Small Molecule Substrates Research Articles

Formation of Hydride, Formyl, Hydroxymethyl, Dimetal Ketone, and Ethylene-Bridged Species from Small-Molecule Substrate Reactions with Rhodium Complexes of an N4 Nonmacrocyclic Ligand

Reactions of the rhodium(II) dimer [(dbpb)Rh]2 (1) ((dbpb)H2 (2) = 4,5-dimethyl-1,2-bis((4-(1-butylpentyl))pyridine-2-carboxamido)benzene) with H2, CO, and CH2CH2 produce a rhodium(III) hydride, (dbpb)Rh−H (3), dirhodium ketone, (dbpb)Rh−C(O)−Rh(dbpb) (4), and an ethylene-bridged complex, (dbpb)Rh−CH2CH2−Rh(dbpb) (5), respectively. The rhodium(III) hydride (3) reacts with CO and H2CO to produce formyl, (dbpb)Rh−CHO (6), and hydroxymethyl, (dbpb)Rh−CH2OH (7) complexes. Equilibrium thermodynamic studies for reactions of 1 with hydrogen and ethene and the observed substrate reactions of 1 and 3 indicate that the (dbpb)Rh−H and (dbpb)Rh−C bond dissociation enthalpies are comparable to those for rhodium porphyrin complexes.

Modeling of Axial-Flow Hollow Fiber Cell Culture Bioreactors

A review of the mathematical modeling of axial-flow hollow fiber cell bioreactors is presented. The models cover the range of diffusion-limited to convective-flow dominant nutrient transport conditions. Mass transport limitations inherent in the axial-flow bioreactor design are illustrated with model calculations. Simulation results further demonstrate the importance of convective nutrient transport to the cell growth region. These model developments were restricted to the transport and consumption of small substrate molecules in dilute solution. Finally, convection-enhanced hollow fiber bioreactor designs and alternative schemes for operating axial-flow bioreactors are discussed.

Altering substrate specificity at the heme edge of cytochrome c peroxidase.

Two mutants of cytochrome c peroxidase (CCP) are reported which exhibit unique specificities toward oxidation of small substrates. Ala-147 in CCP is located near the delta-meso edge of the heme and along the solvent access channel through which H2O2 is thought to approach the active site. This residue was replaced with Met and Tyr to investigate the hypothesis that small molecule substrates are oxidized at the exposed delta-meso edge of the heme. X-ray crystallographic analyses confirm that the side chains of A147M and A147Y are positioned over the delta-meso heme position and might therefore modify small molecule access to the oxidized heme cofactor. Steady-state kinetic measurements show that cytochrome c oxidation is enhanced 3-fold for A147Y relative to wild type, while small molecule oxidation is altered to varying degrees depending on the substrate and mutant. For example, oxidation of phenols by A147Y is reduced to less than 20% relative to the wild-type enzyme, while Vmax/e for oxidation of other small molecules is less affected by either mutation. However, the "specificity" of aniline oxidation by A147M, i.e., (Vmax/e)/Km, is 43-fold higher than in wild-type enzyme, suggesting that a specific interaction for aniline has been introduced by the mutation. Stopped-flow kinetic data show that the restricted heme access in A147Y or A147M slows the reaction between the enzyme and H202, but not to an extent that it becomes rate limiting for the oxidation of the substrates examined. The rate constant for compound ES formation with A147Y is 2.5 times slower than wild-type CCP. These observations strongly support the suggestion that small molecule oxidations occur at sites on the enzyme distinct from those utilized by cytochrome c and that the specificity of small molecule oxidation can be significantly modulated by manipulating access to the heme edge. The results help to define the role of alternative electron transfer pathways in cytochrome c peroxidase and may have useful applications in improving the specificity of peroxidase with engineered function.

An engineered cation site in cytochrome c peroxidase alters the reactivity of the redox active tryptophan.

The crystal structures of cytochrome c peroxidase and ascorbate peroxidase are very similar, including the active site architecture. Both peroxidases have a tryptophan residue, designated the proximal Trp, located directly adjacent to the proximal histidine heme ligand. During the catalytic cycle, the proximal Trp in cytochrome c peroxidase is oxidized to a cation radical. However, in ascorbate peroxidase, the porphyrin is oxidized, not the proximal Trp, despite the close similarity between the two peroxidase active site structures. A cation located approximately 8 A from the proximal Trp in ascorbate peroxidase but absent in cytochrome c peroxidase is thought to be one reason why ascorbate peroxidase does not form a Trp radical. Site-directed mutagenesis has been used to introduce the ascorbate peroxidase cation binding site into cytochrome c peroxidase. Crystal structures show that mutants now bind a cation. Electron paramagnetic resonance spectroscopy shows that the cation-containing mutants of cytochrome c peroxidase no longer form a stable Trp radical. The activity of the cation mutants using ferrocytochrome c as a substrate is < 1% of wild type levels, while the activity toward a small molecule substrate, guaiacol, increases. These results demonstrate that long range electrostatic effects can control the reactivity of a redox active amino acid side chain and that oxidation/reduction of the proximal Trp is important in the oxidation of ferrocytochrome c.

A three-dimensional protein model for human cytochrome P450 2D6 based on the crystal structures of P450 101, P450 102, and P450 108.

Cytochromes P450 (P450s) constitute a superfamily of phase I enzymes capable of oxidizing and reducing various substrates. P450 2D6 is a polymorphic enzyme, which is absent in 5-9% of the Caucasian population as a result of a recessive inheritance of gene mutations. This deficiency leads to impaired metabolism of a variety of drugs. All drugs metabolized by P450 2D6 contain a basic nitrogen atom, and a flat hydrophobic region coplanar to the oxidation site which is either 5 or 7 A away from the basic nitrogen atom. The aim of this study was to build a three-dimensional structure for the protein and more specifically for the active site of P450 2D6 in order to determine the amino acid residues possibly responsible for binding and/ or catalytic activity. Furthermore, the structural features of the active site can be implemented into the existing small molecule substrate model, thus enhancing its predictive value with respect to possible metabolism by P450 2D6. As no crystal structures are yet available for membrane-bound P450s (such as P450 2D6), the crystal structures of bacterial (soluble) P450 101 (P450cam), P450 102 (P450BM3), and P450 108 (P450terp) have been used to build a three-dimensional model for P450 2D6 with molecular modeling techniques. Several important P450 2D6 substrates were consecutively docked into the active site of the protein model. The energy optimized positions of the substrates in the protein agreed well with the original relative positions of the substrates within the substrate model. This confirms the usefulness of small molecule models in the absence of structural protein data. Furthermore, the derived protein model indicates new leads for experimental validation and extension of the substrate model.

Compensation effects in the complexation reactions of α‐ and β‐cyclodextrin

AbstractAnalysis of temperature dependent equilibrium constant data for the complexation of α‐cyclodextrin with approximately 70 different substrates demonstrates the existence of a general compensation effect. Enthalpies (kJ mol−1) and entropies (J K−1 mol−1) of complexation are related by ΔH = −5·70(±0·05) + [421·1(±1·2)]ΔS. A few substrates deviate from the general effect, possibly owing to reorganization of internal hydrogen bonding interactions. Complexation reactions involving β‐cyclodextrin are found to divide into two groups. Eleven small molecule or ion substrates undergo isoentropic complexations with ΔS = −5·7±2 J K−1 mol−1. Fourteen larger substrate species exhibit a compensation effect with ΔH = −23·5(±0·2) + [634(±26)]ΔS. The compensation behavior of the two cyclodextrins is discussed in terms of bonding mechanisms and cavity size.

In vitro oxidative damage to tissue-type plasminogen activator: a selective modification of the biological functions

Tissue-type plasminogen activator (t-PA) is an important component of the blood fibrinolytic system responsible for thrombus dissolution. It is often required to function under oxidative stress, and exogenous t-PA is used clinically in the treatment of acute myocardial infarction (AMI). The aim of this study was to examine alterations in the residual activity of t-PA pre-treated with certain oxidants. Recombinant t-PA (rt-PA) and native t-PA (nt-PA) were pre-treated with freshly generated hypochlorous acid (HOCl) and chloramine T at varying concentrations. The amidolytic activity, the plasminogenolytic activity and the fibrin-binding affinity were then examined using chromogenic assays based on S-2288 and S-2251. The amidolytic activity of t-PA was surprisingly found to be rather sensitive (IC50 1 and 12 mumol/1, respectively), and the plasminogenolytic activity rather resistant to pre-treatment with HOCl and chloramine T. The fibrin binding study of treated t-PA revealed substantial loss of binding to CNBr-digested fibrinogen (FDP-CNBr). The velocity of t-PA in reaction with plasminogen remained the same as non-treated t-PA. The possible mechanisms of this asymmetrical oxidative modification of the biological functions are also discussed. (1) The catalytic activity of t-PA and the binding affinity for its large-molecule substrate plasminogen, rather than the small-molecule substrate S-2288, are highly resistant to oxidative damage; (2) the fibrin-binding affinity of t-PA can be selectively and asymmetrically damaged by exposure to these oxidants. Thus it is possible that the characteristic advantage of thrombus selectivity of t-PA in both spontaneous thrombolysis and thrombolytic therapy may be diluted in circumstances where toxic and reactive oxidants exist.

Random mutagenesis of staphylococcal nuclease and phage display selection

Multiple cycles of mutagenesis and phage display selection have been investigated as a method for obtaining enzymes with altered catalytic properties. A library of staphylococcal nuclease mutants displayed on phage was created by errorprone PCR mutagenesis and selected for binding to thymidine- or guanosine-containing substrate analogs. After discarding non-binders, the binding mutants were then subjected to further mutagenesis and selection rounds. After four mutagenesis and selection cycles, the catalytic properties of some of the resulting nucleases were studied and one nuclease with nine accumulated mutations was found to have a two-fold reduction in k cat for DNA hydrolysis, but a two-fold increase in k cat/ K m for hydrolysis of a thymidine containing small molecule substrate. The possibility of this technique for in vitro evolution of enzyme properties is discussed.

Molecular recognition of receptor sites using a genetic algorithm with a description of desolvation

Understanding the principles whereby macromolecular biological receptors can recognise small molecule substrates or inhibitors is the subject of a major effort. This is of paramount importance in rational drug design where the receptor structure is known (the "docking" problem). Current theoretical approaches utilise models of the steric and electrostatic interaction of bound ligands and recently conformational flexibility has been incorporated. We report results based on software using a genetic algorithm that uses an evolutionary strategy in exploring the full conformational flexibility of the ligand with partial flexibility of the protein, and which satisfies the fundamental requirement that the ligand must displace loosely bound water on binding. Results are reported on five test systems showing excellent agreement with experimental data. The design of the algorithm offers insight into the molecular recognition mechanism.

An Enzymatic Activity in Bovine Brain That Catalyzes the Reversal of the C-Terminal Methyl Esterification of Protein Phosphatase 2A

A novel protein methyltransferase has been recently described that catalyzes the esterification of the C-terminal leucine residue of the catalytic subunit of protein phosphatase 2A in a variety of eucaryotic cells. This reaction can potentially modulate the phosphatase′s activity, subunit interactions, or interactions with specific phosphoprotein substrates. We present evidence here that the methylation reaction is reversible and that an enzymatic activity is present in bovine brain cytosol that catalyzes the hydrolysis of the methyl ester. We show that this activity is sensitive to inhibition by the serine-hydrolase inhibitor phenylmethanesulfonyl fluoride but is not affected by the small molecule substrate analog N-acetyl-L-leucine methyl ester. These results suggest that protein methylation and demethylation reactions can be utilized in eucaryotic cells to modulate enzyme activity in a parallel fashion to protein phosphorylation and dephosphorylation reactions.

Pseudomonas exotoxin A mutants. Replacement of surface-exposed residues in domain III with cysteine residues that can be modified with polyethylene glycol in a site-specific manner.

Pseudomonas exotoxin A (PE) is composed of three structural and functional domains. Domain Ia is responsible for cell recognition, domain II for translocation of PE across the cell membrane, and domain III for ADP-ribosylation of elongation factor 2. To investigate the role of the amino acids exposed on the surface of domain III, we replaced 15 of these, generating 29 different mutants at positions 412, 416, 418, 490, 513, 516, 522, 551, 576, 590, 599, 604, 606, 607 and 608. All but one mutant retained substantial ADP-ribosylation and cytotoxic activities. Modification of proteins with monomethoxy-polyethylene glycol (mPEG) prolongs their circulation in the blood stream and reduces their immunogenicity. Unlike PEGylated enzymes acting on small molecule substrates, PEGylated toxins may lose those functions that are based on macromolecular interactions. Therefore, we selectively PEGylated mutant PEs at positions 490, 513, 516, 522, 604, and 606. Most PEs modified by a 5-kDa mPEG via a disulfide or a thioether bond retained high cytotoxic activity. However, when a 20-kDa mPEG was used there was a decrease in cytotoxic activity with the disulfide-bonded molecules being more active. Positions 522 and 604 are good sites for PEGylation, but 490 is not. We also found that PEGylation of PE 522C prolonged its in vivo circulation time in mice.

Role of porins in outer membrane permeability

It is now well recognized that the outer membranes of gram-negative bacteria serve as molecular sieves which permit the passage of small hydrophilic molecules of sizes below a given cutoff, i.e., the exclusion limit (21, 27). This sieving property, which permits uptake of small substrate molecules but excludes potentially harmful enzymes and other large hydrophilic molecules, is due to a class of proteins called porins (21). Porins form trans-outermembrane, water-filled channels, the dimensions of which determine the exclusion limit. An indication of their importance in bacteria is the observaton that Spirochaeta aurantia, an organism which apparently lacks lipopolysaccharide in its outer membrane (also called its outer sheath) (A. Kropinski and E. P. Greenberg, personal communication), nevertheless contains a major porin protein (19) whose properties are closely related to those of, e.g., Escherichia coli (7). Porins have now been identified and characterized for 32 species of bacteria (R. E. W. Hancock, in M. Inouye, ed., Bacterial Outer Membranes as Model Systems, in

ChemInform Abstract: Organometallic Lanthanide Chemistry

Publisher Summary This chapter discusses organolanthanide chemistry. Organolanthanide chemistry currently is one of the most rapidly developing areas of organometallic chemistry. The lanthanide elements are differentiated from other metallic elements by the fact that their valence electrons are in 4f orbitals. The highly ionic, trivalent organolanthanide complexes appeared to have little potential to interact with small-molecule substrates that provide such a rich chemistry for the transition metals. The synthesis, structure, and physical and chemical properties of organolanthanide complexes containing only polyhaptocyclopentadienyl and cyclooctatetraenyl metal–carbon bonds are covered in other reviews. For many years, the only fully characterized homoleptic organolanthanide complex and the only structurally characterized Ln–C species of any type was the tetrakis complex. One of the most recent and rapidly developing areas of organolanthanide chemistry involves species containing Ln–H bonds. The first evidence for the existence of organolanthanide hydride molecules arose from a study of catalytic hydrogenation by lanthanide metal vapor reaction products. There are two low-valent oxidation states available to the lanthanides under normal conditions: the +2 oxidation state and the formally zero oxidation state found in the elemental metals. The pentamethylcyclopentadienyl ligand, C5Me5, has proven to be of great importance in organometallic chemistry. The thesis that the lanthanide elements offer something unique to organometallic chemistry has been proven in a variety of ways.

A Brownian dynamics simulation of enzyme–substrate encounters at the surface of a colloidal particle

The numerical technique of Brownian dynamics is used to model diffusion-controlled encounters between a small spherical enzyme particle and substrate molecules (‘hot spots’) distributed randomly on the surface of a larger spherical colloidal particle. Reaction occurs when any spot gets within a certain specified distance from the enzyme's active site. Specimen results are presented for the number and distribution of reacted spots as a function of time. The simulation gives information on the effect on the kinetics of interparticle hydrodynamic interactions, the effective size of the active site, and the range of available diffusional space around the colloidal particle.

Contributions of Histochemistry to the Development of the Proteolytic Enzyme Detection System in Diagnostic Medicine 12.

Although the place of enzyme-catalyzed reactions in clinical biochemistry has proven to be of diagnostic value since the late 1950s (16,17), it is only within the last 6 years that methods for determining the activity of proteases have been introduced into the clinical laboratory. Proteolytic enzymes are assuming increasing significance in biological regulation. An understanding of their physiological and pathological roles has come from both biochemical and histochemical standpoints. Proteases, unlike most hydrolases, the general class of enzymes to which they belong, are highly specific and precisely controlled. Furthermore, when this precise control of proteolysis breaks down, the consequences are usually serious. It has long been recognized by physiological chemists that many disease states represent abnormalities of protein synthesis or degradation resulting from disturbances in the activity of proteases or their inhibitors. First attempts at assaying for specific proteases by preparing synthetic substrates were made by Fruton and Bergmann in the 1930s. They found that crude preparations from bovine spleen and partly purified preparations of pepsin and trypsin could hydrolyze small synthetic substrate molecules with free amines. The task for assaying protease activity was greatly simplified when in 1950, Neurath and Schwert demonstrated that amino acid esters were very sensitive substrates. Such substrates were used by Sherry and Troll (58) and associates in the mid-1950s to study clotting and fibrinolytic enzymes. It was Gomori, in 1954, who used chloroacetyl-/3-naphthylamine with ammonia to obtain glycine and D,L-alanyl-/3-naphthyl-

Binuclear complexes of macrocyclic schiff base ligands as hosts for small substrate molecules

Reaction of 2,6-diacetylpyridine or 2,5-diformylpyridine with a variety of diprimary amines in the presence of large alkaline earth metal ions acting as templates yields complexes of [2 + 2 ] macrocyclic Schiff bases in good yield. The single template ion may be replaced by Cu(II), and other transition metal ions, to afford binuclear complexes in most cases. The bi-Cu(II) complexes may act as receptors for one or two small bridging substrate molecules or ions including OH −, OR −, halide, N − 3, NCS −, NCSe −, pyrazolate, imidazolate, and pyrazine. Anti-ferromagnetic superexchange coupling has been recognized and measured by means of measurements of magnetic susceptibility and e.s.r. spectra. X-ray crystal structures of several of the complexes are described. The relationships between the macrocycle cavity size and the nature and mode of attachment of the bridging substrate are discussed. Some di-μ-hydroxo and alkoxo bi-Cu(II) complexes undergo ready reduction to bi-Cu(I) with accompanying oxidative coupling of available substrate molecules. The structure of a tetranuclear Cu(I) cluster containing phenylacetylide is described.

Microencapsulation of Chloroplast Particles

Chloroplast and photosystem I particles were encapsulated in small spheres (about 20 mum diameter) with an artificial membrane built up by cross-linking amino groups of protamine with toluenediisocyanate. The artificial membrane was permeable to small substrate and product molecules but not to soluble proteins. Photosystem I activity was retained by the encapsulated chloroplast particles. Washed photosystem I particles were encapsulated with the soluble proteins, ferredoxin, and ferredoxin-NADP oxidoreductase, and the microcapsules photoreduced NADP using ascorbate plus dichlorophenolindophenol as the electron donor. The photosystem I particles were also encapsulated with hydrogenase from Chromatium and a very low rate of photoevolution of hydrogen was obtained. The results show that chloroplast membrane fragments can be encapsulated with soluble proteins that couple transfer reactions to the primary photochemical apparatus.

Many particle systems with binding: Phase transition and chemical reaction

With the help of some formal considerations and some approximations it is shown that binding of small substrate molecules to macromolecules is a phenomenon which can be interpreted either as phase transition or as chemical reaction. The given method can be used to calculate the temperature dependence of saturation curves, e.g. for the hemoglobinoxygen system. So it might be possible to decide which model of the macromolecule is the most realistic one.

Specificity of thrombin: I. Esterolytic properties of thrombin, plasma, trypsin, and chymotrypsin with N β-substituted guanidino derivatives of p-nitrophenyl- p′-guanidinobenzoate

Thrombin exhibits a restricted specificity, relative to plasmin, trypsin, and chymotrypsin, for a series of derivatives of the titrant substrate p-nitrophenyl- p′-guanidinobenzoate (NPGB). Substitution on the beta guanidino nitrogen of NPGB with an n-butyl, n-hexyl, cyclo-hexyl, or benzyl residue does not prevent the esterolytic cleavage of these derivatives but does markedly alter their substrate properties with the four enzymes investigated. All four enzymes cleave NPGB at equivalent concentrations by releasing p-nitrophenol as pre-steady-state burst reactions followed by its steady-state production. Both chymotrypsin and trypsin similarly display burst reactions with the derivatives at corresponding concentrations. The acyl-enzyme intermediates formed with chymotrypsin, however, are more stable for the derivatives than for NPGB, and those formed with trypsin are less stable. In contrast, plasmin and thrombin exhibit incomplete burst reactions with the derivatives at these concentrations. Except for the cyclo-hexyl derivative, with which plasmin does not react, the derivatives relative to NPGB were cleaved faster by plasmin than by thrombin. These cleavages with thrombin, moreover, were competitively inhibited by benzamidine. Kinetic data obtained for thrombin further indicated that the substituent groups of derivatives hindered the initial formation of enzyme-substrate complexes. These results suggest that thrombin and, most likely, plasmin have restricted primary binding-site regions for small molecule substrates which do not readily accommodate bulky substituent groups. In addition, increasing concentrations of glycerol were found to greatly alter the esterolytic properties of thrombin for the compounds studied. This effect was demonstrated by increased deacylation rates with NPGB and by decreased cleavage rates with the n-butyl derivative.

Cocoonases of silkworm moths: Catalytic properties and biological function

The cocoonases from several silkmoths and bovine trypsin have similar reactivities towards several small-molecule substrates and inhibitors. These include ester and amide substrates, a substrate analogue (TLCK), and a phosphate ester inhibitor (DFP). Trypsin-like substrate activation was also observed with one cocoonase. The enzymes from Antheraea polyphemus and Bombyx mori react with labelled DFP and give, on acid hydrolysis, patterns of labelled peptides similar to those from labelled DIP trypsin and chymotrypsin, thus indicating considerable homology in the active site region of the cocoonases and the pancreatic proteinases. Trypsin and A. polyphemus cocoonase have qualitatively similar but quantitatively different action on polypeptides, with trypsin being more effective. Cocoonase is necessary for effective escape of silkmoths from the cocoon, which can be accomplished from the anterior end with ease and, in a few instances, from the posterior end with difficulty.