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Biomedical subjects

G H Hoa

Publications and source records attributed to G H Hoa.

At least 19 recordsLinked to original sources
PubMed1997

Epitope mapping of cytochrome P450cam (CYP101).

Eighteen linear antigenically active sites were revealed in cytochrome P450 from Pseudomonas putida (P450cam) by hexapeptide scanning. These sites occupy about 31% of the protein sequence. Hexapeptide epitope sequences of P450cam are not found in other cytochromes P450. However, several cytochromes P450 contain shorter fragments of P450cam epitope sequences which may cause weak immune cross-reactions. P450cam antigenic determinants are located generally at the boundaries of secondary structure elements. Mapping of P450cam antigenic determinants on the three-dimensional structure of this protein reveals 14 highly water-accessible antigenic sites and only 1 site (No. 322-327, QMLSGL) which is inaccessible to water. Several functionally important sites and amino acid residues of P450cam are localized within revealed linear epitopes or very close to them. These sites include substrate-binding regions, residues responsible for the putidaredoxin interaction (Arg72, Arg112, Lys314, and Arg364), heme binding (Gln108, Arg112, Asp297, Arg299, and Cys357), and proton translocation (Lys178, Arg186, and Glu366).

Amino Acid Sequence↗
PubMed1997

Mobility of norbornane-type substrates and water accessibility in cytochrome P-450cam.

The behaviour of norbornane-type substrates bound to oxidised cytochrome P-450cam (CYP 101) in 60% (w/w) glycerol-containing phosphate buffer was investigated using electronic absorption spectroscopy. The high-pressure dependence study revealed that the value of the spin-state reaction-volume change decreased from -70 to -22.8 cm3/mol with decreasing high-spin state content from 99 to 63%. Simultaneously, the values for the enthalpy and entropy determined from the low-temperature dependence of the spin-state transition decreased from 73.7 to 24.3 kJ/mol and from 310.4 to 88.9 J/mol K, respectively. Under our experimental conditions the pH-value of the buffer remained at low temperatures and high pressures in the range of pH 7-8, in which no pH-value-induced spin-state conversion occurred. Therefore, the secondary effect of the temperature and pressure-induced pH change can be disregarded as being responsible for the observed spin-state transition effects. Substrate dissociation constants were determined. From the temperature-jump experiments (297 K to 180 K) we found a higher mobility in the active site for the substrates in the sequence (1R)-camphor, (1S)-camphor, camphane, (1R)- and (1S)-camphorquinone, norcamphor, and norbornane. Our findings can be explained by the incomplete fit of the methyl groups of the norbornane-type substrate to the protein, in particular to the I-helix, predominantly determining the substrate mobility and water accessibility to the protein.

Binding Sites↗
PubMed1996

Probing the heme iron coordination structure of pressure-induced cytochrome P420cam.

Cytochrome P450cam was subjected to high pressures of 2.2 kbar, converting the enzyme to its inactive form P420cam. The resultant protein was characterized by electron paramagnetic resonance, magnetic circular dichroism, circular dichroism, and electronic absorption spectroscopy. A range of exogenous ligands has been employed to probe the coordination structure of P420cam. The results suggest that conversion to P420cam involves a conformational change which restricts the substrate binding site and/or alters the ligand access channel. The reduction potential of P420cam is essentially the same in the presence or absence of camphor (-211 +/- 10 and -210 +/- 15 mV, respectively). Thus, the well-documented thermodynamic regulation of enzymatic activity for P450cam in which the reduction potential is coupled to camphor binding is not found with P420cam. Further, cyanide binds more tightly to P420cam (Kd = 1.1 +/- 0.1 mM) than to P450cam (Kd = 4.6 +/- 0.2 mM), reflecting a weakened iron-sulfur ligation. Spectral evidence reported herein for P420cam as well as results from a parallel investigation of the spectroscopically related inactive form of chloroperoxidase lead to the conclusion that a sulfur-derived proximal ligand is coordinated to the heme of ferric cytochrome P420cam.

Camphor 5-Monooxygenase↗
PubMed1996

Structural changes in cytochrome P-450cam effected by the binding of the enantiomers (1R)-camphor and (1S)-camphor.

A comparative study of the enantiomeric substrate [(1R)-camphor- and (1S)-camphor)-bound cytochrome P-450cam concerns the spin-state equilibrium, substrate dissociation, the thermal unfolding of the protein structure, and the subconformer equilibria observed in the infrared spectra of the carbon monoxide (CO) complex of cytochrome P-450cam. The behavior of the different conformational equilibria in dependence on temperature, pressure, pH-value, cosolvent, and cation binding led us to suggest that (1S)-camphor is more loosely and less optimally bound in the heme pocket, which facilitates the access of solvent molecules into the heme-iron environment. The spin reaction volume difference measured using the high pressure technique is smaller by 16 +/- 9 cm3/mol for (1S)-camphor-bound P-450cam compared to the (1R)-camphor-bound P-450cam, which might indicate a higher water content in the protein and in the heme environment in the (1S)-camphor complex. The half-transition temperature of the thermal unfolding of 53.8 degrees C for the (1S)-camphor-bound oxidized cytochrome P-450cam is one degree lower than the value for the (1R)-camphor-bound protein (54.8 degrees C). In the reduced, CO-bound form of cytochrome P-450cam at 290 K the (1S)-camphor complex reveals another CO stretch vibration population distribution with slightly higher frequencies [1940.2 cm-1 (major band) and 1946.3 cm-1 (minor band)] compared to the (1R)-camphor complex [1939.7 cm-1 (major band) and 1930 cm-1 (minor band)]. A loosening of the contact between the iron-bound CO ligand and amino acids of the I-helix, probably induced by compensating effects of the increased water content, is suggested. Assuming the carbon monoxide complex as a model for the dioxygen complex, the more loosened binding of (1S)-camphor, therefore the increased water accessibility, and the weaker contact of the iron ligand to the I-helix might explain the higher amount of uncoupling of the cytochrome P-450 reaction cycle compared to that when (1R)-camphor is used as substrate.

Binding Sites↗
PubMed1995

High-pressure-induced transitions in microsomal cytochrome P450 2B4 in solution: evidence for conformational inhomogeneity in the oligomers.

Pressure-induced changes in ferric P450 2B4 (LM2) were studied as a function of benzphetamine concentration (0.05 divided by 2 mM) and state of aggregation of the hemoprotein in solution. Application of factor analysis to the spectral changes in the Soret region allowed us to resolve two particular pressure-induced processes in 2B4 oligomers. The first process was identified as the conversion of the low-spin P450 into the P420 state. At 25 degrees C it was followed by decay (bleaching) of about 50% of the newly formed P420. The second process was a pressure-induced high- to low-spin shift. Both transitions were reversible, except the hemoprotein bleaching. The amplitude of the P450-->P420 transition accounted for 67 +/- 5% of the total hemoprotein content. Furthermore, the fraction of the hemoprotein exposed to spin equilibrium was not affected by the P450-->P420 conversion and was estimated to be only about 31 +/- 5% of the total hemoprotein content. After the dissociation of the oligomers by 0.2% Triton N-101, the inhomogeneity vanished: 95% of the monomers were involved in the P450-->P420 transition (delta V degrees = -86 ml/mol) followed by intense bleaching of the hemoprotein. This agrees with our earlier observations on the reduced carbonyl complex of P450 2B4 and suggests some conformational difference between subunits in P450 LM2 oligomers. The parameters of the P450-->P420 conversion (delta V degrees = -32 ml/mol, P1/2 = 1560 bar) show no dependency on the substrate concentration. Analysis of the pressure-induced spin shift versus benzphetamine concentration shows this transition to be caused mainly by changes in the spin equilibrium of both substrate-bound (delta V degrees = -49 ml/mol) and substrate-free (delta V degrees = -21 ml/mol) hemoprotein, whereas the substrate binding step itself has a very weak pressure dependency (delta V degrees = -8 ml/mol).

Algorithms↗
PubMed1995

Second derivative spectroscopy of enolase at high hydrostatic pressure: an approach to the study of macromolecular interactions.

Second derivative spectroscopy in the ultraviolet region of proteins has been used to study the polarity of the regions surrounding tyrosine residues. We show here that it can also be a tool to study the degree to which proteins associate and that it can be effectively combined with hydrostatic pressure in order to evaluate equilibrium dissociation constants and reaction volumes. Hydrostatic pressure causes yeast enolase to dissociate. Clear changes in the second derivative spectra of enolase were observed as pressure was increased. At enolase concentrations of about 20 microM, the midpoint of the transition is about 1800 bar. All aspects of the transition are reversible up to 2700 bar. It is likely that the transition observed is the result of enolase dimers dissociating into monomers. The second derivative spectra indicate that one or more tyrosine residues is in an unusually polar environment in the dimer, an environment that is less polar in the monomer. Three tyrosines (6, 11, 130) are near the dimer interface. Tyrosines 6 and 11 are pointing into the water-filled crevice between the subunits and are close to several immobilized waters. All three are close to a network of intersubunit salt bridges and hydrogen bonds. We believe that the average tyrosine polarity in the dimer reflects the exposure of these tyrosines to immobilized water and the fixed dipole of the salt bridge. The water in the crevice between the subunits should be more mobile in the monomer; the salt bridge does not exist in the monomer.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrostatic Pressure↗
PubMed1995

Antagonistic effects of hydrostatic pressure and osmotic pressure on cytochrome P-450cam spin transition.

The combined effects of hydrostatic pressure and osmotic pressure, generated by polyols, on the spin equilibrium of fenchone-bound cytochrome P-450cam were investigated. Hydrostatic pressure indices a high spin to low spin transition, whereas polyols induce the reversed reaction. Of the four solutes used, glycerol, glucose, stachyose, and sucrose, only the last two would act on the spin transition by osmotic stress. The spin volume changes measured by both techniques are different, 29 and -350 ml/mol for hydrostatic pressure and osmotic pressure, respectively. It suggests that even if the two are perturbing water molecules, different properties are probed. From the volume change induced by osmotic stress, 19 water molecules are deduced that would be implicated in the spin transition of the fenchone-bound protein. This result suggests that water molecules other than the well defined ones located in the active site play a key role in modulating the spin equilibrium of cytochrome P-450cam.

Camphor 5-Monooxygenase↗
PubMed1994

Effects of monovalent cations on cytochrome P-450 camphor. Evidence for preferential binding of potassium.

Binding of monovalent cations of increasing ionic radius to ferric cytochrome P-450cam was measured. Potassium has the highest affinity for the cation binding site observed in the X-ray crystallographic structure with Kdcat = 12 mM, compared with the smaller cation lithium, (Kdcat = 37 mM) and the larger cation cesium (Kd cat = 20 mM). Coupling between cation binding and camphor binding is established by the observation of a linear relationship between the corresponding binding free energies. Potassium binding favours a conformational change of tyrosine 96 which increases the affinity of the protein for camphor and fully dehydrates the active site.

Binding Sites↗
PubMed1994

High-pressure effects on beta-lactoglobulin interactions with ligands studied by fluorescence.

The effects of pressure (0.1 MPa to 400 MPa) on intrinsic fluorescence of beta-lactoglobulin and on its binding of retinol and cis-parinaric acid have been studied at neutral and acid pHs. In neutral pH, fluorescence emission spectra of beta-lactoglobulin tryptophanes are characterized by an irreversible 14 nm red-shift indicating pressure-induced folding changes. The intensity of the fluorescence of retinol in beta-lactoglobulin-retinol complex is enhanced by a pressure increase up to 150 MPa. It decreases at higher pressures and disappears altogether at 300 MPa. beta-Lactoglobulin-retinol complex does not reassociate after decompression at neutral pH. At acid pH condition, the fluorescence quenching by pressure of beta-lactoglobulin tryptophans is coupled with a 2 nm spectral shift and is fully reversible demonstrating almost complete restoration of globulin folding. The evolution of retinol fluorescence in beta-lactoglobulin-retinol complex is also entirely reversible between 0.1 MPa and 400 MPa and the complex never dissociates in the studied pressure range. beta-lactoglobulin-cis-parinaric acid complexes at neutral and acid pH values dissociate irreversibly at 200 MPa and 350 MPa, respectively.

Binding Sites↗
PubMed1993

Responses of two protein-protein complexes to solvent stress: does water play a role at the interface?

We have analyzed the stability of the cytochrome c-cytochrome b5 and cytochrome c-cytochrome c oxidase complexes as a function of solvent stress. High concentrations of glycerol were used to displace the two equilibria. Glycerol promotes complex formation between cytochrome c and cytochrome b5 but inhibits that between cytochrome c and cytochrome c oxidase. The results with cytochrome b5 and cytochrome c were expected; the association of this complex is largely entropy driven. Our interpretation is that the cytochrome c-cytochrome b5 complex excludes water. The results with the cytochrome c oxidase and cytochrome c couple were not expected. We interpret them to mean that either glycerol is binding to the oxidase, thereby displacing the cytochrome c, or that water is required at this protein-protein interface. A requirement for substantial quantities of water at the interface of some protein complexes is logical but has been reported only once.

Animals↗
PubMed1992

Substrate analogue induced changes of the CO-stretching mode in the cytochrome P450cam-carbon monoxide complex.

The CO-stretching mode of the carbon monoxide ligand in reduced cytochrome P450cam, in the absence or presence of camphor and in the presence of nine different camphor analogues, was measured at room temperature using Fourier transform infrared spectroscopy. Substrate-free cytochrome P450cam--CO reveals a broad, slightly structured band resulting from an overlap of several stretching mode signals. The multitude of the signals indicates that cytochrome P450 exists in a dynamic equilibrium of several conformational substates. Binding of camphor or camphor analogues strongly influences this equilibrium. For substrate analogues which are not able to form a hydrogen bond to the hydroxyl group of tyrosine 96, the CO-stretching band is rather broad and asymmetric. In contrast, substrate analogues with one quinone group which form a hydrogen bond to the Tyr96 OH induce a shift and a sharpening of the CO-stretching mode band. For substrate analogues with two hetero groups, the infrared spectrum is slightly asymmetric or a minor band appears. Sterical hindrance, substrate mobility, and protein flexibility finally determine the position and width of the CO-stretching mode signals.

Camphor↗
PubMed1992

A critical role of protein-bound water in the catalytic cycle of cytochrome P-450 camphor.

The rates of NADH oxidation during the hydroxylation of camphor by cytochrome P-450cam were followed in the presence of co-solvents used to increase the osmotic pressure surrounding the protein-bound water. As a result, the measured Vmax decreases independently of the perturbant tested. Roughly 28 molecules of water, involved during the catalytic cycle, are deduced from the variation of Vmax as a function of osmotic pressure. These molecules, in part, could be those present in the cytochrome P-450cam-putidaredoxin interface.

Camphor 5-Monooxygenase↗
PubMed1992

High pressure induced inactivation of ferrous cytochrome P-450 LM2 (IIB4) CO complex: evidence for the presence of two conformers in the oligomer.

The effect of high pressure on the spectral properties of cytochrome P-450 LM2(Fe2+)-CO complex was studied. The application of high pressure was shown to induce the conversion of cytochrome P-450 to P-420. In the solution when P-450 was oligomeric only about 65% of the total converted to P-420. The remaining portion of cytochrome P-450 was stable at pressures up to 6 kbar. When P-450 was incorporated into membranes or when it was succinylated, the proportion of the pressure sensitive fraction was slightly higher (about 75%). Dissociation of P-450 oligomers into monomers was made by addition of 0.2% Triton N-101. Monomers were the most sensitive to pressure; they could be completely converted to P-420. These results have been interpreted as evidence for the existence of two different conformers of P-450 LM2, which differ in pressure stability. Splitting between these two states appears to be a result of the oligomeric organization of cytochrome P-450 in solution and in the membrane.

Animals↗
PubMed1992

Cytochrome c and cytochrome c oxidase interactions: the effects of ionic strength and hydrostatic pressure studied with site-specific modifications of cytochrome c.

Seven cytochromes c, in which individual lysines have been modified to the propylthiobimane derivatives, have been prepared. These derivatives were also converted to the porphyrin cytochromes c by treatment with HF. The properties of both types of modified proteins were studied in their reactions with cytochrome c oxidase. The results show that lysines 25, 27, 60, 72, and 87 do not contribute a full charge to the binding interaction with the oxidase. These five residues, with the exception of the lysine-60 derivative, on the front surface of the protein and contain the solvent-accessible edge of the heme prosthetic group. By contrast, lysines 8 and 13 at the top of the front surface do contribute a full charge to the binding interaction with the oxidase. The removal of the positive charge on any one lysine weakens the binding to cytochrome c oxidase by at least 1 kcal (1 cal = 4.1868 J). The presence of bimane at lysines 13 and 87 clearly forces the separation of the cytochrome c and oxidase, but this does not occur with the other complexes. The bimane-modified lysine-13 protein, and to a lesser extent that modified at lysine 8, show the interesting effect of enhanced complex formation with cytochrome c oxidase when subjected to pressure, possibly because of entrapment of water at the newly created interface of the complex. Our observations indicate that the two proteins of the cytochrome c - cytochrome oxidase complex have preferred, but not obligatory, spatial orientations and that interaction occurs without either protein losing significant portions of its hydration shell.

Animals↗
PubMed1990

A nontraditional role for water in the cytochrome c oxidase reaction.

The passage of electrons through cytochrome c oxidase is directly related to the activity of water. Reducing the activity in a system containing reductant, oxygen, and cytochrome oxidase blocks electron transfer between reduced cytochrome a and oxidized cytochrome a3. The extent of the block is directly related to the osmotic pressure of the system, implying that the protein shell of the oxidase acts as a semipermeable membrane that excludes osmotic perturbants but not water. It appears that approximately 10 water molecules must enter and leave the oxidase in order for internal electron transfer to occur.

Animals↗
PubMed1990

A reactor permitting injection and sampling for steady state studies of enzymatic reactions at high pressure: tests with aspartate transcarbamylase.

A high pressure reactor for steady state studies of enzymes is described. It allows injection, stirring, and sampling without release of the pressure (up to at least 400 MPa). Thus, either substrate or enzyme can be injected to initiate an enzyme-catalyzed reaction whose progress can then be followed by measurements on samples taken from the reactor. The dead time of sampling is 10-15 s, which allows reactions with pseudo-first-order rate constants smaller than about 1 min-1 to be monitored. It can be used for any enzymatic reaction; unlike previously described high pressure apparatus, it is not limited to the study of enzymes whose activity can be directly followed by spectrophotometry. The use and reliability of this reactor is demonstrated by tests with aspartate transcarbamylase. The activity of this enzyme is enhanced by pressures of the order of 120 MPa.

Aspartate Carbamoyltransferase↗
PubMed1987

P-450 binding to substrates camphor and linalool versus pressure.

The spin equilibrium of two bacterial cytochrome P-450 enzymes are compared by their visible spectra versus temperature and pressure. P-450 from Pseudomonas linalool shows a much weaker dependence on pressure than P-450 from P. putida which has camphor as substrate. The linalool system denatures at a higher pressure (3 kbar) than the camphor system (1 kbar) and shows a weaker dependence on external solvent conditions. The camphor system shows evidence of the binding of a second substrate molecule which reverses the effect of the first on the spin equilibrium. A model involving two substrate molecules is an alternative explanation of the apparent saturation with camphor of the spin equilibrium.

Acyclic Monoterpenes↗