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S Narasimhulu

Publications and source records attributed to S Narasimhulu.

At least 19 recordsLinked to original sources
PubMed2001

Temperature-jump relaxation kinetics of substrate-induced spin-state transition in cytochrome P450 (comparison of the wild-type and C334A mutant P450(CAM) and P450(2B4)).

The kinetics of binding of the substrate camphor to the cytochrome P450(CAM) and the C334A mutant as well as the kinetics of binding of benzphetamine to the wild-type P450(2B4) have been studied by the temperature-jump relaxation technique in order to distinguish between the two models for substrate-induced spin-state transition. These models are the bimolecular model in which spin-state transition occurs in parallel with substrate binding, and the two-step spin-equilibrium model in which substrate binding is a separate step preceding the spin-state transition. With all three P450s, the relaxation rate versus concentration data were linear as predicted by the bimolecular model and inconsistent with the spin-equilibrium model, which predicts a curve reaching saturation. With all three P450s, the relaxation rate versus concentration data exhibited maxima. These results are considered to resolve the controversy in favor of the bimolecular model for substrate-induced spin-state transition. In addition, the results suggest that the bimolecular model may be applicable to other P450s as well.

Amino Acid Substitution↗
PubMed1998

Interactions of substrate and product with cytochrome P450: P4502B4 versus P450cam.

In the present study, two P450s (P4502B4 and P450cam) have been examined with regard to their interactions with their substrates and products utilizing the characteristic spectral perturbations as criteria for their binding. The results indicate that although there are differences between the two P450s (E) in regard to their precise interactions with their substrates (S) and products (P), the spectral titration data were consistent with the two-site model--E + S<-->ES (K1), E + P<-->EP (K2); EP + S<-->ESP (K3); ES + P<-->ESP (K4) in which S and P bind to E forming ESP. The data were inconsistent with the two-site model in which S and P compete for the same site. As required by the two-site model, the relationship K2K3 = K1K4 was maintained with both P450s at all product concentrations tested, although K3 and K4 decreased considerably when product concentration was increased. The relationship K3 >> K4 was also maintained, indicating that with both enzymes' ESP is formed predominantly by binding of S to EP rather than binding of P to ES, and that ESP dissociates predominantly to ES and P rather than EP and S. In other words, binding of S to EP facilitates the dissociation of P. This indicates that the relative parameter values are compatible for ESP to have functional significance. The possible role of ESP in controlling catalytic rate and catalytic efficiency is discussed.

Animals↗
PubMed1996

Interactions of substrate and product with cytochrome P450 2B4.

Interactions of the substrate(s) benzphetamine and the product (P) desmethylbenzphetamine with cytochrome P450 2B4 were studied by difference spectrophotometry. A two-sites model in which site 1 binding, causing Type I transition (low- to high-spin) must precede site 2 binding, causing Type II transition (high- to low-spin), gave an acceptable fit to the spectral titration data. The equilibrium association constant of substrate for site 1 (K1) was greater than that for site 2 (K2), and the K2 for the product was greater than K1, indicating that the substrate binds preferentially to site 1 and the product prefers site 2. In addition, competition between P and a strong Type II ligand (1-benzylimidazole) and a noncompetitive type of interaction between S and the same Type II ligand was observed. This indicates that P binds to the same site as the Type II ligand and S binds to a different site. The observed high-spin maxima for both P (EP1HSmax) and S (ES1HSmax) were similar to those calculated using the K1 and K2 values obtained from the curve-fitting procedure, indicating that the equilibrium concentration of the high-spin species is controlled entirely by K1 and K2. Simultaneous presence of the substrate and product decreased K1 of the substrate and K2 of the product, indicating that there is interaction between the substrate-preferred and the product-preferred sites. A possible functional significance of the differences in the site preferences of the substrate and product is discussed.

Animals↗
PubMed1993

Substrate-induced spin-state transition in cytochrome P450LM2: a temperature-jump relaxation study.

The kinetics of the benzphetamine-P450LM2 binding reaction were studied by the T-jump relaxation technique, using the substrate-induced type I spectral change (which reflects transformation of the heme from the low- to the high-spin state) as the criterion for binding. The reciprocal relaxation time (kobs) exhibited a linear dependence on [E]eq+[S]eq. The kinetically determined dissociation equilibrium constant (68 +/- 10 microM) and that determined by direct titration of the spectral change (61 +/- 4 microM) were very similar. These results indicate that the substrate-induced spin-state transition follows a simple biomolecular binding mechanism; that is, the substrate-induced low- to high-spin transition reflects substrate binding.

Animals↗
PubMed1993

On the model controversy for substrate-induced spin-state transition in cytochrome P450: (a new perspective).

Three models have been proposed for substrate-induced spin-state transition in cytochrome P450. These are referred to as two-, three- and four-state models. In this communication the three models are reviewed with respect to their experimental basis and their ability to accommodate the results reported on the effects of substrates on spin-state and reduction of various P450's. In addition, a new perspective is presented.

Animals↗
PubMed1991

Inhibition of substrate binding to the adrenal cytochrome P450C-21 by acrylamide and its implications for solvent accessibility of the binding site in the microsomes.

The present study offers evidence indicating that acrylamide, a highly polar molecule and an efficient quencher of tryptophanyl fluorescence, inhibits substrate binding to P450C-21 in bovine adrenocortical microsomes, in a competitive manner similar to that in the purified enzyme. Resolution of the fluorescence-quenching data revealed an acrylamide quenching constant (K2 = 9.9 M, that is, the association constant for the quencher-fluorophore complex) that was similar to the reciprocal of its inhibition constant (1/Ki = Ka = 8.3 +/- 0.9 M) for substrate binding. The substrate inhibited the fluorescence quenching by acrylamide as indicated by its concentration-dependent decrease in K2. The inhibition was in accordance with partial competition. These results are essentially similar to those previously observed in the purified lipid-free enzyme. In addition, the substrate dissociation, acrylamide inhibition, and fluorescence-quenching constants and the tryptophanyl fluorescence maximum (340-342 nm) were essentially the same in the microsomes and the lipid-free purified enzyme. These results indicate that the substrate-binding site of P450C-21 and the concerned tryptophan are accessible to the highly polar molecule in the microsomal membranes, similar to that in the lipid-free purified enzyme. This implies that the substrate-binding site is not shielded by lipids in such a way that only the substrate in the lipid phase can gain access to the binding site. This conclusion is consistent with the currently favored model, for membrane topology of mammalian P450 enzymes, in which P450 is anchored to the membrane through a short N-terminal sequence while the remaining portion of the molecule is exposed to polar environment.

17-alpha-Hydroxyprogesterone↗
PubMed1991

On the solvent accessibility of substrate binding site of cytochrome P450C-21 in bovine adrenocortical microsomes.

The present study offers evidence indicating that acrylamide a polar molecule inhibits substrate-binding to P450C-21 in a competitive manner and quenches tryptophanyl fluorescence in bovine adrenocortical microsomes, similar to that in the purified lipid-free enzyme. Resolution of tryptophanyl fluorescence of the microsomes revealed an acrylamide quenching constant (K2 = 9.9M, is the association constant for the quencher-fluorophore complex) which was similar to the reciprocal of its inhibition constant (1/Kj = Ka = 8.3 +- 0.9M) for substrate-binding. The substrate inhibited the fluorescence quenching by acrylamide which was in accordance with partial competition. In addition the substrate dissociation, acrylamide inhibition and fluorescence quenching constants and tryptophanyl fluorescence maximum (340-342nm) were essentially the same in the microsomes and the purified enzyme. These results suggest that, similar to that in the purified enzyme, a tryptophan in a polar environment in the membrane-bound P450, may serve as a reporter group for the substrate binding site and the site in the membrane-bound enzyme, is accessible to the substrate in aqueous phase.

Acrylamide↗
PubMed1990

Binding of substrates to cytochrome P450 enzymes: mathematical artifacts in the application of difference spectrophotometry.

Investigation of the difference absorption spectrum has been a common method for studying many aspects of hemoproteins such as cytochrome P450. The difference spectrophotometric technique, as applied in studies on binding of substrates to cytochrome P450, has resulted in certain mathematical artifacts which have been mistaken for real changes in binding equilibria. The purpose of this paper is to point out these artifacts.

Binding Sites↗
PubMed1989

Heterogeneity of the bovine adrenal steroid 21-hydroxylase.

The results presented indicate that purified cytochrome P-45021 which migrated upon SDS gel electrophoresis essentially as a single band, is further separable into different species by ion-exchange chromatography. The P-450 eluted from the CM-Sephadex column at different points along the buffer concentration gradient, exhibited significant differences in (1) the 21-hydroxylation of 17 alpha-OH-progesterone compared to progesterone and (2) the Type I spectral change produced by 17 alpha-OH-progesterone compared to that due to delta 4-androstenedione. These results indicate that the purified P-450 which appeared homogeneous contains different species differing in net charge and steroid preferences for 21-hydroxylation and binding. The ratio, 21-hydroxylation of 17 alpha-OH-progesterone/progesterone ranged between 2.6 and 0.86 suggesting that the purified preparation is a mixture of 17 alpha-OH-progesterone preferring and progesterone preferring species. Possible molecular bases for the heterogeneity of the 21-hydroxylase are discussed.

Adrenal Cortex↗
PubMed1988

Quenching of tryptophanyl fluorescence of bovine adrenal P-450C-21 and inhibition of substrate binding by acrylamide.

Quenching of the tryptophanyl fluorescence of cytochrome P-450C-21 by acrylamide and its relationship to substrate binding are investigated by using steady-state and time-resolved data. The average collisional quenching constant was 0.4 M whereas the quenching constant for the total fluorescence was 10.8 +/- 0.9 M. This indicates that the quenching is essentially static. The quencher inhibited the binding of the substrate apparently competitively. The inhibition constant was 0.092 M, giving rise to an association constant of 10.9 M which is remarkably similar to the static quenching constant. It is suggested that tryptophan(s) may represent a key to the substrate-binding site in P-450C-21.

Acrylamide↗
PubMed1985

Adrenal microsomal hydroxylating system: purification and substrate binding properties of cytochrome P-450C-21.

The substrate-cytochrome P-450C-21 binding reaction has been investigated in detail by using the purified cytochrome. The apparent substrate dissociation constant (KDapp) depended on the enzyme concentration, indicating that the binding reaction does not follow simple two-component mass action equilibrium. However, the binding data fit reasonably well to a model in which the P-450C-21 exists in a monomer-dimer equilibrium and the substrate does not bind to the dimer. The intrinsic dissociation constant (K1) and the dissociation constant for the dimerization reaction (K2) were calculated from the titration data by a pattern search procedure. K1 and K2 were found to be essentially independent of the enzyme concentration, indicating the appropriateness of the assumed model. In the present study, all factors that increased the dissociation of the dimer, as indicated by an increase in K2, decreased KDapp so that it approached the intrinsic constant K1. These results suggest that there is mutual interaction of the substrate binding and self-association reactions of cytochrome P-450C-21 in the purified preparation.

Adrenal Cortex↗
PubMed1985

Interaction of PGBx and peroxides with cytochrome c and inhibition of lipid peroxidation.

PGBx, a derivative of prostaglandin B1, stimulated the oxidation of cytochrome c in the presence of H2O2. Although the reaction was nonenzymatic, the apparent activation energies of 12 and 4.9 kcal above and below the transition at 21.5 degrees C were similar to those for oxidation by cytochrome oxidase. Depletion of H2O2 and oxidation of cytochrome c followed similar time courses, suggesting that H2O2 was consumed in the reaction. PGBx was a specific requirement, but organic hydroperoxides (ethyl and T-butyl) could replace H2O2. Low concentrations of ethyl or t-butyl hydroperoxide initially stimulated the oxidation of cytochrome c; this stimulation disappeared before completion of the oxidation, but was restored when the hydroperoxide concentration was renewed, suggesting that these hydroperoxides were probably also consumed in the reaction. The concentration of PGBx (8.9 microM) required for half-maximum stimulation of the oxidation was similar to the apparent Kd for its dissociation from oxidized cytochrome c (6.8 microM). Binding data and CD spectra suggested that a 1:1 complex between cytochrome c and PGBx was formed, altering the conformation of the heme region. This conformational change caused a shift of the Soret absorption peak from 410 to 406 nm and may be responsible for the enhanced oxidizability of the cytochrome c by H2O2. Cytochrome c inhibited lipid peroxidation in microsomes, an effect enhanced by the addition of PGBx. In the absence of lipid peroxidation, cytochrome c and PGBx stimulated NADPH oxidation via NADPH-cytochrome c reductase. Thus the inhibition of lipid peroxidation by cytochrome c and PGBx may involve either the removal of hydroperoxides or deviation of electron transfer away from the pathway for lipid peroxidation.

Animals↗
PubMed1979

Constraint on the substrate cytochrome P-450 binding reaction in bovine adrenocortical microsomes at physiological temperature.

The addition of cholate to the microsomes at 37.5 degrees C resulted in a striking decrease in the apparent substrate dissociation constant (K's) and its temperature dependency. The microsomal membranes depleted of 80% of the lipids preserved the temperature dependency of the Ks and exhibited breaks in the Van't Hoff plot at the characteristic temperature of the lipids phase transition. The results indicate that the cytochrome P-450 is considerably restrained from expressing its maximum substrate binding potential at physiological temperature. In addition, the results indicate that the majority of the lipids apparently do not play a significant role in imposing constraint on the substrate-cytochrome -450 binding reaction and in the temperature dependency of the Ks.

Adrenal Cortex↗
PubMed1978

Bovine adrenocortical microsomal hydroxylase and thermotropic transition. Substrate-cytochrome P-450 binding reaction versus substrate hydroxylation.

The effect of temperature on steroid C-21 hydroxylation and substrate-cytochrome P-450 binding reaction under turnover conditions (NADPH + O2 are investigated. The Arrhenius activity plot exhibited a single break, while the van 't Hoff plot of the substrate dissociation constant (Ks) exhibited four breaks between 10 and 40 degrees C which corresponded to the characteristic temperatures of the lipids' phase transitions. Unlike the case of the Ks value, the detergent Triton X-114 was without effect on the Arrhenius activity plot. This indicates that the single break in the case of the enzyme activity is distinct from but not necessarily independent of the multiple breaks inthe case of the Ks. At physiologic temperature and concentration of the substrate, the free energy (--9.5 kcal/mol) of the substrate-cytochrome binding reaction is more than sufficient to account for the apparent activation energy (6.6 kcal/mol) of the overall hydroxylation. This suggests that the substrate-cytochrome P-450 binding reaction has the potential of being a source of energy for the overall reaction.

Adrenal Cortex↗
PubMed1977

Thermotropic transitions in fluidity of bovine adrenocortical microsomal membrane and substrate-cytochrome P-450 binding reaction.

Temperature dependence of the substrate-cytochrome P-450 binding reaction is investigated in the adrenocortical microsomes using 17-hydroxyprogesterone as the substrate. The apparent substrate dissociation constant ("Ks") increases with increase in temperature. Van't Hoff's plot of "Ks" shows breaks at 21 and 31 degrees C. These temperatures correlate well with the lipid phase transition temperatures as determined by the technique of fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene as the probe. The results indicate that the affinity of the substrate depends upon the temperature. In addition the results suggest that changes in the physical state of the lipids is a factor responsible for the temperature dependency of the reaction.

Adrenal Glands↗