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PubMed1985

Biosynthesis of monoterpenes: demonstration of a geranyl pyrophosphate:(-)-bornyl pyrophosphate cyclase in soluble enzyme preparations from tansy (Tanacetum vulgare).

Tansy (Tanacetum vulgare L.) produces an essential oil containing the optically pure monoterpene ketone, (-)-camphor, as a major constituent. A soluble enzyme preparation from immature leaves of this plant converts the acyclic precursor [1-3H]geranyl pyrophosphate to the bicyclic monoterpene alcohol borneol in the presence of MgCl2, and oxidizes a portion of the borneol to camphor in the presence of a pyridine nucleotide. The identity of the major biosynthetic product as borneol was confirmed by chemical oxidation to camphor and crystallization of the derived oxime to constant specific radioactivity. The stereochemistry of the borneol was verified as the (-)-(1S,4S) isomer by oxidation to camphor, conversion to the corresponding ketal with D-(-)-2,3-butanediol, and separation of diastereoisomers by radio-gas-liquid chromatography. When enzyme reaction mixtures were treated with a mixture of acid phosphatase and apyrase, following an initial ether extraction of labeled borneol, additional quantities of borneol were generated, indicating the presence of a phosphorylated derivative of borneol. This water-soluble metabolite was prepared by large-scale enzyme incubations with [1-3H]geranyl pyrophosphate (plus phosphatase inhibitor), and the identity of the initial cyclization product was established as (-)-bornyl pyrophosphate by direct ion-exchange chromatographic analysis and enzymatic hydrolysis. The pathway for the formation of (-)-(1S,4S)-camphor was therefore identical to that previously demonstrated for the (+)-(1R,4R) isomer, involving cyclization of geranyl pyrophosphate to bornyl pyrophosphate, hydrolysis of this intermediate to borneol, and oxidation of the alcohol to the ketone. The labeling pattern of the product derived from [1-3H2, U-14C]geranyl pyrophosphate was determined by oxidation of the biosynthetic borneol to camphor and selective removal of tritium by exchange of the alpha hydrogens at C3 of the ketone. This labeling pattern was identical to that observed previously for the (+) isomer, suggesting the same mechanism of cyclization, but of opposite enantiospecificity. Some properties of the antipodal (+)- and (-)-bornyl pyrophosphate cyclases were compared.

Camphor↗
PubMed2003

Phenotypic characterization of overexpression or deletion of the Escherichia coli crcA, cspE and crcB genes.

The authors have previously shown that overexpression of the Escherichia coli K-12 crcA, cspE and crcB genes protects the chromosome from decondensation by camphor. In this study they examine the phenotypic consequences of deleting or overexpressing crcA, cspE and crcB. Overexpressing crcA, cspE and crcB increases supercoiling levels of plasmids in wild-type cells and in temperature-sensitive (Ts) gyrase mutants, suppresses the sensitivity of gyrase and topoisomerase IV (topo IV) Ts mutants to nalidixic acid, makes gyrase and topo IV Ts mutants more resistant to camphor and corrects the nucleoid morphology defects in topo IV Ts mutants. Overexpression of crcA, cspE and crcB results in a slight (2.2-fold) activation of the rcsA gene. Deleting crcA, cspE and crcB is not lethal to cells but results in an increase in sensitivity to camphor. Deletion of crcA, cspE and crcB exacerbates the nucleoid morphology defects of the topo IV Ts mutants. When the individual crcA, cspE or crcB genes were tested for their effects on camphor resistance and regulation of rcsA, cspE alone conferred 10-fold camphor resistance and 1.7-fold activation of rcsA. These activities were augmented when crcB was overexpressed with cspE (100-fold camphor resistance and 2.1-fold induction of rcsA).

Base Sequence↗
PubMed1984

Novel reactivity of cytochrome P-450-CAM. Methyl hydroxylation of 5,5-difluorocamphor.

The interaction of the camphor hydroxylating P-450 isolated from Pseudomonas putida grown on camphor (P-450-CAM) with 5,5-difluorocamphor, a substrate analog in which the two methylene hydrogens at the normal site of hydroxylation have been replaced with fluorine, has been examined. This compound binds tightly to the enzyme with a dissociation constant and UV-visible absorption spectrum identical to that observed with d-camphor. In the presence of the reconstituted P-450-CAM system, 5,5-difluorocamphor is metabolized at a rate approximately one-third the rate of the physiological substrate, d-camphor, resulting in the formation of a hydroxylated product with a molecular weight of 204 as well as a minor (less than 3%) hydroxylated product of molecular weight 184. Isotopically labeled molecular oxygen (18O2) is incorporated into the major product while labeled oxygen from water (H218O) is not incorporated, clearly indicating that the hydroxyl oxygen originates from dioxygen. Proton NMR characterization (400 MHz) of the major product has led to its assignment as 5,5-difluoro-9-hydroxy-camphor, with supporting structural evidence provided by the mass spectral fragmentation pattern. The formation of 9-hydroxylated product represents the first example of methyl hydroxylation catalyzed by cytochrome P-450-CAM, indicates a change in regio-selectivity when the normal site of reaction is blocked, and supports the hypothesis that the delivery of the oxygen atom occurs from the exo side of the camphor molecule.

Camphor↗
PubMed1981

Interaction of 5-bromocamphor with cytochrome P-450 cam. Production of 5-ketocamphor from a mixed spin state hemoprotein.

Camphor is stereospecifically hydroxylated by the soil bacterium Pseudomonas putida at the 5-exo position by a cytochrome P-450 mixed function oxidase system consisting of a flavoprotein reductase; putidaredoxin, an iron-sulfur oxidation-reduction transport-effector protein; and the P-450 hemoprotein. We have studied the interaction of a substrate analog of camphor, 5-exo-bromocamphor, with this cytochrome P-450 mixed function oxidase system in order to probe the molecular mechanisms of electron transport and catalytic substrate oxygenation. 5-exo-Bromocamphor was found to bind tightly in a 1:1 complex with P-450 with a dissociation constant of 2.9 microM, very near that for the normal camphor substrate. Contrary to camphor, however, the 5-exo-bromocamphor-bound material exhibits only a 46% population of the high spin form of the macromolecule. Measurement of the oxidation-reduction potential of the bromocamphor-P-450 complex yields a value of E0' = -246 mV, intermediate between that of substrate-free (-300 mV) and camphor-bound (-173 mV) cytochrome. These data are interpreted in terms of a thermodynamic linkage model relating spin, substrate, and oxidation-reduction equilibria in the P-450 monoxygenase system. 5-exo-Bromocamphor is readily metabolized by the P-450cam mixed function oxidase to 5-ketocamphor at rates and yields similar to that of the normal substrate, camphor, suggesting abstraction of the endo-hydrogen of 5-exo-bromocamphor and oxygen addition to produce a transient 5-bromo-5-hydroxycamphor intermediate. Implications of these chemical findings for the chemical mechanisms of P-450-catalyzed oxygenations and the flexibility of substrates at the active site of the macromolecule are discussed.

Camphor↗
PubMed1988

The roles of active site hydrogen bonding in cytochrome P-450cam as revealed by site-directed mutagenesis.

The role of the active site hydrogen bond of cytochrome P-450cam has been studied utilizing a combination of site-directed mutagenesis and substrate analogues with altered hydrogen bonding capabilities. Cytochrome P-450cam normally catalyzes the regiospecific hydroxylation of the monoterpene camphor. The x-ray crystal structure of this soluble bacterial cytochrome P-450 (Poulos, T. L., Finzel, B. C., Gunsalus, I. C., Wagner, G. C., and Kraut, J. (1985) J. Biol. Chem. 260, 16122-16128) indicates a specific hydrogen bond between tyrosine 96 and the carbonyl moiety of the camphor substrate. The site-directed mutant in which tyrosine 96 has been changed to a phenylalanine and the substrate analogues thiocamphor and camphane have been used to probe this interaction in several aspects of catalysis. At room temperature, both the mutant enzyme with camphor and the wild type enzyme with thiocamphor bound result in 59 and 65% high-spin ferric enzyme as compared to the 95% high spin population obtained with native enzyme and camphor as substrate. The equilibrium dissociation constant is moderately increased, from 1.6 microM for the wild type protein to 3.0 and 3.3 microM for wild type-thiocamphor and mutant-camphor complexes, respectively. Camphane bound to cytochrome P-450cam exhibits a larger decrease in high spin fraction (45%) and a correspondingly larger KD (46 microM), suggesting that the carbonyl moiety of camphor plays an important steric role in addition to its interaction as a hydrogen bond acceptor. The absolute regioselectivity of the mutant enzyme, and of the wild type enzyme with thiocamphor, is lost resulting in production of several hydroxylated products in addition to the 5-exo-hydroxy isomer. Based on rates of NADH oxidation, comparison of the substrate specificity for these systems (kcat/KD) indicates a 5- and 7-fold decrease in specificity for the mutant enzyme and thiocamphor-wild type complex, respectively. The replacement of the cytochrome P-450cam active site tyrosine with phenylalanine does not affect the branching ratio of monooxygenase versus oxidase chemistry or peroxygenase activity (Atkins, W.M., and Sligar, S.G. (1987) J. Am. Chem. Soc. 109, 3754-3760).

Chemical Phenomena↗
PubMed1996

[Building up of an animal model of conditioned immunosuppression and analysis of its possible mechanism].

In the present study, camphor odor and intraperitoneal (ip) injection of cyclophosphamide (CY) were used as conditional and unconditional stimulus, respectively, in mice. Mice were exposed to camphor odor for 1 h in their cage in a closed area followed by an ip injection of CY (75 mg.kg-1). This association trial session was repeated once on the next day. Delayed type hypersensitivity response (DTH) was induced as follows: six days after the second association trial session the mice were sensitized by smearing dinitrochlorobenezene (DNCB) on their abdominal skin. The mice were challenged by smearing DNCB on the left ear 5 days after the antigen sensitization. The left and right ears were removed 24 h after the challenge and weighed, the weight ratio of left/right ears was calculated for identification of the response. The ratio was 1.30 +/- 0.113 (+/- s, P < 0.001), indicating that the challenged ear was heavier than the other and DTH was induced. In the unconditioned response (UCR) group, CY (75 mg.kg-1) was given 24 h prior to the challenge and the ratio was 1.09 +/- 0.024 (P < 0.001) indicating that DTH was suppressed by unconditional stimulus (CY). In the conditioned response (CR) group mice were reexposed to camphor odor 24 h prior to the challenge and normal saline was injected instead of CY. The ratio was 1.13 +/- 0.074 (P < 0.001), indicating that DTH was also suppressed by conditional stimulus (camphor odor). These results show that a conditioned immunosuppressive response was induced. In the experiment, many other groups, including unconditioned response group, CYE group and camphor control group, were described in more details in the text. In order to further analyse the mechanisms of the conditioned response, the blood from the mice in CR group was obtained 6 h after reexposure to camphor odor and the serum was injected to normal mice 6 h prior to the challenge. DTH was found to be suppressed significantly when compared with the mice injected with normal serum. The conditioned serum was dialyzed against a membrane with a 10,000 molecular weight cut off. The suppressive activity of the conditioned serum disappeared, suggesting that the molecular weight of the suppressive element in the serum was probably less than 10,000 kDa.

Animals↗
PubMed2005

The influence of substrate on the spectral properties of oxyferrous wild-type and T252A cytochrome P450-CAM.

To probe whether the nature of the substrate can directly influence the spectral properties of oxyferrous cytochrome P450-CAM, the complex has been investigated in the absence and in the presence of the natural substrate (1R)-camphor (camphor) and of several camphor analogs. The oxyferrous complex of T252A P450-CAM, a mutant lacking the hydroxyl group that forms a hydrogen bond to the heme iron-coordinated dioxygen, has also been studied to gauge the influence of this hydrogen bond. UV-visible absorption and magnetic circular dichroism (MCD) spectra of these oxyferrous adducts prepared and stabilized at -40 degrees C in 60% (v/v) ethylene glycol are generally similar, exhibiting absorption bands at approximately 355, approximately 420, approximately 554, and approximately 585 nm (shoulder) and a characteristic MCD trough at approximately 585 nm. The MCD spectrum of camphor-bound oxyferrous P450-CAM is similar to that of the substrate-free oxyferrous enzyme, but the spectrum of the oxyferrous enzyme differs detectably in the presence of substrate analogs. The spectra of the oxyferrous T252A mutant and wild-type enzyme are overall similar except for Soret band position blue shifts by 2-6 nm for the mutant. 5-Methylenylcamphor (epoxidation substrate) appears to have an anomalous binding mode for the mutant compared with that for the wild-type enzyme. The present results indicate that the structures of the camphor analogs can sensitively influence the physical (spectroscopic) properties of the P450 dioxygen complex and could also affect its reactivity. The ability of substrate to modulate the reactivity of P450 intermediates could be a relevant factor in explaining the remarkable diversity of reactions catalyzed by the enzyme.

Binding Sites↗
PubMed2002

Specific and non-specific effects of potassium cations on substrate-protein interactions in cytochromes P450cam and P450lin.

Substrate binding to cytochrome P450cam is generally considered to be a two-step process. The first step corresponds to the entrance of the substrate, camphor, into the heme pocket. The second step corresponds to a spin transition (low spin-->high spin) of the iron in the protein-substrate complex. This spin transition is related to the mobility of the substrate inside the active site [Biochim Biophys Acta 1338 (1997) 77]. Potassium cations (K(+)) have a specific effect on the spin equilibrium. This is generally attributed to the K(+) ion-induced conformational change of tyrosine 96, the hydroxyl group of which is hydrogen bonded to the keto group of camphor and results in optimum substrate orientation and reduced mobility of this substrate in the active site. In the present paper, we show that K(+) not only affects the substrate-Tyr 96 couple, but acts more globally since K(+) effects are also observed in the Tyr96Phe mutant as well as in complexes with camphor-analogues. Large compounds, that fit well in the heme pocket and bind with higher affinity than camphor, display high spin contents that are less dependent on the presence of K(+). In contrast, K(+) has a significant effect on the high spin content of substrate-cytochrome P450cam complexes with looser interactions. We conclude that large compounds with higher affinities than camphor have more van der Waals contacts with the active site residues. Their mobilities are then reduced and less dependent on the presence of K(+). In this study, we also explored, for comparison, the K(+) effect on the spin transition state of another member of the P450 superfamily, cytochrome P450lin. This effect is not as strong as those observed for cytochrome P450cam. Even though the spin equilibrium does not change dramatically in the presence of K(+) or Na(+), the value of the dissociation constant (K(d)) for linalool binding is significantly affected by ionic strength. Analysis of the thermodynamic parameters for the linalool binding strongly suggests that, similarly to our previous finding for cytochrome P450cam, electrostatic gates participate in the control of substrate access.

Amino Acid Sequence↗
PubMed2001

Putidaredoxin-cytochrome P450cam interaction.

Cytochrome P450cam (P450cam) catalyzes the monooxygenation of D-camphor. During the enzymatic reaction, oxyferrous, D-camphor-bound P450cam forms a binary complex with reduced putidaredoxin as an obligatory reaction intermediate. We have found that reduced putidaredoxin undergoes EPR-detectable conformational changes upon formation of the intermediate complex and also upon formation of a binary complex with CO- or NO-ferrous, D-camphor-bound P450cam. The structural changes in putidaredoxin are almost identical irrespective of the ligand bound to P450cam, and distinct from and significantly larger than those induced by unliganded ferrous P450cam. The binary complex formation also induce conformational alterations in the CO- and NO-ferrous, D-camphor-bound P450cam, thereby evoking simultaneous changes in the structure of the two proteins. A molecular basis and roles of such structural changes in the D-camphor monooxygenation are discussed.

Bacterial Proteins↗
PubMed2002

Spectroscopic studies of peroxyacetic acid reaction intermediates of cytochrome P450cam and chloroperoxidase.

It is generally assumed that the putative compound I (cpd I) in cytochrome P450 should contain the same electron and spin distribution as is observed for cpd I of peroxidases and catalases and many synthetic cpd I analogues. In these systems one oxidation equivalent resides on the Fe(IV)=O unit (d(4), S=1) and one is located on the porphyrin (S'=1/2), constituting a magnetically coupled ferryl iron-oxo porphyrin pi-cation radical system. However, this laboratory has recently reported detection of a ferryl iron (S=1) and a tyrosyl radical (S'=1/2), via Mössbauer and EPR studies of 8 ms-reaction intermediates of substrate-free P450cam from Pseudomonas putida, prepared by a freeze-quench method using peroxyacetic acid as the oxidizing agent [Schünemann et al., FEBS Lett. 479 (2000) 149]. In the present study we show that under the same reaction conditions, but in the presence of the substrate camphor, only trace amounts of the tyrosine radical are formed and no Fe(IV) is detectable. We conclude that camphor restricts the access of the heme pocket by peroxyacetic acid. This conclusion is supported by the additional finding that binding of camphor and metyrapone inhibit heme bleaching at room temperature and longer reaction times, forming only trace amounts of 5-hydroxy-camphor, the hydroxylation product of camphor, during peroxyacetic acid oxidation. As a control we performed freeze-quench experiments with chloroperoxidase from Caldariomyces fumago using peroxyacetic acid under the identical conditions used for the substrate-free P450cam oxidations. We were able to confirm earlier findings [Rutter et al., Biochemistry 23 (1984) 6809], that an antiferromagnetically coupled Fe(IV)=O porphyrin pi-cation radical system is formed. We conclude that CPO and P450 behave differently when reacting with peracids during an 8-ms reaction time. In P450cam the formation of Fe(IV) is accompanied by the formation of a tyrosine radical, whereas in CPO Fe(IV) formation is accompanied by the formation of a porphyrin radical.

Binding Sites↗
PubMed1992

Resonance Raman investigations of Escherichia coli-expressed Pseudomonas putida cytochrome P450 and P420.

High-resolution resonance Raman spectra of the ferric, ferrous, and carbonmonoxy (CO)-bound forms of wild-type Escherichia coli-expressed Pseudomonas putida cytochrome P450cam and its P420 form are reported. The ferric and ferrous species of P450 and P420 have been studied in both the presence and absence of excess camphor substrate. In ferric, camphor-bound, P450 (mos), the E. coli-expressed P450 is found to be spectroscopically indistinguishable from the native material. Although substrate binding to P450 is known to displace water molecules from the heme pocket, altering the coordination and spin state of the heme iron, the presence of camphor substrate in P420 samples is found to have essentially no effect on the Raman spectra of the heme in either the oxidized or reduced state. A detailed study of the Raman and absorption spectra of P450 and P420 reveals that the P420 heme is in equilibrium between a high-spin, five-coordinate (HS,5C) form and low-spin six-coordinate (LS,6C) form in both the ferric and ferrous oxidation states. In the ferric P420 state, H2O evidently remains as a heme ligand, while alterations of the protein tertiary structure lead to a significant reduction in affinity for Cys(357) thiolate binding to the heme iron. Ferrous P420 also consists of an equilibrium between HS,5C and LS,6C states, with the spectroscopic evidence indicating that H2O and histidine are the most likely axial ligands. The spectral characteristics of the CO complex of P420 are found to be almost identical to those of a low pH of Mb. Moreover, we find that the 10-ns transient Raman spectrum of the photolyzed P420 CO complex possesses a band at 220 cm-1, which is strong evidence in favor of histidine ligation in the CO-bound state. The equilibrium structure of ferrous P420 does not show this band, indicating that Fe-His bond formation is favored when the iron becomes more acidic upon CO binding. Raman spectra of stationary samples of the CO complex of P450 reveal VFe-CO peaks corresponding to both substrate-bound and substrate-free species and demonstrate that substrate dissociation is coupled to CO photolysis. Analysis of the relative band intensities as a function of photolysis indicates that the CO photolysis and rebinding rates are faster than camphor rebinding and that CO binds to the heme faster when camphor is not in the distal pocket.

Bacterial Proteins↗
PubMed1990

The formation of cytochrome P-450 from cytochrome P-420 is promoted by spermine.

This paper is concerned with camphor-bound bacterial cytochrome P-450 and processes that alter its spin-state equilibrium and influence its transition to the nonactive form, cytochrome P-420, as well as its renaturation to the native camphor-bound cytochrome P-450. Spermine, a polycation carrying a charge of 4 +, and potassium, a monovalent cation, were shown to differently cause an increase of high-spin content of camphor-bound cytochrome P-450. The spermine-induced spin transition saturates around 75% of the high spin; a further addition of KCl to the spermine-containing sample shifted the spin state to 95% of the high spin. The volume change of these spin transitions as measured by the use of high pressure indicated an excess of -40 mL/mol for the sample containing potassium as compared to that containing spermine. These results suggest that the proposed privileged site for potassium has not been occupied by spermine and that pressure forces both the camphor and the potassium ion from its sites, allowing solvent movement into the protein as well as ordering of solvent by the excluded camphor and potassium. Cytochrome P-420 was produced from cytochrome P-450 by hydrostatic pressure in the presence of potassium, spermine, and cysteine. Potassium cation shows a bigger effect on the stability of cytochrome P-450 than spermine or cysteine, as revealed by a higher value of the pressure of half-inactivation, P1/2, and a bigger inactivation volume change. However, potassium cation did not promote renaturation of cytochrome P-420 to cytochrome P-450 while the presence of spermine did.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed2004

Formulation design and optimization of mouth dissolve tablets of nimesulide using vacuum drying technique.

The purpose of this research was to develop mouth dissolve tablets of nimesulide. Granules containing nimesulide, camphor, crospovidone, and lactose were prepared by wet granulation technique. Camphor was sublimed from the dried granules by exposure to vacuum. The porous granules were then compressed. Alternatively, tablets were first prepared and later exposed to vacuum. The tablets were evaluated for percentage friability, wetting time, and disintegration time. In the investigation, a 32 full factorial design was used to investigate the joint influence of 2 formulation variables: amount of camphor and crospovidone. The results of multiple linear regression analysis revealed that for obtaining a rapidly disintegrating dosage form, tablets should be prepared using an optimum concentration of camphor and a higher percentage of crospovidone. A contour plot is also presented to graphically represent the effect of the independent variables on the disintegration time and percentage friability. A checkpoint batch was also prepared to prove the validity of the evolved mathematical model. Sublimation of camphor from tablets resulted in superior tablets as compared with the tablets prepared from granules that were exposed to vacuum. The systematic formulation approach helped in understanding the effect of formulation processing variables.

Administration, Oral↗
PubMed1985

The 2.6-A crystal structure of Pseudomonas putida cytochrome P-450.

The crystal structure of Pseudomonas putida cytochrome P-450cam in the ferric, camphor bound form has been determined and partially refined to R = 0.23 at 2.6 A. The single 414 amino acid polypeptide chain (Mr = 45,000) approximates a triangular prism with a maximum dimension of approximately 60 A and a minimum of approximately 30 A. Twelve helical segments (A through L) account for approximately 40% of the structure while antiparallel beta pairs account for only approximately 10%. The unexposed iron protoporphyrin IX is sandwiched between two parallel helices designated the proximal and distal helices. The heme iron atom is pentacoordinate with the axial sulfur ligand provided by Cys 357 which extends from the N-terminal end of the proximal (L) helix. A substrate molecule, 2-bornanone (camphor), is buried in an internal pocket just above the heme distal surface adjacent to the oxygen binding site. The substrate molecule is held in place by a hydrogen bond between the side chain hydroxyl group of Tyr 96 and the camphor carbonyl oxygen atom in addition to complementary hydrophobic contacts between the camphor molecule and neighboring aliphatic and aromatic residues. The camphor is oriented such that the exo-surface of C5 would contact an iron bound, "activated" oxygen atom for stereoselective hydroxylation.

Amino Acid Sequence↗
PubMed1978

Magnetic circular dichroism of Pseudomonas putida cytochrome P-450 in near infrared region.

Magnetic circular dichroism spectra of oxidized, reduced and carbonmonoxy reduced forms of cytochrome P-450 from D-camphor grown Pseudomonas putida (P-450cam) were studied in the near infrared region (650 to 1200 nm) at various temperatures in the presence of D-camphor. Oxidized P-450cam with camphor exhibited positive (+) and negative (-) magnetic CD bands at 825 and 970 nm, respectively, and both of them were assigned to Faraday B terms. The magnetic CD spectrum of reduced P-450cam in the presence of D-camphor exhibited at least five components in the region between 650 to 1175 nm and one of them at 760 nm showed considerably smaller magnitude than that of the corresponding band of deoxymyoglobin. These results were interpreted to mean that the heme-iron in both oxidized and reduced P-450cam has a ligand field symmetry lower than C4v, i.e. a strong rhombic character of the heme in cytochrome P-450. Carbonmonoxide complex of reduced P-450cam exhibited no detectable magnitude of magnetic CD in the near infrared region but showed CD bands at 710 (-) and 850 (+) nm. The results were compared and discussed with those of carbonmonoxy hemoglobin and myoglobin. In addition, temperature dependent changes in the spin state of oxidized P-450cam from high to low by decrease of temperature were observed by measuring both magnetic CD and absorption spectra in the near ultraviolet and visible regions (300 to 650 nm), provided that the temperature of the sample was varied slowly (approximately 3 degrees C/min) between room and liquid nitrogen temperature in a 0.03 M phosphate buffer (pH 7.2) containing a saturated amount of D-camphor and 70% (v/v) glycerol. The significance of this phenomenon is also discussed.

Circular Dichroism↗
PubMed2000

In-vitro inhibition of human erythrocyte acetylcholinesterase by salvia lavandulaefolia essential oil and constituent terpenes.

Sage (Salvia spp) is reputed in European herbal encyclopaedias to enhance memory, and current memory-enhancing/anti-dementia drugs are based on enhancing cholinergic activity by inhibiting cholinesterase. In this study the effects of Salvia lavandulaefolia Vahl. (Spanish sage) essential oil and some of its constituent terpenes on human erythrocyte acetylcholinesterase were examined in-vitro. The main constituents in the essential oil batch used for analysis of cholinesterase inhibition were camphor (27%), 1,8-cineole (13%), alpha- and beta-pinene (10-15%) and bornyl acetate (10%) with other minor constituents (1% or less) including geraniol, limonene, linalool, terpineol and gamma-terpinene. Using the Ellman spectrophotometric method, kinetic analysis was conducted on the interaction of the essential oil and the main monoterpenoids, camphor, 1,8-cineole and alpha-pinene. IC50 values were obtained for the essential oil, 1,8-cineole and alpha-pinene and were 0.03 microL [corrected] mL(-1), 0.67 mM and 0.63 mM, respectively. Camphor and other compounds tested (geraniol, linalool and gamma-terpinene) were less potent (camphor IC50: >10mM). The essential oil, alpha-pinene, 1,8-cineole and camphor were found to be uncompetitive reversible inhibitors. These findings suggest that if the inhibitory activity of the essential oil is primarily due to the main inhibitory terpenoid constituents identified, there is a major synergistic effect among the constituents. Since no single constituent tested was particularly potent, it remains to be determined whether these in-vitro cholinesterase inhibitory activities are relevant to in-vivo effects of the ingestion of S. lavandulaefolia essential oil on brain acetylcholinesterase activity.

Acetylcholinesterase↗
PubMed1990

Solvation of the active site of cytochrome P450-cam.

Energetically favorable water binding sites in the substrate pocket of cytochrome P450-cam have been predicted by a molecular mechanics method. Binding sites corresponding to all the experimentally observed water sites in this region of the enzyme were located. The calculations also indicate the presence of two further water binding sites. One of these is located in a hydrophobic region of the protein where a water molecule would not bind tightly to the substrate-free enzyme. However, in the substrate-bound enzyme, a water molecule in this region could donate a hydrogen bond of optimum geometry to the carbonyl oxygen atom of the camphor substrate and could therefore contribute to the correct positioning of the camphor substrate for 5-exo-hydroxylation. These calculations also suggest that a steric analogue of camphor, containing an alkyl group which could prevent a water molecule from binding in this region, might inhibit cytochrome P450-cam by forming a more stable enzyme-ligand complex than camphor itself.

Binding Sites↗
PubMed2004

Role of substrate on the conformational stability of the heme active site of cytochrome P450cam: effect of temperature and low concentrations of denaturants.

The effect of 1R-camphor on the conformational stability of the heme active site of cytochrome P450cam has been investigated. The absorption spectra of the heme moiety showed the presence of two hitherto unknown intermediates formed at low urea concentrations or during small temperature perturbations. The corresponding thermodynamic parameters were obtained by global fitting of the experimental data to a generalized sequential unfolding model at different wavelengths, which showed that the active conformation of the enzyme is stabilized by binding of the substrate at the active site. Circular-dichroism spectra of the enzyme in the visible- and far-UV region were studied to identify the critical range of denaturant concentration and the temperature at which the tertiary structure around the heme center was affected with almost no change in the secondary structure of the enzyme. This critical range of urea concentration was 0-2.8 M in the presence of camphor and 0-1.5 M in the absence of camphor. The tertiary structure of the enzyme was found to undergo conformational change in the temperature range 20-60 degrees C in the presence of the substrate and 20-47 degrees C in its absence. The spectral assignments of the intermediate species of the heme active site with the intact secondary structure of the enzyme were made by deconvolution of the Soret absorption spectra, and the results were analyzed to determine stabilization of the heme active-site geometry by 1R-camphor. Results showed that subtle conformational changes due to melting of the tertiary contacts in the active site lead to formation of intermediates which are coordinatively similar to the native enzyme. Analogous intermediate species might be responsible for leakage in the redox catalytic cycle of the enzyme.

Binding Sites↗