Changes In Catalytic Efficiency Research Articles

A new Mn(Ⅱ)-based metal-organic framework: synthesis, characterization, ferrimagnetic behavior and catalytic conversion of CO2 to cyclic carbonates

A new three-dimensional (3D) metal–organic framework (MOF) [Mn3(bqca)2(DMF)4]n (1) is constructed based on a tricarboxylate ligand 2,3-di-(4-carboxyphenyl)-quinoline-6-carboxylic acid (H3bqca) through solvothermal reaction. The structure, composition and stability of the complex were fully characterized by single crystal X-ray diffraction, powder X-ray diffraction, infrared spectrum and thermogravimetric analysis. The results show that 1 has a 3D microporous framework and exhibits high thermal stability up to 450 ℃. Direct-current magnetic susceptibility reveals that 1 is a rare ferrimagnetic homospin system. Importantly, 1 can effectively catalyze the cycloaddition reaction of CO2 with epoxides to generate cyclic carbonates with high yields and selectivity, and it can also be recycled at least five times without a noticeable change in catalytic efficiency.

Functional analysis of metalloenzymes from human gut microbiota and their role in ulcerative colitis.

Metalloenzymes produced by gut microbiota play an essential role in various physiological processes, and maintains homeostasis of gastrointestinal tract. Our study includes functional analysis of microbial metalloenzymes using metagenomics and metatranscriptomics data from Inflammatory Bowel Disease Multiomics Database. The distance matrix calculated by using metalloenzymes data produced significant results for bacterial taxonomy, with higher variance compared to HMP analysis in both Western and Indian population. Differential gene expression analysis revealed altered expression of ulcerative colitis (UC) associated enzymes, increased folds changes in Prevotella and Megamonas transcripts; whereas, low transcripts of Alistipes genera. Further, docking and simulation studies performed on screened UC-associated enzymes revealed change in catalytic efficiency and ligand interacting residues. The β-diversity using microbes containing metalloenzymes suggests considering small group of specific genes or enzymes for understanding the diversity between ulcerative colitis and healthy individuals. The docking and differential gene expression analysis collectively suggest the probable role of metalloenzymes and few UC-associated enzymes in the severity of ulcerative colitis.

Rational mutagenesis of Thermobifida fusca cutinase to modulate the enzymatic degradation of polyethylene terephthalate.

Thermobifida fusca cutinase (TfCut2) is a carboxylesterase (CE) which degrades polyethylene terephthalate (PET) as well as its degradation intermediates [such as oligoethylene terephthalate (OET), or bis-/mono-hydroxyethyl terephthalate (BHET/MHET)] into terephthalic acid (TPA). Comparisons of the surfaces of certain CEs (including TfCut2) were combined with docking and molecular dynamics simulations involving 2HE-(MHET)3, a three-terephthalate OET, to support the rational design of 22 variants with potential for improved generation of TPA from PET, comprising 15 single mutants (D12L, E47F, G62A, L90A, L90F, H129W, W155F, ΔV164, A173C, H184A, H184S, F209S, F209I, F249A, and F249R), 6 double mutants [H129W/T136S, A173C/A206C, A173C/A210C, G62A/L90F, G62A/F209I, and G62A/F249R], and 1 triple mutant [G62A/F209I/F249R]. Of these, nine displayed no activity, three displayed decreased activity, three displayed comparable activity, and seven displayed increased (~1.3- to ~7.2-fold) activity against solid PET, while all variants displayed activity against BHET. Of the variants that displayed increased activity against PET, four displayed more activity than G62A, the most-active mutant of TfCut2 known till date. Of these four, three displayed even more activity than LCC (G62A/F209I, G62A/F249R, and G62A/F209I/F249R), a CE known to be ~5-fold more active than wild-type TfCut2. These improvements derived from changes in PET binding and not changes in catalytic efficiency.

Recognition of Divergent Viral Substrates by the SARS-CoV-2 Main Protease.

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease (COVID-19), is an ideal target for pharmaceutical inhibition. Mpro is conserved among coronaviruses and distinct from human proteases. Viral replication depends on the cleavage of the viral polyprotein at multiple sites. We present crystal structures of SARS-CoV-2 Mpro bound to two viral substrate peptides. The structures show how Mpro recognizes distinct substrates and how subtle changes in substrate accommodation can drive large changes in catalytic efficiency. One peptide, constituting the junction between viral nonstructural proteins 8 and 9 (nsp8/9), has P1′ and P2′ residues that are unique among the SARS-CoV-2 Mpro cleavage sites but conserved among homologous junctions in coronaviruses. Mpro cleaves nsp8/9 inefficiently, and amino acid substitutions at P1′ or P2′ can enhance catalysis. Visualization of Mpro with intact substrates provides new templates for antiviral drug design and suggests that the coronavirus lifecycle selects for finely tuned substrate-dependent catalytic parameters.

Using sulfate-functionalized Hf-based metal–organic frameworks as a heterogeneous catalyst for solvent-free synthesis of pyrimido[1,2-a]benzimidazoles via one-pot three-component reaction

Functionalized metal–organic frameworks have recently received significant attention in heterogeneous catalysis applications due to their high catalytic activity. In this work, a sulfate-functionalized Hf-cluster-based metal–organic framework was prepared via sulfation of a Hf-MOF, named Hf-BTC, constructed by Hf6 clusters and 1,3,5-tricarboxylate linkers. The Hf-BTC-SO4 material was consequently demonstrated to be an efficiently reusable superacid catalyst for a one-pot three-component reaction of pyrimido[1,2-a]benzimidazoles synthesis. The reaction catalyzed by the sulfated Hf-BTC could be carried out under mild and solvent-free conditions and give superior performance in a wide range of substrates. According to detailed investigation, the good catalytic performance of the sulfated-functionalized MOF likely originates from the high-porosity framework and the high active sites of the functionalized clusters. Importantly, the catalyst could be easy to recover and reuse the functionalized framework several times with minor changes in catalytic efficiency.

Nitric Oxide Synthase Oxygenase in Thin Layer-By-Layer Polymeric Films: Does the pH at Which the Film Is Built Affect the Direct Electron Transfer or the Related NOS-Mediated Catalysis?

We prepared thin films containing nitric oxide synthase (NOS) oxygenase enzymes and polyethyleneimine (PEI) using the layer-by-layer film building strategy. The charge-based layer-by-layer electrostatic adsorption allows for assembly of multi-component protein/PEI films. During the film building process, we used NOS oxygenase enzyme from solutions at two distinct pHs (pH=7.0 and pH=8.6) to study the effect of pH on enzyme loading in the thin polymeric film. We studied direct electron transfer to NOS oxygenase in the two kinds of films at physiologic pH. We show that NOS oxygenase enzymes maintain their overall integral structure in PEI films in both pH conditions, and exhibit relatively fast but similar electron transfer rates. Using thin film coulometry, we show that the enzyme loading from pH 8.6 is almost 4-times more than the amount loaded in films prepared using NOS oxygenase solutions at pH=7.0.To closely examine the effect of pH during the layer-by-layer film building on the trapped NOS oxygenase enzymes, we studied the catalytic reduction of exogenous nitric oxide analyte mediated by NOS oxygenases in PEI films. We used this property to compare and contrast the catalytic activity of the films of NOS enzymes prepared at pH 7.0 and those prepared at pH 8.6. Our results show that there is a distinct increase in catalytic activity of enzymes adsorbed from pH 8.6 compared to those prepared at pH=7.0. We also show that the measured differential loading amnounts in the two kinds of films cannot explain the observed increase in mediated catalytic activity. In this regard, we show that NOS oxygenase films constructed at pH 8.6 exhibit significantly higher catalytic efficiency (kcat /KM) compared to films constructed at pH 7.0. To better understand the change in catalytic activity, we used detailed Fourier Transform Infrared (FTIR) spectroscopic analysis of the thin films to closely examine the structural integrity of the trapped NOS oxygenases in the two sets of films. Deconvolution methods using FTIR data suggest that although NOS oxygenase enzymes maintain their overall structure, we observe distinct pH-dependent conformational changes, which includes a decrease in α-helix contributions in the NOS protein adsorbed at pH 8.6. The observed changes in the secondary structure apparently add to the disordering of protective loops that affect a hydrophobic pocket trap that is critical for NO binding during catalysis.This finding is in line with the observed change in NO affinity for the trapped NOS enzymes as evidenced by the distinct change in catalytic efficiency of NOS oxygenases in the two sets of films. This work shows that while the electron transfer to NOS enzymes in PEI films is not affected by pH in which the layer-by-layer film fabrication occurs, the subtle pH-induced backbone changes provides a structural basis for the higher specific catalytic electrochemical reduction of NO by NOS oxygenases in PEI films constructed at pH 8.6.

A missense variant remote from the active site impairs stability of human phosphoglucomutase 1.

Missense variants of human phosphoglucomutase 1 (PGM1) cause the inherited metabolic disease known as PGM1 deficiency. This condition is categorised as both a glycogen storage disease and a congenital disorder of glycosylation. Approximately 20 missense variants of PGM1 are linked to PGM1 deficiency, and biochemical studies have suggested that they fall into two general categories: those affecting the active site and catalytic efficiency, and those that appear to impair protein folding and/or stability. In this study, we characterise a novel variant of Arg422, a residue distal from the active site of PGM1 and the site of a previously identified disease-related variant (Arg422Trp). In prior studies, the R422W variant was found to produce insoluble protein in a recombinant expression system, precluding further in vitro characterisation. Here we investigate an alternative variant of this residue, Arg422Gln, which is amenable to experimental characterisation presumably due to its more conservative physicochemical substitution. Biochemical, crystallographic, and computational studies of R422Q establish that this variant causes only minor changes in catalytic efficiency and 3D structure, but is nonetheless dramatically reduced in stability. Unexpectedly, binding of a substrate analog is found to further destabilise the protein, in contrast to its stabilising effect on wild-type PGM1 and several other missense variants. This work establishes Arg422 as a lynchpin residue for the stability of PGM1 and supports the impairment of protein stability as a pathomechanism for variants that cause PGM1 deficiency. SYNOPSIS: Biochemical and structural studies of a missense variant far from the active site of human PGM1 identify a residue with a key role in enzyme stability.

Air‐stable zirconium (IV)‐salophen perfluorooctanesulfonate as a highly efficient and reusable catalyst for the synthesis of 3,4‐dihydropyrimidin‐2‐(1H)‐ones/thiones under solvent‐free conditions

An air‐stable complex of zirconium (IV)‐salophen perfluorooctanesulfonate (1) was successfully synthesized by reacting Zr (salphen)Cl2and C8F17SO3Ag. Complex1was characterized and studied by different techniques (NMR, IR, HRMS, TG‐DSC, conductivity and acidity measurements), and was found to be air‐stable, water tolerant, thermally stable and strongly Lewis‐acidic. Complex1exhibited high catalytic efficiency for the synthesis of 3,4‐dihydropyrimidin‐2‐(1H)‐ones/thiones via the Biginelli reaction of aldehydes, 1,3‐dicarbonyl compounds and urea/thiourea under solvent‐free conditions. Furthermore, complex1could be reused 5 times with minimal changes in catalytic efficiency. Compared with previously reported methods, the important features of this protocol are the solvent‐free conditions, the short reaction times, the wide substrate compatibility, the high efficiency and the good reusability.

Air-stable μ-oxo-bridged binuclear titanium(IV) salophen perfluorooctanesulfonate as a highly efficient and recyclable catalyst for the synthesis of bis(indolyl) methane derivatives

An air-stable μ-oxo-bridged binuclear Lewis acid of titanium(IV) salophen perfluorooctanesulfonate [{Ti(salophen)H2O}2O][OSO2C8F17]2 (1) was successfully synthesized by the reaction of TiIV(salophen)Cl2 with AgOSO2C8F17 and characterized by different techniques such as IR, NMR and HRMS. TG-DSC analysis showed that this complex was thermally stable at 220 °C. Meanwhile, it exhibited good solubility in polar organic solvents and good ionic dissociation in CH3CN solution. Fluorescence spectra and Hammett indicator results indicated that the complex had relatively strong acidity. And it showed high catalytic efficiency for the synthesis of bis(indolyl) methane derivatives through Friedel-crafts reaction of aldehyde/ketone with indoles. Moreover, it can be reused five times with minimal changes in catalytic efficiency.

A Tail of Kinase Regulation: How C‐termini Modulate CK1 Substrate Phosphorylation

CK1 enzymes signal in a variety of important cellular pathways, including DNA damage repair, mitotic checkpoint signaling, circadian rhythm, Wnt signaling, endocytosis, and neurodegenerative disease progression. Like other multifunctional kinases, CK1 must be regulated in space and time in order to target specific subsets of its substrates in each of the pathways it participates in. However, CK1 is generally regarded as a “rogue” kinase, which is constitutively active, ubiquitous throughout cells and tissues, and unregulated except by autoinhibition.CK1 enzymes are known to autophosphorylate their C‐terminal non‐catalytic tails, which are proposed to inhibit their activity by acting as pseudosubstrates. This model would require a phosphorylation‐dependent intramolecular interaction between the C‐terminus and the kinase domain, but we are unaware of any evidence demonstrating such an event. Furthermore, this proposed mechanism of autoinhibition has not been tested in vivo in any organism.We have identified six serine and threonine autophosphorylation sites on the C‐terminus of Schizosaccharomyces pombe Hhp1, one of two soluble CK1 enzymes in this organism, and are testing candidate sites on Hhp2 and the human homologues CK1□/□. When these sites are specifically phosphorylated, the Hhp1 C‐terminus binds the kinase domain via a low‐affinity electrostatic interaction. At concentrations above the Kd of this interaction, the phosphorylated C‐terminus inhibits Hhp1 kinase activity, while mutations that abolish phosphorylation increase the activity of the full‐length kinase.Structural studies have identified two conserved basic patches on the CK1 kinase domain that are hypothesized to interact with the phosphorylated C‐terminus; however, we show that the tail instead interacts with the substrate binding pocket. The physiological substrate Sid4 has a higher affinity than the tail for the Hhp1 kinase domain, and the model substrate casein can out‐compete the phosphorylated C‐terminus for binding to the kinase domain. Our data support a new model of CK1 activation in which the presence of high‐affinity substrates is sufficient to displace the low‐affinity tail and relieve autoinhibition without the need for prior dephosphorylation of CK1.Because the C‐termini of CK1 family members are responsible for most of the sequence variation between isoforms, we hypothesize that these different tails may differ in affinity for the conserved kinase domain, leading to displacement by different cohorts of substrates. Thus, this mechanism may also explain variations in substrate specificities between CK1 enzymes. In support of this model, truncating the tail of Hhp1 or Hhp2 eliminates substrate‐specific differences in catalytic efficiency. We are currently using phosphorylation site mutants of hhp1 to confirm that these changes in catalytic efficiency are dependent on autophosphorylation and to investigate the significance of autophosphorylation in vivo.Support or Funding InformationSNC was supported by the Integrated Biological Systems Training in Oncology Program (2T32CA119925). This work was supported by NIH GM112989 to KLG.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of metal ions (Cr3+, Co2+, Ni2+, Cu2+ and Zn2+) substitution on the structural, magnetic and catalytic properties of substituted Co–Mn ferrites synthesized by sol–gel route

Manganese substituted cobalt ferrites (CoMn x Fe2−x O4, x = 0.2, 0.4, 0.6, 0.8 and 1.0) synthesized using sol gel autocombustion method were used as photocatalysts for the degradation of both cationic and anionic dyes i.e Safranine-O and Remazol Brilliant yellow. CoMn0.4Fe1.6O4 exhibited maximum photocatalytic activity among all the synthesized ferrites. To further study the effect of metal ion doping on catalytic performance, substitution with different metal ions (Cr3+, Co2+, Ni2+, Cu2+ and Zn2+) was done in CoMn0.4Fe1.6O4 lattice. The structural and magnetic properties of prepared samples were investigated using powder X-ray diffraction, Fourier transform infrared spectroscopy and vibrating sample magnetometer. The average crystallite size of the synthesized nanoparticles was observed to be in the range of 39–52 nm. The saturation magnetization of the samples was found to decrease with the introduction of all metal cations (except Co2+) in the Co–Mn lattice. The changes in the catalytic activity were tested for degradation of Safranine-O and Remazol Brilliant yellow. Amongst the synthesized CoMn0.4M x Fe1.6−x O4, Cu substituted Co–Mn ferrite exhibited maximum photocatalytic efficiency. All the magnetic nanoferrites were easily recovered by using an external magnet and could be reused without any remarkable change in catalytic efficiency.

Characterization of the cinnamoyl-CoA reductase (CCR) gene family in Populus tomentosa reveals the enzymatic active sites and evolution of CCR.

Two distinct cinnamoyl-coenzyme A reductases (CCRs) from Populus tomentosa were cloned and studied and active sites in CCRs were further identified based on sequence divergence, molecular simulation, and site-directed mutants. Cinnamoyl-coenzyme A (CoA) reductase (CCR) is the first committed gene in the lignin-specific pathway and plays a role in the lignin biosynthesis pathway. In this study, we cloned 11 genes encoding CCR or CCR-like proteins in Populus tomentosa. An enzymatic assay of the purified recombinant P. tomentosa (Pto) CCR and PtoCCR-like proteins indicated that only PtoCCR1 and PtoCCR7 had detectable activities toward hydroxycinnamoyl-CoA esters. PtoCCR1 exhibited specificity for feruloyl-CoA, with no detectable activity for any other hydroxycinnamoyl-CoA esters. However, PtoCCR7 catalyzed p-coumaroyl-CoA, caffeoyl-CoA, feruloyl-CoA, and sinapoyl-CoA with a preference for feruloyl-CoA. Site-directed mutations of selected amino acids divergent between PtoCCR1 and 7, combined with modeling and docking, showed that A132 in CCR7 combined with the catalytic triad might comprise the catalytic center. In CCR7, L192, F155, and H208 were identified as the substrate-binding sites, and site-directed mutations of these amino acids showed obvious changes in catalytic efficiency with respect to both feruloyl-CoA and sinapoyl-CoA. Mutant F155Y exhibited greater catalytic efficiency for sinapoyl-CoA compared with that of wild-type PtoCCR7. Finally, recent genome duplication events provided the foundation for CCR divergence. This study further identified the active sites in CCRs and the evolutionary process of CCRs in terrestrial plants.

Mn porphyrins immobilized on non-modified and chloropropyl-functionalized mesoporous silica SBA-15 as catalysts for cyclohexane oxidation

In this work, solid catalysts were prepared by the heterogenization of Mn porphyrins (MnP) on mesoporous silica SBA-15 by two simple strategies: (1) immobilization of the three cationic isomers of Mn(III) N-methylpyridiniumporphyrins (MnTM-X-PyPCl5, X=2, 3,4) on mesoporous silica SBA-15 by electrostatic interaction; and (2) anchoring of the three neutral isomers of Mn(III) N-pyridylporphyrins (MnT-X-PyPCl, X=2, 3, 4) on chloropropyl-functionalized mesoporous silica SBA-15Cl via covalent bonding. SBA-15 and SBA-15Cl solids were characterized by FTIR, TGA-DTA, elemental analysis, SAXS, textural analysis, SEM, TEM, and 13C CPMAS NMR (for SBA-15Cl only). The resulting MnP-supported materials with MnP loadings of 0.3% w/w were characterized by diffuse reflectance UV/VIS spectroscopy and explored as catalysts for hydroxylation of cyclohexane using iodosylbenzene as oxygen donor. Changes on SBA-15Cl silanol and CCl bands on FTIR spectra, and UV/VIS analyses confirm the porphyrin immobilization. The heterogenized catalysts were more efficient and selective toward alcohol than the corresponding homogeneous systems. The covalently immobilized catalysts SBA-15Cl/MnT-X-PyPCl (X=2, 3, 4) showed total oxidation yields and selectivities slightly higher than those of SBA-15/MnTM-X-PyPCl5. In both solid systems, the efficiency and selectivity remained essentially unaffected by the immobilized MnP isomer. The strong interaction between MnP and the mesoporous silica, in both SBA-15 and SBA-15Cl systems, was verified by the low-leaching of MnP from the materials even after exhaustive washings. The solid catalysts were recovered and reused in recycling studies showing high resistance against oxidative destruction with no significant changes in catalytic efficiency upon recycling.

Silver-containing microemulsion as a high-efficient and recyclable catalytic system for hydration of alkynes

A silver-catalyzed highly efficient and regioselective synthesis of ketones from a wide range of alkynes is described. The reaction is dramatically accelerated by its performance in aqueous emulsion, which is self-assembled by the addition of silver perfluorooctanesulfonate (1) and perfluorooctanesulfonic acid (PFOS) to water. The reaction is conducted under convenient conditions with broad substrate scope, including a variety of aromatic and aliphatic terminal alkynes and internal alkynes. Furthermore, the air- and light-stable silver catalytic microemulsion can be reused for 4 times with minimal change in catalytic efficiency.

Towards Tricking a Pathogen’s Protease into Fighting Infection: The 3D Structure of a Stable Circularly Permuted Onconase Variant Cleavedby HIV-1 Protease

Onconase® is a highly cytotoxic amphibian homolog of Ribonuclease A. Here, we describe the construction of circularly permuted Onconase® variants by connecting the N- and C-termini of this enzyme with amino acid residues that are recognized and cleaved by the human immunodeficiency virus protease. Uncleaved circularly permuted Onconase® variants are unusually stable, non-cytotoxic and can internalize in human T-lymphocyte Jurkat cells. The structure, stability and dynamics of an intact and a cleaved circularly permuted Onconase® variant were determined by Nuclear Magnetic Resonance spectroscopy and provide valuable insight into the changes in catalytic efficiency caused by the cleavage. The understanding of the structural environment and the dynamics of the activation process represents a first step toward the development of more effective drugs for the treatment of diseases related to pathogens expressing a specific protease. By taking advantage of the protease’s activity to initiate a cytotoxic cascade, this approach is thought to be less susceptible to known resistance mechanisms.

S-Nitrosoglutathione covalently modifies cysteine residues of human carbonyl reductase 1 and affects its activity

Carbonyl reductase 1 (CBR1 or SDR21C1) is a ubiquitously-expressed, cytosolic, monomeric, and NADPH-dependent enzyme. CBR1 participates in apoptosis, carcinogenesis and drug resistance, and has a protective role in oxidative stress, cancer and neurodegeneration. S-Nitrosoglutathione (GSNO) represents the newest addition to its diverse substrate spectrum, which includes a wide range of xenobiotics and endogenous substances. GSNO has also been shown to covalently modify and inhibit CBR1. The aim of the present study was to quantify and characterize the resulting modifications. Of five candidate cysteines for modification by 2mM GSNO (positions 26, 122, 150, 226, 227), the last four were analyzed using MALDI-TOF/TOF mass spectrometry and then quantified using the Selected Reaction Monitoring Approach on hyphenated HPLC with a triple quadrupole mass spectrometer. The analysis confirmed GSNO concentration-dependent S-glutathionylation of cysteines at positions 122, 150, 226, 227 which was 2–700times higher compared to wild-type CBR1 (WT-CBR1). Moreover, a disulfide bond between neighboring Cys-226 and Cys-227 was detected. We suggest a role of these two cysteines as a redox-sensitive cysteine pair. The catalytic properties of wild-type and enzyme modified with 2mM GSNO were also investigated by steady state kinetic experiments with various substrates. GSNO treatment of CBR1 resulted in a 2–5-fold decrease in kcat with menadione, 4-benzoylpyridine, 2,3-hexanedione, daunorubicin and 1,4-naphthoquinone. In contrast, the same treatment increased kcat for substrates containing a 1,2-diketo group in a ring structure (1,2-naphthoquinone, 9,10-phenanthrenequinone, isatin). Except for 9,10-phenanthrenequinone, all changes in kcat were at least in part compensated for by a similar change in Km, overall yielding no drastic changes in catalytic efficiency. The findings indicate that GSNO-induced covalent modification of cysteine residues affects the kinetic mechanism of CBR1 both in terms of substrate binding and turnover rate, probably by covalent modification of Cys-226 and/or Cys-227.

The bacterial ribonuclease P holoenzyme requires specific, conserved residues for efficient catalysis and substrate positioning

RNase P is an RNA-based enzyme primarily responsible for 5′-end pre-tRNA processing. A structure of the bacterial RNase P holoenzyme in complex with tRNAPhe revealed the structural basis for substrate recognition, identified the active site location, and showed how the protein component increases functionality. The active site includes at least two metal ions, a universal uridine (U52), and P RNA backbone moieties, but it is unclear whether an adjacent, bacterially conserved protein loop (residues 52–57) participates in catalysis. Here, mutagenesis combined with single-turnover reaction kinetics demonstrate that point mutations in this loop have either no or modest effects on catalytic efficiency. Similarly, amino acid changes in the ‘RNR’ region, which represent the most conserved region of bacterial RNase P proteins, exhibit negligible changes in catalytic efficiency. However, U52 and two bacterially conserved protein residues (F17 and R89) are essential for efficient Thermotoga maritima RNase P activity. The U52 nucleotide binds a metal ion at the active site, whereas F17 and R89 are positioned >20 Å from the cleavage site, probably making contacts with N−4 and N−5 nucleotides of the pre-tRNA 5′-leader. This suggests a synergistic coupling between transition state formation and substrate positioning via interactions with the leader.

The Evolution of Cefotaximase Activity in the TEM β-Lactamase

The development of a molecular-level understanding of drug resistance through β-lactamase is critical not only in designing newer-generation antibacterial agents but also in providing insight into the evolutionary mechanisms of enzymes in general. In the present study, we have evaluated the effect of four drug resistance mutations (A42G, E104K, G238S, and M182T) on the cefotaximase activity of the TEM-1 β-lactamase. Using computational methods, including docking and molecular mechanics calculations, we have been able to correctly identify the relative order of catalytic activities associated with these four single point mutants. Further analyses suggest that the changes in catalytic efficiency for mutant enzymes are correlated to structural changes within the binding site. Based on the energetic and structural analyses of the wild-type and mutant enzymes, structural rearrangement is suggested as a mechanism of evolution of drug resistance through TEM β-lactamase. The present study not only provides molecular-level insight into the effect of four drug resistance mutations on the structure and function of the TEM β-lactamase but also establishes a foundation for a future molecular-level analysis of complete evolutionary trajectory for this class of enzymes.

Cysteine dioxygenase: a robust system for regulation of cellular cysteine levels

Cysteine catabolism in mammals is dependent upon cysteine dioxygenase (CDO), an enzyme that adds molecular oxygen to the sulfur of cysteine, converting the thiol to a sulfinic acid known as cysteinesulfinic acid (3-sulfinoalanine). CDO is one of the most highly regulated metabolic enzymes responding to diet that is known. It undergoes up to 45-fold changes in concentration and up to 10-fold changes in catalytic efficiency. This provides a remarkable responsiveness of the cell to changes in sulfur amino acid availability: the ability to decrease CDO activity and conserve cysteine when cysteine is scarce and to rapidly increase CDO activity and catabolize cysteine to prevent cytotoxicity when cysteine supply is abundant. CDO in both liver and adipose tissues responds to changes in dietary intakes of protein and/or sulfur amino acids over a range that encompasses the requirement level, suggesting that cysteine homeostasis is very important to the living organism.

Glycosaminoglycans Facilitate Procathepsin B Activation through Disruption of Propeptide-Mature Enzyme Interactions

Lysosomal cysteine cathepsin B participates in numerous diverse cellular processes. In acquiring its activity, the proregion, which blocks the substrate-binding site in the proenzyme, needs to be cleaved off. Here we demonstrate that polyanionic polysaccharides, glycosaminoglycans (GAGs), can accelerate the autocatalytic removal of the propeptide and subsequent activation of cathepsin B. We show that naturally occurring GAGs such as chondroitin sulfates and heparin, as well as the synthetic analog dextran sulfate, accelerate the processing in a concentration-dependent manner. Heparin oligosaccharides down to the size of tetrasaccharides were efficient in accelerating the procathepsin B processing, whereas disaccharides were without effect. Further, the ability of the GAGs to accelerate procathepsin B processing was sensitive to increasing NaCl concentrations, indicating that electrostatic interaction between the GAGs and procathepsin B are operative in the accelerating effect. Also the processing of the catalytic procathepsin B mutant by wild type cathepsin B was enhanced in the presence of GAGs, suggesting that GAGs induce a conformational change in procathepsin B, converting it into a better substrate. Site-directed mutagenesis showed that His(28), Lys(39), and Arg(40), located within the procathepsin B propeptide, have significant roles in the acceleration of procathepsin B activation induced by short GAGs. Because procathepsin B and GAGs often co-localize in vivo, we propose that GAGs may play a physiological role in the activation of procathepsin B.