs to subtilase S8 family of proteases, comprising 27 aa secretion signal, 83 aa prosequence and 269 aa mature BLAP. The amino acids Asp 141, His 171 and Ser 324 form catalytic triad, while Ile 214, Leu 233 and Asn 267 are other active site moieties. Recombinant alkaline protease (rBLAP) is a monomeric protein of 39.0±1.0kDa, and it is active over broad pH (8–12) and temperature (30–60°C) ranges, with optima at pH 12.8 and 50°C. rBLAP is stimulated by SDS, Co2+, Ca2+, β-ME, and inhibited by Hg2+ and PMSF. The rBLAP is compatible with commercial detergents, useful in silk degumming and silver recovery from the used photographic films and a potent biocontrol agent for arresting the development of eggs of the nematode Meloidogyne incognita.

Protonation states of the key active site residues and structural dynamics of the glmS riboswitch as revealed by molecular dynamics.

The glmS catalytic riboswitch is part of the 5'-untranslated region of mRNAs encoding glucosamine-6-phosphate (GlcN6P) synthetase (glmS) in numerous gram-positive bacteria. Binding of the cofactor GlcN6P induces site-specific self-cleavage of the RNA. However, the detailed reaction mechanism as well as the protonation state of the glmS reactive form still remains elusive. To probe the dominant protonation states of key active site residues, we carried out explicit solvent molecular dynamic simulations involving various protonation states of three crucial active site moieties observed in the available crystal structures: (i) guanine G40 (following the Thermoanaerobacter tengcongensis numbering), (ii) the GlcN6P amino/ammonium group, and (iii) the GlcN6P phosphate moiety. We found that a deprotonated G40(-) seems incompatible with the observed glmS active site architecture. Our data suggest that the canonical form of G40 plays a structural role by stabilizing an in-line attack conformation of the cleavage site A-1(2'-OH) nucleophile, rather than a more direct chemical role. In addition, we observe weakened cofactor binding upon protonation of the GlcN6P phosphate moiety, which explains the experimentally observed increase in K(m) with decreasing pH. Finally, we discuss a possible role of cofactor binding and its interaction with the G65 and G1 purines in structural stabilization of the A-1(2'-OH) in-line attack conformation. On the basis of the identified dominant protonation state of the reaction precursor, we propose a hypothesis of the self-cleavage mechanism in which A-1(2'-OH) is activated as a nucleophile by the G1(pro-R(p)) nonbridging oxygen of the scissile phosphate, whereas the ammonium group of GlcN6P acts as the general acid protonating the G1(O5') leaving group.

Nucleophile activation in PD…(D/E)xK metallonucleases: An experimental and computational pK a study

Metallonucleases conduct metal-dependent nucleic acid hydrolysis. While metal ions serve in multiple mechanistic capacities in these enzymes, precisely how the attacking water is activated remains unclear for those lacking an obvious general base. All arguments hinge on appropriate pK as for active site moieties very close to this species, and measurement of the pK a of a specific water molecule is difficult to access experimentally. Here we describe a computational approach for exploring the local electrostatic influences on the water-derived nucleophile in metallonucleases featuring the common PD…(D/E)xK motif. We utilized UHBD to predict the pK as of active site groups, including that of a water molecule positioned to act as a nucleophile. The pK a of a Mg(II)-ligated water molecule hydrogen bonded to the conserved Lys70 in a Mg(II)–PvuII enzyme complex was calculated to be 6.5. The metal and the charge on the Lys group were removed in separate experiments; both resulted in the elevation of the pK a of this water molecule, consistent with contributions from both moieties to lowering this pK a. This behavior is preserved among other PD…(D/E)xK metallonucleases. pK as extracted from the pH dependence of the single turnover rate constant are compared to previous experimental data and the above predicted pK as.

Functional and Mechanistic Analyses of Biomimetic Aminoacyl Transfer Reactions in de Novo Designed Coiled Coil Peptides via Rational Active Site Engineering

Ribosomes and nonribosomal peptide synthetases (NRPSs) carry out instructed peptide synthesis through a series of directed intermodular aminoacyl transfer reactions. We recently reported the design of coiled-coil assemblies that could functionally mimic the elementary aminoacyl loading and intermodular aminoacyl transfer steps of NRPSs. These peptides were designed initially to accelerate aminoacyl transfer mainly through catalysis by approximation by closely juxtaposing four active site moieties, two each from adjacent noncovalently associated helical modules. In our designs peptide self-assembly positions a cysteine residue that is used to covalently capture substrates from solution via transthiolesterification (substrate loading step to generate the aminoacyl donor site) adjacent to an aminoacyl acceptor site provided by a covalently tethered amino acid or modeled by the epsilon-amine of an active site lysine. However, through systematic functional analyses of 48 rationally designed peptide sequences, we have now determined that the substrate loading and intermodular aminoacyl transfer steps can be significantly influenced (up to approximately 103-fold) by engineering changes in the active site microenvironment through amino acid substitutions and variations in the inter-residue distances and geometry. Mechanistic studies based on 15N NMR and kinetic analysis further indicate that certain active site constellations furnish an unexpectedly large pK(a) depression (1.5 pH units) of the aminoacyl-acceptor moiety, helping to explain the observed high rates of aminoacyl transfer in those constructs. Taken together, our studies demonstrate the feasibility of engineering efficient de novo peptide sequences possessing active sites and functions reminiscent of those in natural enzymes.

Hirudin Binding Reveals Key Determinants of Thrombin Allostery

Thrombin exists in two allosteric forms, slow (S) and fast (F), that recognize natural substrates and inhibitors with significantly different affinities. Because under physiologic conditions the two forms are almost equally populated, investigation of thrombin function must address the contribution from the S and F forms and the molecular origin of their differential recognition of ligands. Using a panel of 79 Ala mutants, we have mapped for the first time the epitopes of thrombin recognizing a macromolecular ligand, hirudin, in the S and F forms. Hirudin binding is a relevant model for the interaction of thrombin with fibrinogen and PAR1 and is likewise influenced by the allosteric S-->F transition. The epitopes are nearly identical and encompass two hot spots, one in exosite I and the other in the Na+ site at the opposite end of the protein. The higher affinity of the F form is due to the preferential interaction of hirudin with Lys-36, Leu-65, Thr-74, and Arg-75 in exosite I; Gly-193 in the oxyanion hole; and Asp-221 and Asp-222 in the Na+ site. Remarkably, no correlation is found between the energetic and structural involvements of thrombin residues in hirudin recognition, which invites caution in the analysis of protein-protein interactions in general.

Reflections on Edsall's carbonic anhydrase: paradoxes of an ultra fast enzyme

John Edsall's investigations of human erythrocyte carbonic anhydrase, a zinc metalloenzyme that powerfully catalyzes the reversible hydration of carbon dioxide, highlighted a conundrum regarding the correct hydration product. The measured kinetic parameters could not be reconciled with the choice of carbonic acid, since its bimolecular recombination rate with enzyme would exceed the diffusion limit. The alternate choice of bicarbonate obviated the recombination rate problem but required that the active site deprotonation exceed the diffusion-limited maximum rate by an even greater extent. This paradox was resolved in favor of bicarbonate when the unsuspected role of buffer species indirectly deprotonating the enzyme was finally proposed, spurring numerous investigations to verify the hypothesis. Edsall's laboratory also reported the accidental discovery of the first competitive inhibitor, imidazole. This opened new avenues to understanding the binding of the CO2 substrate and stimulated many investigations on this inhibitor. Paramagnetic NMR and crystallographic studies demonstrated that the only other known competitive inhibitor, phenol, apparently shared this unusual binding site. Despite enormous progress since Edsall's retirement, particularly the use of site-directed mutagenesis approaches, the precise interactions of carbon dioxide and bicarbonate with specific active site moieties remain as elusive today as when Edsall first considered these questions.

Auto-inactivation by cleavage within the dimer interface of kaposi’s sarcoma-associated herpesvirus protease

An autolysis site of functional and structural significance has been mapped within the dimer interface of Kaposi’s sarcoma-associated herpesvirus protease. Cleavage 27 residues from the C terminus of the 230 amino acid residue, 25 kDa protein was observed to cause a loss of dimerization and proteolytic activity, even though no active site moieties were lost. Gel-filtration chromatography and analytical ultracentrifugation were used to analyze the changes in oligomerization upon autolysis. The selective auto-disruption of this essential protein-protein interface by proteolytic cleavage resulted in a 60 % loss in mean residue ellipticity by circular dichroism as well as a 20 % weaker, 10 nm red-shifted intrinsic protein fluorescence emission spectrum. These apparent conformational changes induced a strict inhibition of enzymatic activity. An engineered substitution at the P1′ position of this cleavage site attenuated autolysis by the enzyme and restored wild-type dimerization. In addition to retaining full proteolytic activity in a continuous fluorescence-based enzyme assay, this protease variant allowed the determination of the enzyme’s dimerization dissociation constant of 1.7 (±0.9) μM. The structural perturbations observed in this enzyme may play a role in viral maturation, and offer general insight into the allosteric relationship between the dimer interface and active site of herpesviral proteases. The functional coupling between oligomerization and activity presented here may allow for a better understanding of such phenomena, and the design of an enzyme variant stabilized to autolysis should further the structural and mechanistic characterization of this viral protease.