Study Of Catalytic Mechanism Research Articles

Investigation of Menaquinone Biosynthesis in Mycobacterium Tuberculosis: Catalytic Mechanism and Inhibition Studies of MenB

The lipid soluble redox cofactor menaquinone is an essential component of the mycobacterial respiratory chain and selective inhibitors of menaquinone biosynthesis are promising lead compounds for the treatment of latent tuberculosis (TB) infections. Menaquinone is synthesized from chorismate by the action of at least 7 enzymes including 1,4‐dihydroxynaphthoyl‐CoA synthase (MenB), which catalyzes an intramolecular Claisen (Dieckmann) condensation to form 1,4‐dihydroxynaphthoyl‐CoA from O‐succinylbenzoyl‐CoA. Currently we are comparing and contrasting the mechanism of the TB MenB enzyme with that of MenB from E. coli as well as with other members of the crotonase superfamily including 2‐ketocyclohexanecarboxyl‐CoA hydrolase (BadI), which catalyzes a retroDieckmann reaction. Crystallographic and kinetic data indicate that conserved catalytic residues play similar roles in the reactions catalyzed by MenB and BadI. However, the studies also suggest subtle differences in the mechanisms of the reactions catalyzed by the TB and E. coli MenB enzymes. In separate studies we recently identified several μM inhibitors of MenB using high‐throughput screening at the NRPB. These compounds are promising leads for developing novel inhibitors of menaquinone biosynthesis.

Acyl-CoA dehydrogenases. A mechanistic overview.

Acyl-CoA dehydrogenases constitute a family of flavoproteins that catalyze the alpha,beta-dehydrogenation of fatty acid acyl-CoA conjugates. While they differ widely in their specificity, they share the same basic chemical mechanism of alpha,beta-dehydrogenation. Medium chain acyl-CoA dehydrogenase is probably the best-studied member of the class and serves as a model for the study of catalytic mechanisms. Based on medium chain acyl-CoA dehydrogenase we discuss the main factors that bring about catalysis, promote specificity and determine the selective transfer of electrons to electron transferring flavoprotein. The mechanism of alpha,beta-dehydrogenation is viewed as a process in which the substrate alphaC-H and betaC-H bonds are ruptured concertedly, the first hydrogen being removed by the active center base Glu376-COO- as an H+, the second being transferred as a hydride to the flavin N(5) position. Hereby the pKa of the substrate alphaC-H is lowered from > 20 to approximately 8 by the effect of specific hydrogen bonds. Concomitantly, the pKa of Glu376-COO- is also raised to 8-9 due to the decrease in polarity brought about by substrate binding. The kinetic sequence of medium chain acyl-CoA dehydrogenase is rather complex and involves several intermediates. A prominent one is the molecular complex of reduced enzyme with the enoyl-CoA product that is characterized by an intense charge transfer absorption and serves as the point of transfer of electrons to the electron transferring flavoprotein. These views are also discussed in the context of the accompanying paper on the three-dimensional properties of acyl-CoA dehydrogenases.

Myeloperoxidase up-regulates the catalytic activity of inducible nitric oxide synthase by preventing nitric oxide feedback inhibition.

Kinetic and structure analysis of inducible nitric oxide synthase (iNOS) revealed that, in addition to the increase of iNOS expression in inflamed areas, the major pathway causing overproduction of NO is destabilization of the iNOS-nitrosyl complex(es) that form during steady-state catalysis. Formation of such a complex allows iNOS to operate at only a fraction (20-30%) of its maximum activity. Thus, bioavailability of NO scavengers at sites of inflammation may play an essential role in up-regulation of the catalytic activity of iNOS, by preventing the catalytic activity inhibition that is attributed to nitrosyl complex formation. Myeloperoxidase (MPO), a major NO scavenger, is a pivotal enzyme involved in leukocyte-mediated host defenses. It is thought to play a pathogenic role under circumstances such as acute inflammatory tissue injury and chronic inflammatory conditions. However, a detailed understanding of the interrelationship between iNOS and MPO at sites of inflammation is lacking. We used direct spectroscopic, HPLC, and selective NO-electrode measurements to determine the interdependent relationship that exists between iNOS and MPO and the role of the MPO/H2O2 system in up-regulating the catalytic activity of iNOS that occurs at sites of inflammation. Scavenging free NO from the iNOS milieu by the MPO/H2O2 system subsequently restores the full capacity of iNOS to convert L-arginine to product (NO), as judged by the increase in the rates of citrulline and nitrite/nitrate production. Studies of iNOS catalytic mechanisms and function are essential to a more fundamental understanding of these factors, which govern iNOS-dependent processes in human health and disease.

UV Raman studies of adsorbed oxygen and NOx species on Pt/γ-alumina catalysts

Raman spectra at 244nm are obtained in situ from several Pt/γ-Al2O3 catalysts under gas flows containing O2, CO, NO2, and H2 at temperatures from 20 to 600°C. With O2 or NO2, a broad peak at 571cm−1 is observed which we attribute to the Pt–O stretch mode of atomic oxygen. This marks the first direct spectroscopic observation of this fundamental species on a real catalyst. The peak can be easily removed with CO or H2, even at room temperature, and the process is completely reversible. With NO2 exposure, several peaks associated with surface NOx species also appear, the most prominent of which is a nitrate line at 1048cm−1. Under reaction conditions, at 350°C with a stoichiometric CO/O2/N2 gas stream, the steady-state O coverage on the Pt particles is about half of the maximum coverage achieved with only an O2 flow. This observation paves the way for future in situ studies of catalytic mechanisms addressing the role of atomic O as an intermediate.

A study of catalytic mechanism of a mammalian Histidine Decarboxylase

A study of catalytic mechanism of a mammalian Histidine Decarboxylase

In situ MAS NMR spectroscopy study of catalytic reaction mechanisms

In situ magic-angle spinning (MAS) NMR spectroscopy has been recently applied successfully to study catalytic reaction mechanisms in batch-like and continuous-flow conditions. The characteristics of both methods are highlighted. Examples taken from our own studies of the activation of propane at low temperature on Ga/H-ZSM-5, the alkylation of benzene with propane on Ga/H-ZSM-5, and the activation of n-butane at low temperature on Pt/H-Theta-1 illustrate how batch-like conditions in situ MAS NMR spectroscopy with the help of strategically 13C-labelled reagents contributes to the understanding of catalytic reaction mechanisms and of the dynamics and reactivity of surface intermediates and active sites.

Characterization and crystallization of an active N-terminally truncated form of the Escherichia coli glycogen branching enzyme.

The prokaryotic glycogen branching enzymes (GBE) can be divided into two groups on the basis of their primary structures: the first group of enzymes, which includes GBE from Escherichia coli, is characterized by a long N-terminal extension that is absent in the enzymes of the second group. The extension consists of approximately 100 amino-acid residues with unknown function. In order to characterize the function of this region, the 728 amino-acid residue, full-length E. coli GBE, and a truncated form (nGBE) missing the first 107 amino-acid residues were overexpressed in E. coli. Both enzymes were purified to homogeneity by a simple purification procedure involving ammonium sulphate precipitation, ion-exchange chromatography, and a second ammonium sulphate precipitation. Purified full-length enzyme was poorly soluble and formed aggregates, which were inactive, at concentrations above 1 mg.mL-1. In contrast, the truncated form could be concentrated to 6 mg.mL-1 without any visible signs of aggregation or loss of activity on concentration. The ability to overexpress nGBE in a highly soluble form has allowed us to produce diffracting crystals of a branching enzyme for the first time. A comparison of the specific activities of purified GBE and nGBE in assays where amylose was used as substrate demonstrated that nGBE retained approximately half of the branching activity of full-length GBE and is therefore a suitable model for the study of the enzymes' catalytic mechanism.

7] Methods for studying glucosylceramide synthase

Glucosylceramide synthase (GCS), also referred to as “glucocerebroside synthase,” and ceramide glucosyltransferase, glucosylates ceramide uses UDP-glucose (UDP-Glc) as a hexose donor. Glucosylceramide (GlcCer) is the precursor of most plasma membrane glycosphingolipids. Thus, the regulation of the expression and activity of GCS has relevance to numerous biological and pathological processes—for example, development, differentiation, tumorigenesis, and host/pathogen interactions—in which glycosphingolipids appear to play a role. Inhibitors of GCS are being investigated as candidate drugs for the treatment of cancer. Identification of GCS as an integral membrane protein of the Golgi complex poses questions concerning the mechanisms of targeting and localization of this enzyme. Thus, it is important to develop techniques for the study of GCS expression, activity, catalytic mechanism, and localization. The purification of measurable quantities of GCS for studies of secondary structure, catalytic mechanism, inhibitor design, and Golgi localization is probably only possible by the overexpression of GCS in a bacterial or eukaryotic cell system. The design of fusion proteins in which GCS is fused to tags (e.g., polyhistidine) for purification or to large hydrophilic carrier proteins for better solubility and decreased aggregation will likely facilitate GCS purification.

Application of low temperature IR spectroscopy for studies of catalyst properties and dynamics on the example of aluminum halide complexes

This paper shows the benefits of applying IR spectroscopy to solid state studies of active labile catalytic complexes, intermediates and reaction mechanisms taking aluminum halide complexes as an example. Aluminum halide complexes with ethylene, hydrogen chloride and organic nitrocompounds were prepared under codeposition of reagents on a cooled surface and then films obtained were studied at 80–150 K. Semi-empirical and ab initio calculations were used to analyze spectral data. A limited molecular mobility and special features of the solid state make it possible to investigate the nature and properties of different types of complexes. In addition, direct observation of catalytic species unstable in solutions becomes possible. Structure and reactivity of a number of molecular and ionic monomer and dimer aluminum halide complexes were studied. Dimer complexes are more active and may catalyze hydrohalogenation of ethylene and dehydration of nitrocompounds even at low temperatures.

Oxidative phosphorylation at the fin de siècle.

Mitochondria produce most of the energy in animal cells by a process called oxidative phosphorylation. Electrons are passed along a series of respiratory enzyme complexes located in the inner mitochondrial membrane, and the energy released by this electron transfer is used to pump protons across the membrane. The resultant electrochemical gradient enables another complex, adenosine 5'-triphosphate (ATP) synthase, to synthesize the energy carrier ATP. Important new mechanistic insights into oxidative phosphorylation have emerged from recent three-dimensional structural analyses of ATP synthase and two of the respiratory enzyme complexes, cytochrome bc1 and cytochrome c oxidase. This work, and new enzymological studies of ATP synthase's unusual catalytic mechanism, are reviewed here.

In Situ Real-Time Studies of Heterogeneous Catalytic Mechanisms at Ambient Pressures As Probed by Surface-Enhanced Raman and Mass Spectroscopies

The utility of surface-enhanced Raman spectroscopy (SERS) as an in situ adsorbate probe of heterogeneous catalytic systems at high gas pressures (here up to 1 atm) in conjunction with mass spectrometry (MS) is demonstrated for reactions on transition-metal catalysts, at temperatures up to 500 °C. This simultaneous SERS−MS approach enables the relationships between the formation of specific adsorbed species (as sensed by SERS) and reaction products (as detected by MS) to be explored on a common (≈1 s) time scale. The low-frequency (<1000 cm-1) spectral region, where metal−adsorbate vibrations are located, is especially informative. The environmentally important reactions of NO reduction by CO or H2 and the oxidation of methanol on transition metals (Rh, Pd, Pt) are considered here. The possible mechanistic significance of surface oxide formation observed during such ambient-pressure catalytic reactions is also discussed.

Application of the superposition principle to the study of CEC, CE, EC and catalytic mechanisms in cyclic chronopotentiometry. Part III

In this paper we have obtained the general analytical equations corresponding to the response of complicated charge transfer processes with coupled homogeneous chemical reactions in cyclic chronopotentiometry by applying the superposition principle. The analysis of different cycles of this response can be used to obtain accurate and contrasted values of kinetic parameters in each complex reaction scheme analysed. These parameters are of great interest for the chemist and the biochemist. These equations are also applicable to cyclic derivative chronopotentiometry which is a very useful method since the response is obtained with peaks in a similar way to cyclic voltammetry but its mathematical treatment is simpler.

Alterations in chemical shifts and exchange broadening upon peptide boronic acid inhibitor binding to alpha-lytic protease.

alpha-Lytic protease, a bacterial serine protease of 198 amino acids (19 800 Da), has been used as a model system for studies of catalytic mechanism, structure-function relationships, and more recently for studies of pro region-assisted protein folding. We have assigned the backbones of the enzyme alone, and of its complex with the tetrahedral transition state mimic N-tert-butyloxycarbonyl-Ala-Pro-boro Val, using double- and triple-resonance 3D NMR spectroscopy on uniformly 15N- and 13C/15N-labeled protein. Changes in backbone chemical shifts between the uncomplexed and inhibited form of the protein are correlated with distance from the inhibitor, the displacement of backbone nitrogens, and change in hydrogen bond strength upon inhibitor binding (derived from previously solved crystal structures). A comparison of the solution secondary structure of the uninhibited enzyme with that of the X-ray structure reveals no significant differences. Significant line broadening, indicating intermediate chemical exchange, was observed in many of the active site amides (including three broadened to invisibility), and in a majority of cases the broadening was reversed upon addition of the inhibitor. Implications and possible mechanisms of this line broadening are discussed.

Binding and Hydrolysis of TNP-ATP by Escherichia coli F1-ATPase

It had previously been suggested that Vmax hydrolysis rate of 2', 3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) by F1-ATPase required filling of only two catalytic sites on the enzyme (Grubmeyer, C., and Penefsky, H. S. (1981) J. Biol. Chem. 256, 3718-3727), whereas recently it was shown that Vmax rate of ATP hydrolysis requires that all three catalytic sites are filled (Weber, J., Wilke-Mounts, S., Lee, R. S. F., Grell, E., and Senior, A. E. (1993) J. Biol. Chem. 268, 20126-20133). To resolve this apparent discrepancy, we measured equilibrium binding and hydrolysis of MgTNP-ATP under identical conditions, using betaY331W mutant Escherichia coli F1-ATPase, in which the genetically engineered tryptophan provides a direct fluorescent probe of catalytic site occupancy. We found that MgTNP-ATP hydrolysis at Vmax rate did require filling of all three catalytic sites, but in contrast to the situation with MgATP, "bisite hydrolysis" of MgTNP-ATP amounted to a substantial fraction (approximately 40%) of Vmax. Binding of MgTNP-ATP to the three catalytic sites showed strong binding cooperativity (Kd1 < 1 nm, Kd2 = 23 nm, Kd3 = 1.4 microM). Free TNP-ATP (i.e. in presence of EDTA) bound to all three catalytic sites with lower affinity but was not hydrolyzed. These data emphasize that the presence of Mg2+ is critical for cooperativity of substrate binding, formation of the very high affinity first catalytic site, and hydrolytic activity in F1-ATPases and that these three properties are strongly correlated.

Engineering surface charge. 2. A method for purifying heterodimers of Escherichia coli glutathione reductase

Two gor genes encoding different mutants of Escherichia coli glutathione reductase have been expressed in the same E. coli cell, leading to the creation of a hybrid form of the enzyme dimer. One of the gor genes carried, in addition to various directed mutations, a 5' extension that encodes a benign penta-arginine "arm" added to the N-terminus of the glutathione reductase polypeptide chain [Deonarain, M.P., Scrutton, N.S., & Perham, R.N. (1992) Biochemistry (preceding paper in this issue)]. This made possible, by means of ion-exchange chromatography or nondenaturing polyacrylamide gel electrophoresis, the facile separation of the hybrid enzyme from the two parental forms. Moreover, the two subunits in the hybrid enzyme could be made to carry different mutations. In this way, glutathione reductases with only one active site per dimer were generated: the effects of replacing tyrosine-177 with glycine in the NADPH-binding site, which greatly diminishes the Km for glutathione and switches the kinetic mechanism from ping-pong to ordered sequential, and of replacing His-439 with glutamine in the glutathione-binding site, which greatly diminishes the Km for NADPH, were both found to be restricted to the one active site carrying the mutations. This system of generating separable enzyme hybrids is generally applicable and should make it possible now to undertake a more systematic study of catalytic mechanism and assembly for the many enzymes with quaternary structure.

Characterization of Escherichia coli ATP synthase .beta.-subunit mutations using a chromosomal delection strain

(1) We constructed Escherichia coli strain JP17 with a deletion in the ATP synthase beta-subunit gene. JP17 is completely deficient in ATP synthase activity and expresses no beta-subunit. Expression of normal beta-subunit from a plasmid restores haploid levels of ATP synthase in membranes. JP17 was shown to be efficacious for studies of beta-subunit mutations. Site-directed mutants were studied directly in JP17. Randomly generated chromosomal mutants were identified by PCR and DNA sequencing, cloned, and expressed in JP17. (2) Eight novel mutations occurring within the putative catalytic nucleotide-binding domain were characterized with respect to their effects on catalysis and structure. The mutations beta C137S, beta G152D, beta G152R, beta E161Q, beta E161R, and beta G251D each impaired catalysis without affecting enzyme assembly or oligomeric structure and are of interest for future studies of catalytic mechanism. The mutations beta D301V and beta D302V, involving strongly conserved carboxyl residues, caused oligomeric instability of F1. However, growth characteristics of these mutants suggested that neither carboxyl side chain is critical for catalysis. (3) The mutations beta R398C and beta R398W rendered ATP synthase resistant to aurovertin, giving strong support to the view that beta R398 is a key residue in the aurovertin-binding site. Neither beta R398C or beta R398W impaired catalysis significantly.

The Determination of Isotopically Labeled Species by FT-IR Difference Spectroscopy in the Study of Catalytic Reaction Mechanisms

The use of FT-IR gas-phase difference spectroscopy for the determination of isotopically labeled species in the study of catalytic reaction mechanisms is illustrated. Applications are based on the study of the mechanism of oxidative coupling of methane over oxide catalysts and include determinations of: (1) deuterated and partially deuterated ethylenes and ethanes from experiments with 2H labeling of the methane; (2) 18O contents of CO and CO2 from experiments with 18O labeling of O2, CO, and CO2; (3) 13C/12C ratios in hydrocarbons and carbon oxides from experiments with 13C labeling of C2 hydrocarbons. The major limitation of the technique is in regions of strong absorption where nonlinear effects make subtraction difficult; however, this limitation could be alleviated by judicious choice of pathlength. This technique is a powerful supplement to measurements based on mass spectrometry.

Interpretation of the rotational spectra of multideuteriated species of but-l-ene for the study of catalytic mechanisms by microwave spectroscopy

Each isotopic species of a given molecules presents a distinct rotational spectrum even if the difference is only in the position of substitution. This exceptional selectivity is taken advantage of in order to study the mechanisms of catalytic reactions, by introducing an isotopic label in the course of the reaction.The synthesis and the spectral interpretation of each isotopic species, which could appear in the final mixture, are two preliminary steps which we can avoid. It is possible to predict precisely the transition frequencies of each species as soon as some of them have been identified.The method described by the authors entails first a geo- metrical structure optimisation and subsquently a prediction of the rotational spectra of all species of interest. The judicious choice of the input information anti the resulting accuracy of the method are discussed.

Inactivation of some pancreatic and leucocyte elastases by peptide chloromethyl ketones and alkyl isocyanates

Several authors recently demonstrated that polymorphonuclear leucocytes from various mammalian species contain elastase-like proteases of rather broad specificity see [l-3]. The enzymes from human leucocytes are supposed to be involved in pathological processes such as acute arthritis, pulmonary emphysema and various inflammatory and immunological reactions [4-81. Synthetic inactivators of these enzymes might have some medical applications apart from being used in studies of biological functions and catalytic mechanism of elastases. Since leucocyte elastase-like proteases contain serine and histidine in the active centre [2,3] site-specific inactivators may be directed towards these two amino acid residues. Powers and Tuhy [9] synthesized a series of alanine chloromethyl ketones that bind irreversibly to the histidine residue in the active centre of pancreatic [9,10] and human granulocyte [ 12,131 elastases. Independently, Brown and Wold [ 14,151 introduced alkyl isocyanates as active site-specific reagents for studying the structure of substrate-binding pocket of serine proteases. They observed that butyl isocyanate preferentially inactivates pancreatic elastase [ 1.51 while we confirmed this for two horse leucocyte elastases [2] . Now we report here comparative studies on the kinetics of inactivation of two highly purified pancreatic elastases and four leucocyte proteases by