Quantitative analysis of the time courses of enzyme-catalyzed reactions

The catalytic properties of enzymes are usually evaluated by measuring and analyzing reaction rates. However, analyzing the complete time course can be advantageous because it contains additional information about the properties of the enzyme. Moreover, for systems that are not at steady state, the analysis of time courses is the preferred method. One of the major barriers to the wide application of time courses is that it may be computationally more difficult to extract information from these experiments. Here the basic approach to analyzing time courses is described, together with some examples of the essential computer code to implement these analyses. A general method that can be applied to both steady state and non-steady-state systems is recommended. (C) 2001 academic Press.

A potential role for isothermal calorimetry in studies of the effects of thermodynamic non-ideality in enzyme-catalyzed reactions

Attention is drawn to the feasibility of using isothermal calorimetry for the characterization of enzyme reactions under conditions bearing greater relevance to the crowded biological environment, where kinetic parameters are likely to differ significantly from those obtained by classical enzyme kinetic studies in dilute solution. An outline of the application of isothermal calorimetry to the determination of enzyme kinetic parameters is followed by considerations of the nature and consequences of crowding effects in enzyme catalysis. Some of those effects of thermodynamic non-ideality are then illustrated by means of experimental results from calorimetric studies of the effect of molecular crowding on the kinetics of catalysis by rabbit muscle pyruvate kinase. This review concludes with a discussion of the potential of isothermal calorimetry for the experimental determination of kinetic parameters for enzymes either in biological environments or at least in media that should provide reasonable approximations of the crowded conditions encountered in vivo. Copyright (C) 2004 John Wiley Sons, Ltd.

Synthesis and stereochemistry of some bicyclic gamma-lactones from parasitic wasps (Hymenoptera : Braconidae). Utility of hydrolytic kinetic resolution of epoxides and palladium(II)-catalyzed hydroxycyclization-carbonylation-lactonization of ene-diols

A palladium(II)-catalyzed hydroxycyclization-carbonylation-lactonization sequence with appropriate pent-4-ene-1,3-diols provides efficient access to the bicyclic gamma -lactones, 5-n-butyl- and 5-n-hexyltetrahydrofuro-[3,2-b]furan-2(3H)-ones (3) and (4), respectively, in both racemic and enantiomeric forms. Some of the substrate pent-4-ene-1,3-diols of high enantiomeric excess (ee) have been derived from racemic terminal epoxides by hydrolytic kinetic resolution (HKR) using cobalt (III)-salen complexes. (9Z,12R)-(+)-Ricinoleic acid also serves as a chiral pool source of other pent-4-ene-1,3-diols. These syntheses and enantioselective gas chromatography confirm the structures and absolute stereochemistry of the lactones in some species of parasitic wasps (Hymenoptera: Braconidae). The highly abundant 5-n-hexyltetrahydrofuro-[3,2-b]furan-2(3H)-one (4) in Diachasmimorpha kraussii and D. longicaudata is of high ee (> 99%) with (3aR,5R,6aR) stereochemistry.

p-cresol as a reversible acylium ion scavenger in solid-phase peptide synthesis

In this work we have defined the nature of the p-cresol and p-thiocresol adducts generated from acylium ions during HF cleavage, following contemporary Boc/benzyl solid-phase peptide synthesis. Contrary to the results in previous reports, we found that both p-cresol and p-thiocresol predominantly form. aryl esters under typical cleavage conditions. Initially we investigated a number of small peptides containing either a single glutamate residue or a C-terminal long-chain amino acid which allowed us to unambiguously characterize the scavenged side products. Whereas, the p-cresol esters are stable at 0 degrees C they rearrange irreversibly at higher temperatures (5-20 degrees C) to form aryl ketones. By contrast, p-thiocresol esters do not undergo a Fries rearrangement but readily undergo further additions of p-thiocresol to form ketenebisthioacetals and trithio ortho esters, even at low temperatures. Importantly, we found by LC/MS and FT-ICR MS analysis that peptides containing p-cresol esters at glutamyl side chains are susceptible to amidation and fragmentation reactions at these sites during standard mild base workup procedures. The significance of these side reactions was further demonstrated in the synthesis of neutrophil immobilization factor, a 26-residue peptide, containing four glutamic acid residues. The side reactions were largely avoided by mild hydrogen peroxide-catalyzed hydrolysis which converted the p-cresol adducts to the free carboxylic acids in near quantitative yield. The choice of p-cresol as a reversible acylium ion scavenger when coupled with the simple workup conditions described is broadly applicable to Boc/benzyl peptide synthesis and will significantly enhance the quality of peptides produced.

Synthesis of hydroperoxide and perketal derivatives of polyunsaturated fatty acids as potential antimalarial agents

Hydroperoxide derivatives of beta-oxa-substituted polyunsaturated fatty acids were prepared by 15-lipoxygenase catalysed oxidation and perketal derivatives of fatty acid hydroperoxides were synthesized. The perketals are more stable than their parent fatty acid hydroperoxides, but less active as antimalarial agents in the in vitro growth inhibition of Plasmodium falciparum. (C) 1998 Elsevier Science Ltd. All rights reserved.

Synthesis of fructans by partially purified fructosyltransferase activities from Lolium rigidum

Sucrose:sucrose fructosyltransferase (SST) activity was partially purified from whole shoots of Lolium rigidum by a combination of affinity chromatography, gel filtration and anion-exchange chromatography. The SST activity co-eluted with some fructan:fructan fructosyltransferase (FFT) and invertase activities and consequently the partially purified preparation was termed the fructosyltransferase (FT) preparation. The SST-like activity in the FT preparation was purified 214-fold and had an apparent molecular mass of 84 000. The FT preparation contained several peptides with an apparent pI of 4.6-4.7. When assayed with sucrose concentrations up to 600 mM, the FT preparation synthesized 1-kestose at all concentrations, and synthesized 6-kestose at concentrations of 150 mM and greater. The K-m of 1-kestose production was 0.2 M. When the FT preparation was assayed at a concentration of activity approximately half that measured in fresh tissue with 100 mM sucrose, 1-kestose, or 6(G)-kestose as substrates, fructans of degree of polymerization (DP) less than or equal to 5 were synthesized. A partially purified FFT activity, free of SST and invertase activities, which synthesized beta-2,1-glycosidic linked oligofructans of DP less than or equal to 6, was combined in vitro with the FT preparation (FFT-FT preparation) to give a ratio of SST:FFT activities similar to that measured in crude enzyme extracts from L. rigidum. The FFT-FT preparation synthesized oligofructans when assayed with 100 mM concentrations of sucrose, 1-kestose or 6(G)-kestose, but was not able to synthesize fructans of DP greater than or equal to 6 even after extended assays of up to 10 h. The FFT-FT preparation was also assayed with 100 mM sucrose with small amounts of concentrated sucrose added periodically during the assay to maintain the substrate concentration. In this assay, the FFT-FT preparation synthesized fructans up to an apparent DP of 17 or greater. The fructans of DP greater than or equal to 6 synthesized in the assay appeared to form two molecular series containing both beta-2,1- and beta-2,6-glycosidic linked fructosyl residues with terminal or internal glucosyl residues. The apparent rate of SST activity in the assay of the FFT-FT preparation was greater than that measured in a similar assay of the FT preparation alone which did not result in fructans with DP greater than or equal to 6. It was concluded that the FFT-FT preparation, when assayed with a continual supply of sucrose, contained a factor which promoted synthesis of fructans of DP greater than or equal to 6 and synthesis of beta-2,B-glycosidic linkages between fructosyl residues.

Enamel matrix derivative induces matrix synthesis by cultured human periodontal fibroblast cells

Background: Periodontal wound healing and regeneration require that new matrix be synthesized, creating an environment into which cells can migrate. One agent which has been described as promoting periodontal regeneration is an enamel matrix protein derivative (EMD). Since no specific growth factors have been identified in EMD preparations, it is postulated that EMD acts as a matrix enhancement factor. This study was designed to investigate the effect of EMD in vitro on matrix synthesis by cultured periodontal fibroblasts. Methods: The matrix response of the cells was evaluated by determination of the total proteoglycan synthesis, glycosaminoglycan profile, and hyaluronan synthesis by the uptake of radiolabeled precursors. The response of the individual proteoglycans, versican, decorin, and biglycan were examined at the mRNA level by Northern blot analysis. Hyaluronan synthesis was probed by identifying the isotypes of hyaluronan synthase (HAS) expressed in periodontal fibroblasts as HAS-2 and HAS-3 and the effect of EMD on the levels of mRNA for each enzyme was monitored by reverse transcription polymerase chain reaction (RTPCR). Comparisons were made between gingival fibroblast (GF) cells and periodontal ligament (PDLF) cells. Results: EMD was found to significantly affect the synthesis of the mRNAs for the matrix proteoglycans versican, biglycan, and decorin, producing a response similar to, but potentially greater than, mitogenic cytokines. EMD also stimulated hyaluronan synthesis in both GF and PDLF cells. Although mRNA for HAS-2 was elevated in GF after exposure to EMD, the PDLF did not show a similar response. Therefore, the point at which the stimulation of hyaluronan becomes effective may not be at the level of stimulation of the mRNA for hyaluronan synthase, but, rather, at a later point in the pathway of regulation of hyaluronan synthesis. In all cases, GF cells appeared to be more responsive to EMD than PDLF cells in vitro. Conclusions: EMD has the potential to significantly modulate matrix synthesis in a manner consistent with early regenerative events.

Getting the adrenaline going: Crystal structure of the adrenaline-synthesizing enzyme PNMT

Background: Adrenaline is localized to specific regions of the central nervous system (CNS), but its role therein is unclear because of a lack of suitable pharmacologic agents. Ideally, a chemical is required that crosses the blood-brain barrier, potently inhibits the adrenaline-synthesizing enzyme PNMT, and does not affect other catecholamine processes. Currently available PNMT inhibitors do not meet these criteria. We aim to produce potent, selective, and CNS-active PNMT inhibitors by structure-based design methods. The first step is the structure determination of PNMT. Results: We have solved the crystal structure of human PNMT complexed with a cofactor product and a submicromolar inhibitor at a resolution of 2.4 Angstrom. The structure reveals a highly decorated methyltransferase fold, with an active site protected from solvent by an extensive cover formed from several discrete structural motifs. The structure of PNMT shows that the inhibitor interacts with the enzyme in a different mode from the (modeled) substrate noradrenaline. Specifically, the position and orientation of the amines is not equivalent. Conclusions: An unexpected finding is that the structure of PNMT provides independent evidence of both backward evolution and fold recruitment in the evolution of a complex enzyme from a simple fold. The proposed evolutionary pathway implies that adrenaline, the product of PNMT catalysis, is a relative newcomer in the catecholamine family. The PNMT structure reported here enables the design of potent and selective inhibitors with which to characterize the role of adrenaline in the CNS. Such chemical probes could potentially be useful as novel therapeutics.

Crystallization of PNMT, the adrenaline-synthesizing enzyme, is critically dependent on a high protein concentration

Phenylethanolamine N-methyltransferase, PNMT, utilizes the methylating cofactor S-adenosyl-L-methionine to catalyse the synthesis of adrenaline. Human PNMT has been crystallized in complex with an inhibitor and the cofactor product S-adenosyl-L-homocysteine using the hanging-drop technique with PEG 6000 and lithium chloride as precipitant. A critical requirement for crystallization was a high enzyme concentration (>90 mg ml(-1)) and cryocrystallography was used for high-quality data measurement. Diffraction data measured from a cryocooled crystal extend to a resolution of 2.3 Angstrom. Cryocooled crystals belong to space group P4(3)2(1)2 and have unit-cell parameters a = b = 94.3, c = 187.7 Angstrom.

Thermodynamic non-ideality as an alternative source of the effect of sucrose on the thrombin-catalyzed hydrolysis of peptide p-nitroanalide substrates

The inhibitory effect of sucrose on the kinetics of thrombin-catalyzed hydrolysis of the chromogenic substrate S-2238 (D-phenylalanyl-pipecolyl-arginoyl-p-nitroanilide) is re-examined as a possible consequence of thermodynamic non-ideality-an inhibition originally attributed to the increased viscosity of reaction mixtures. However, those published results may also be rationalized in terms of the suppression of a substrate-induced isomerization of thrombin to a slightly more expanded (or more asymmetric) transition state prior to the irreversible kinetic steps that lead to substrate hydrolysis. This reinterpretation of the kinetic results solely in terms of molecular crowding does not signify the lack of an effect of viscosity on any reaction step(s) subject to diffusion control. Instead, it highlights the need for development of analytical procedures that can accommodate the concomitant operation of thermodynamic non-ideality and viscosity effects.

Synthesis, stability, antiviral activity, and protease-bound structures of substrate-mimicking constrained macrocyclic inhibitors of HIV-1 protease

Three new peptidomimetics (1-3) have been developed with highly stable and conformationally constrained macrocyclic components that replace tripeptide segments of protease substrates. Each compound inhibits both HIV-1 protease and viral replication (HIV-I, HIV-2) at nanomolar concentrations without cytotoxicity to uninfected cells below 10 mu M. Their activities against HIV-1 protease (K-i 1.7 nM (1), 0.6 nM (2), 0.3 nM (3)) are 1-2 orders of magnitude greater than their antiviral potencies against HIV-1-infected primary peripheral blood mononuclear cells (IC50 45 nM (1), 56 nM (2), 95 nM (3)) or HIV-1-infected MT2 cells (IC50 90 nM (1), 60 nM (2)), suggesting suboptimal cellular uptake. However their antiviral potencies are similar to those of indinavir and amprenavir under identical conditions. There were significant differences in their capacities to inhibit the replication of HIV-1 and HIV-2 in infected MT2 cells, 1 being ineffective against HIV-2 while 2 was equally effective against both virus types. Evidence is presented that 1 and 2 inhibit cleavage of the HIV-1 structural protein precursor Pr55(gag) to p24 in virions derived from chronically infected cells, consistent with inhibition of the viral protease in cells. Crystal structures refined to 1.75 Angstrom (1) and 1.85 Angstrom (2) for two of the macrocyclic inhibitors bound to HIV-1 protease establish structural mimicry of the tripeptides that the cycles were designed to imitate. Structural comparisons between protease-bound macrocyclic inhibitors, VX478 (amprenavir), and L-735,524 (indinavir) show that their common acyclic components share the same space in the active site of the enzyme and make identical interactions with enzyme residues. This substrate-mimicking minimalist approach to drug design could have benefits in the context of viral resistance, since mutations which induce inhibitor resistance may also be those which prevent substrate processing.

Products of Cytochrome P450BioI (CYP107H1)-Catalyzed Oxidation of Fatty Acids

[GRAPHICS] Oxidation of tetradecanoic and hexadecanoic acids by cytochrome P450(Biol) (CYP107H1) produces mainly the 11-, 12-, and 13-hydroxy C-14 fatty acids and the 11- to 15-hydroxy C-16 fatty acids, respectively. In contrast to previous reports, terminal hydroxylation is not observed. The enantiospecificity of fatty acid hydroxylation by P450(Biol) was also determined, and the enzyme was shown to be moderately selective for production of the (R)-alcohols.

Molecular analysis of the pathway for the synthesis of thiol tripeptides in the model legume Lotus japonicus

The thiol tripeptides, glutathione (GSH) and homoglutathione (hGSH), perform multiple roles in legumes, including protection against toxicity of free radicals and heavy metals. The three genes involved in the synthesis of GSH and hGSH in the model legume, Lotus japonicus, have been fully characterized and appear to be present as single copies in the genome. The gamma-glutamylcysteine synthetase (gammaecs) gene was mapped on the long arm of chromosome 4 (70.0 centimorgans [cM]) and consists of 15 exons, whereas the glutathione synthetase (gshs) and homoglutathione synthetase (hgshs) genes were mapped on the long arm of chromosome 1 (81.3 cM) and found to be arranged in tandem, with a separation of approximately 8 kb. Both genes consist of 12 exons of exactly the same size (except exon 1, which is similar). Two types of transcripts were detected for the gshs gene, which putatively encode proteins localized in the plastids and cytosol. Promoter regions contain cis-acting regulatory elements that may be involved in the plant's response to light, hormones, and stress. Determination of transcript levels, enzyme activities, and thiol contents in nodules, roots, and leaves revealed that gammaecs and hgshs are expressed in all three plant organs, whereas gshs is significantly functional only in nodules. This strongly suggests an important role of GSH in the rhizobia-legume symbiosis.

Temperature dependence of the thrombin-catalyzed proteolysis of prothrombin

Measurement of the temperature-dependence of thrombin-catalyzed cleavage of the Arg(155)-Ser(156) and Arg(284)-Thr(285) peptide bonds in prothrombin and prothrombin-derived substrates has yielded Arrhenius parameters that are far too large for classical mechanistic interpretation in terms of a simple hydrolytic reaction. Such a difference from the kinetic behavior exhibited in trypsin- and chymotrypsin-catalyzed proteolysis of peptide bonds is attributed to contributions by enzyme exosite interactions as well as enzyme conformational equilibria to the magnitudes of the experimentally determined Arrhenius parameters. Although the pre-exponential factor and the energy of activation deduced from the temperature-dependence of rate constants for proteolysis by thrombin cannot be accorded the usual mechanistic significance, their evaluation serves a valuable role by highlighting the existence of contributions other than those emanating from simple peptide hydrolysis to the kinetics of proteolysis by thrombin and presumably other enzymes of the blood coagulation system. (C) 2004 Elsevier B.V. All rights reserved.