An electrostatically driven conformational transition is involved in the mechanisms of substrate binding and cooperativity in cytochrome P450eryF

The effect of ionic strength (I) on substrate-induced spin transitions and cooperativity in cytochrome P450eryF was studied. At a saturating concentration of 1-pyrenebutanol (1-PB) increasing ionic strength in the 0.06-1.2 M range promotes the formation of the high-spin state of P450, which fraction increases from 26% at 0.06 M to 75% at 1.2 M. This effect was associated with a considerable decrease in cooperativity as revealed in the 1-PB-induced spin shift. While P450eryF exhibits distinct positive cooperativity (S(50) = 8.3 microM, n = 2.4) with this substrate at low ionic strength (I = 0.06 M), n decreases to 1.2 (S(50) = 3.2 microM) at I = 0.66 M. Increasing ionic strength also increases the distance between the first (effector) molecule of 1-PB and the heme, as detected by the changes in the efficiency of FRET from 1-PB to the heme. The modification of Cys(154) with 7-(diethylamino)-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) largely suppresses these effects of ionic strength and causes a prominent decrease in the cooperativity. The same effect was observed when Cys(154) was substituted with isoleucine. Importantly, Cys(154) is located at the C-terminal end of helix E and is surrounded by salt bridges formed by arginine, glutamate, and aspartate residues located in helices D, E, F, and G. Our results suggest that the binding of the first substrate molecule causes an important conformational transition in the P450eryF that facilitates the substrate-induced spin shift. This transition is apparently accompanied by dissociation or rearrangement of several salt bridges in the proximity of Cys(154) and modulates accessibility and hydration of the heme pocket.     

Resolution of multiple substrate binding sites in cytochrome P450 3A4: the stoichiometry of the enzyme-substrate complexes probed by FRET and Job's titration

To explore the mechanism of homotropic cooperativity in human cytochrome P450 3A4 (CYP3A4) we studied the interactions of the enzyme with 1-pyrenebutanol (1-PB), 1-pyrenemethylamine (PMA), and bromocriptine by FRET from the substrate fluorophore to the heme, and by absorbance spectroscopy. These approaches combined with an innovative setup of titration-by-dilution and continuous variation (Job's titration) experiments allowed us to probe the relationship between substrate binding and the subsequent spin transition caused by 1-PB or bromocriptine or the type-II spectral changes caused by PMA. The 1-PB-induced spin shift in CYP3A4 reveals prominent homotropic cooperativity, which is characterized by a Hill coefficient of 1.8 +/- 0.3 (S50 = 8.0 +/- 1.1 microM). In contrast, the interactions of CYP3A4 with bromocriptine or PMA reveal no cooperativity, exhibiting KD values of 0.31 +/- 0.08 microM and 7.1 +/- 2.3 microM, respectively. The binding of all three substrates monitored by FRET in titration-by-dilution experiments at an enzyme:substrate ratio of 1 reveals a simple bimolecular interaction with KD values of 0.16 +/- 0.09, 4.8 +/- 1.4, and 0.18 +/- 0.09 microM for 1-PB, PMA, and bromocriptine, respectively. Correspondingly, Job's titration experiments showed that the 1-PB-induced spin shift reflects the formation of a complex of the enzyme with two substrate molecules, while bromocriptine and PMA exhibit 1:1 binding stoichiometry. Combining the results of Job's titrations with the value of KD obtained in our FRET experiments, we demonstrate that the interactions of CYP3A4 with 1-PB obey a sequential binding mechanism, where the spin transition is triggered by the binding of 1-PB to the low-affinity site, which becomes possible only upon saturation of the high-affinity site.     

Role of subunit interactions in P450 oligomers in the loss of homotropic cooperativity in the cytochrome P450 3A4 mutant L211F/D214E/F304W

The contribution of conformational heterogeneity to cooperativity in cytochrome P450 3A4 was investigated using the mutant L211F/D214E/F304W. Initial spectral studies revealed a loss of cooperativity of the 1-pyrenebutanol (1-PB) induced spin shift (S(50)=5.4 microM, n=1.0) but retained cooperativity of alpha-naphthoflavone binding. Continuous variation (Job's titration) experiments showed the existence of two pools of enzyme with different 1-PB binding characteristics. Monitoring of 1-PB binding by fluorescence resonance energy transfer from the substrate to the heme confirmed that the high-affinity site (K(D)=0.3 microM) is retained in at least some fraction of the enzyme, although cooperativity is masked. Removal of apoprotein on a second column increased the high-spin content and restored cooperativity of 1-PB binding and of progesterone and testosterone 6beta-hydroxylation. The loss of cooperativity in the mutant is, therefore, mediated by the interaction of holo- and apo-P450 in mixed oligomers.     

Allosteric transitions in cytochrome P450eryF explored with pressure-perturbation spectroscopy, lifetime FRET, and a novel fluorescent substrate, Fluorol-7GA

To establish a direct method for monitoring substrate binding in cytochrome P450eryF applicable at elevated hydrostatic pressures, we introduce a laser dye Fluorol-7GA (F7GA) as a novel fluorescent ligand. The high intensity of fluorescence and the reasonable resolution of the excitation band from the absorbance bands of P450 allowed us to establish highly sensitive binding assays compatible with pressure perturbation. The interactions of F7GA with P450eryF cause an ample spin shift revealing cooperative binding ( S50 = 8.2 +/- 1.3 microM; n = 2.3 +/- 0.1). Fluorescence resonance energy transfer (FRET) experiments suggest the presence of at least two substrate binding sites with apparent K D values in the ranges of 0.1-0.3 and 6-9 microM. Similar to that observed earlier with CYP3A4, increasing hydrostatic pressure does not cause either a complete dissociation of the substrate complexes or a displacement of the spin equilibrium toward the low-spin state. Rather, increased pressure enhances the cooperativity of the F7GA-induced spin shift, so that the Hill coefficient approaches 3 at 2 kbar. Lifetime FRET experiments revealed an important increase in the affinity of the enzyme for F7GA at elevated pressures, suggesting that the binding of the ligand induces a conformational transition associated with an important increase in the level of protein hydration. This transition largely attenuates the solvent accessibility of the heme pocket and causes an unusual stability of the high-spin, substrate-bound enzyme at elevated pressures.     

Mechanism of interactions of alpha-naphthoflavone with cytochrome P450 3A4 explored with an engineered enzyme bearing a fluorescent probe

Design of a partially cysteine-depleted C98S/C239S/C377S/C468A cytochrome P450 3A4 mutant designated CYP3A4(C58,C64) allowed site-directed incorporation of thiol-reactive fluorescent probes into alpha-helix A. The site of modification was identified as Cys-64 with the help of CYP3A4(C58) and CYP3A4(C64), each bearing only one accessible cysteine. Changes in the fluorescence of CYP3A4(C58,C64) labeled with 6-(bromoacetyl)-2-(dimethylamino)naphthalene (BADAN), 7-(diethylamino)-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM), or monobromobimane (mBBr) were used to study the interactions with bromocriptine (BCT), 1-pyrenebutanol (1-PB), testosterone (TST), and alpha-naphthoflavone (ANF). Of these substrates only ANF has a specific effect, causing a considerable decrease in fluorescence intensity of BADAN and CPM and increasing the fluorescence of mBBr. This ANF-binding event in the case of the BADAN-modified enzyme is characterized by an S50 of 18.2 +/- 0.7, compared with the value of 2.2 +/- 0.3 for the ANF-induced spin transition, thus revealing an additional low-affinity binding site. Studies of the effect of TST, 1-PB, and BCT on the interactions of ANF monitored by changes in fluorescence of CYP3A4(C58,C64)-BADAN or by the ANF-induced spin transition revealed no competition by these substrates. Investigation of the kinetics of fluorescence increase upon H2O2-dependent heme depletion suggests that labeled CYP3A4(C58,C64) is represented by two conformers, one of which has the fluorescence of the BADAN and CPM labels completely quenched, presumably by photoinduced electron transfer from the neighboring Trp-72 and/or Tyr-68 residues. The binding of ANF to the newly discovered binding site appears to affect the interactions of the label with the above residue(s), thus modulating the fraction of the fluorescent conformer.     

Allosteric mechanisms in cytochrome P450 3A4 studied by high-pressure spectroscopy: pivotal role of substrate-induced changes in the accessibility and degree of hydration of the heme pocket

Allosteric mechanisms in human cytochrome P450 3A4 (CYP3A4) in oligomers in solution or monomeric enzyme incorporated into Nanodiscs (CYP3A4ND) were studied by high-pressure spectroscopy. The allosteric substrates 1-pyrenebutanol (1-PB) and testosterone were compared with bromocriptine (BCT), which shows no cooperativity. In both CYP3A4 in solution and CYP3A4ND, we observed a complete pressure-induced high-to-low spin shift at pressures of <3 kbar either in the substrate-free enzyme or in the presence of BCT. In addition, both substrate-free and BCT-bound enzyme revealed a pressure-dependent equilibrium between two states with different barotropic parameters designated R for relaxed and P for pressure-promoted conformations. This pressure-induced conformational transition was also observed in the studies with 1-PB and testosterone. In CYP3A4 oligomers, the transition was accompanied by an important increase in homotropic cooperativity with both substrates. Surprisingly, at high concentrations of allosteric substrates, the amplitude of the spin shift in both CYP3A4 in solution and Nanodiscs was very low, demonstrating that hydrostatic pressure induces neither substrate dissociation nor an increase in the heme pocket hydration in the complexes of the pressure-promoted conformation of CYP3A4 with 1-PB or testosterone. These findings suggest that the mechanisms of interactions of CYP3A4 with 1-PB and testosterone involve an effector-induced transition that displaces a system of conformational equilibria in the enzyme toward the state(s) with decreased solvent accessibility of the active site so that the flux of water into the heme pocket is impeded and the high-spin state of the heme iron is stabilized.     

NMR studies of ligand binding to P450(eryF) provides insight into the mechanism of cooperativity

Cytochrome P450's (P450's) catalyze the oxidative metabolism of most drugs and toxins. Although extensive studies have proven that some P450's demonstrate both homotropic and heterotropic cooperativity toward a number of substrates, the mechanistic and molecular details of P450 allostery are still not well-established. Here, we use UV/vis and heteronuclear nuclear magnetic resonance (NMR) spectroscopic techniques to study the mechanism and thermodynamics of the binding of two 9-aminophenanthrene (9-AP) and testosterone (TST) molecules to the erythromycin-metabolizing bacterial P450(eryF). UV/vis absorbance spectra of P450(eryF) demonstrated that binding occurs with apparent negative homotropic cooperativity for TST and positive homotropic cooperativity for 9-AP with Hill-equation-derived dissociation constants of K(S) = 4 and 200 microM, respectively. The broadening and shifting observed in the 2D-{1H,15N}-HSQC-monitored titrations of 15N-Phe-labeled P450(eryF) with 9-AP and TST indicated binding on intermediate and fast chemical exchange time scales, respectively, which was consistent with the Hill-equation-derived K(S) values for these two ligands. Regardless of the type of spectral perturbation observed (broadening for 9-AP and shifting for TST), the 15N-Phe NMR resonances most affected were the same in each titration, suggesting that the two ligands "contact" the same phenylalanines within the active site of P450(eryF). This finding is in agreement with X-ray crystal structures of bound P450(eryF) showing different ligands occupying similar active-site niches. Complex spectral behavior was additionally observed for a small collection of resonances in the TST titration, interpreted as multiple binding modes for the low-affinity TST molecule or multiple TST-bound P450(eryF) conformational substates. A structural and energetic model is presented that combines the energetics and structural aspects of 9-AP and TST binding derived from these observations.     

Temperature jump relaxation kinetics of the P-450cam spin equilibrium

The ferric spin-state equilibrium and relaxation rate of cytochrome P-450 has been examined with temperature jump spectroscopy using a number of camphor analogues known to induce different mixed spin states in the substrate-bound complexes [Gould, P., Gelb, M., & Sligar, S. G. (1981) J. Biol. Chem. 256, 6686]. All temperature-induced spectral changes were monophasic, and the spin-state relaxation rate reached a limiting value at high substrate concentrations. The ferric spin equilibrium constant, Kspin, is defined in terms of the rate constants k1 and k-1 via Kspin = k1/k-1 = [P-450(HS)]/[P-450(LS)] where HS and LS represent high-spin (S = 5/2) and low-spin (S = 1/2) ferric iron, respectively, and the spectrally observed spin-state relaxation rate by kobsd = k1 + k-1. A strong correlation between the fraction of high-spin species and the rate constant, k-1, is observed. For a 3 degrees C temperature jump (from 10 to 13 degrees C), the 23% high-spin tetramethylcyclohexanone complex (Kd = 45 +/- 20 microM) is characterized by a ferric spin relaxation rate of kobsd = 1990 s-1, while the rates for the d-fenchone (41% high spin, Kd = 42 +/- 10 microM) and kobsd = 1990 s-1, while the rates for the d-fenchone (41% high spin, Kd = 42 +/- 10 microM) and camphoroquinone (75% high spin, Kd = 15 +/- 5 microM) complexes are 1430 and 346 s-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that cysteine 298 is in the active site of tryptophan indole-lyase

Escherichia coli tryptophan indole-lyase (tryptophanase) mutants, with cysteine residues 294 and 298 selectively replaced by serines, have been prepared by site-directed mutagenesis. Both mutant enzymes are highly active for beta-elimination reactions measured with both L-tryptophan and S-(o-nitrophenyl)-L-cysteine. The Cys-294----Ser mutant enzyme is virtually identical to the wild type with respect to pyridoxal phosphate binding (KCO = 2 microM), cofactor absorption spectrum (lambda max = 420 and 337 nm) and pH dependence (pK alpha = 7.3), pH profile for catalysis, and rate of bromopyruvic acid inactivation. In contrast, the Cys-298----Ser mutant enzyme exhibits a reduced affinity for pyridoxal phosphate (KCO = 6 microM), a shift in the cofactor absorption spectrum to 414 nm and an altered pK alpha = 8.5, an alkaline shift in the pH profile for catalysis, and resistance to inactivation of the apoenzyme by bromopyruvic acid. The C298S mutant enzyme (wherein cysteine 298 is altered to serine) also undergoes an isomerization to an unreactive state upon storage at 4 degrees C. These results demonstrate that the sulfhydryl groups of Cys-294 and Cys-298 are catalytically nonessential. However, these data suggest that Cys-298 is located within or very near the active site of the enzyme and is the reactive cysteine residue previously observed by others.     

High-pressure investigations of cytochrome P-450 spin and substrate binding equilibria

The effects of high pressure (1-2000 bar) on the spin state and substrate binding equilibria in cytochrome P-450 have been determined. The high-spin (S = 5/2) to low spin (S = 1/2) transition of the ferric hemoprotein was monitored by uv-visible spectroscopy at various substrate concentrations. Increasing hydrostatic pressure on a sample of substrate-bound cytochrome P-450 resulted in a decrease in the high-spin fraction as monitored by a Soret maxima at 391 nm and an increase in the low-spin 417-nm region of the spectrum. These pressure-induced optical changes were totally reversible for all pressures below 800 bar and were found to correspond to simple substrate dissociation from the enzyme. High levels of the normally metabolized substrate, d-camphor, corresponding to a 99.9% saturation of the hemoprotein active site (50 mM Tris-Cl, 100 mM KCl, pH 7.2) completely prevented the pressure-induced high-spin to low-spin transition that is observed at less than saturating substrate concentrations. A gradual increase in the formation of the inactive P-420 form of the cytochrome was noted if the pressure of the sample was increased above 800 bar. These pressure-linked spectral changes were used to determine the microscopic volume change accompanying substrate binding, which was found to be -47.0 +/- 2 ml/mol (pH 7.2) which represents a substantial change for a ligand dissociation reaction. The observed volume change for camphor binding decreases to -30.6 +/- 2 ml/mol at pH 6.0, suggesting the involvement of a linked proton equilibrium. Various substrate analogs of camphor induce varying degrees of low-spin to high-spin shift upon binding to ferric cytochrome P-450 (3). The volume changes for the dissociation of these substrates were very similar to those obtained with camphor. The conformational changes associated with a shift from high- to low-spin ferric iron appear to be small in comparison to the overall macroscopic changes in volume accompanying substrate binding to the enzyme.     

The Role of Serine-246 in Cytochrome P450eryF-Catalyzed Hydroxylation of 6-Deoxyerythronolide B

A strongly conserved threonine residue in the I-helix of cytochrome P450 enzymes participates in a proton delivery system for binding and cleavage of dioxygen molecules. 6-Deoxyerythronol ide B hydroxylase (P450eryF) is unusual in that the conserved threonine residue is replaced by alanine in this enzyme. On the basis of crystal structures of substrate-bound P450eryF, it has been proposed that the C-5 hydroxyl group of the substrate and serine-246 of the enzyme form hydrogen bonds with water molecules 519 and 564, respectively. This hydrogen bonding network constitutes the proton delivery system whereby P450eryF maintains its catalytic activity in the absence of a threonine hydroxyl group in the conserved position. To further assess the role in the proton delivery system of hydroxyl groups around the active site, three mutant forms of P450eryF (A245S, S246A, and A245S/S246A) were constructed and characterized. In each case, decreased catalytic activity and increased uncoupling could be correlated with changes in the hydrogen bonding environment. These results suggest that Ser-246 does indeed participate in the proton shuttling pathway, and also support our previous hypothesis that the C-5 hydroxyl group of the substrate participates in the acid-catalyzed dioxygen bond cleavage reaction. Copyright 2000 Academic Press.     

Aurothiomalate inhibits transformed growth by targeting the PB1 domain of protein kinase Ciota

We recently identified the gold compound aurothiomalate (ATM) as a potent inhibitor of the Phox and Bem1p (PB1)-PB1 domain interaction between protein kinase C (PKC) iota and the adaptor molecule Par6. ATM also blocks oncogenic PKCiota signaling and the transformed growth of human lung cancer cells. Here we demonstrate that ATM is a highly selective inhibitor of PB1-PB1 domain interactions between PKCiota and the two adaptors Par6 and p62. ATM has no appreciable inhibitory effect on other PB1-PB1 domain interactions, including p62-p62, p62-NBR1, and MEKK3-MEK5 interactions. ATM can form thio-gold adducts with cysteine residues on target proteins. Interestingly, PKCiota (and PKCzeta) contains a unique cysteine residue, Cys-69, within its PB1 domain that is not present in other PB1 domain containing proteins. Cys-69 resides within the OPR, PC, and AID motif of PKCiota at the binding interface between PKCiota and Par6 where it interacts with Arg-28 on Par6. Molecular modeling predicts formation of a cysteinyl-aurothiomalate adduct at Cys-69 that protrudes into the binding cleft normally occupied by Par6, providing a plausible structural explanation for ATM inhibition. Mutation of Cys-69 of PKCiota to isoleucine or valine, residues frequently found at this position in other PB1 domains, has little or no effect on the affinity of PKCiota for Par6 but confers resistance to ATM-mediated inhibition of Par6 binding. Expression of the PKCiota C69I mutant in human non-small cell lung cancer cells confers resistance to the inhibitory effects of ATM on transformed growth. We conclude that ATM inhibits cellular transformation by selectively targeting Cys-69 within the PB1 domain of PKCiota.     

The role of serine-246 in cytochrome P450eryF-catalyzed hydroxylation of 6-deoxyerythronolide B

A strongly conserved threonine residue in the I-helix of cytochrome P450 enzymes participates in a proton delivery system for binding and cleavage of dioxygen molecules. 6-Deoxyerythronolide B hydroxylase (P450eryF) is unusual in that the conserved threonine residue is replaced by alanine in this enzyme. On the basis of the crystal structures of substrate-bound P450eryF, it has been proposed that the C-5 hydroxyl group of the substrate and serine-246 of the enzyme form hydrogen bonds with water molecules 519 and 564, respectively. This hydrogen bonding network constitutes the proton delivery system whereby P450eryF maintains its catalytic activity in the absence of a threonine hydroxyl group in the conserved position. To further assess the role in the proton delivery system of hydroxyl groups around the active site, three mutant forms of P450eryF (A245S, S246A, and A245S/S246A) were constructed and characterized. In each case, decreased catalytic activity and increased uncoupling could be correlated with changes in the hydrogen bonding environment. These results suggest that Ser-246 does indeed indirectly participate in the proton shuttling pathway, and also strongly support our previous hypothesis that the C-5 hydroxyl group of the substrate participates in the acid-catalyzed dioxygen bond cleavage reaction.     

Effect of glutathione on homo- and heterotropic cooperativity in cytochrome P450 3A4

Glutathione (GSH) exerted a profound effect on the oxidation of 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) and 7-benzyloxyquinoline (BQ) by human liver microsomes as well as by CYP3A4-containing insect cell microsomes (Baculosomes). The cooperativity in O-debenzylation of both substrates is eliminated in the presence of 1-4 mM GSH. Addition of GSH also increased the amplitude of the 1-PB induced spin shift with purified CYP3A4 and abolished the cooperativity of 1-PB or BFC binding. Changes in fluorescence of 6-bromoacetyl-2-dimethylaminonaphthalene attached to the cysteine-depleted mutant CYP3A4(C58,C64) suggest a GSH-induced conformational changes in proximity of α-helix A. Importantly, the K_S value for formation of the GSH complex and the concentrations in which GSH decreases CYP3A4 cooperativity are consistent with the physiological concentrations of GSH in hepatocytes. Therefore, the allosteric effect of GSH on CYP3A4 may play an important role in regulation of microsomal monooxygenase activity in vivo.     

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).     

Determination of disulfide bond assignment of human vitamin K-dependent gamma-glutamyl carboxylase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Vitamin K-dependent gamma-glutamyl carboxylase is a 758 amino acid integral membrane glycoprotein that catalyzes the post-translational conversion of certain protein glutamate residues to gamma-carboxyglutamate. Carboxylase has ten cysteine residues, but their form (sulfhydryl or disulfide) is largely unknown. Pudota et al. in Pudota, B. N., Miyagi, M., Hallgren, K. W., West, K. A., Crabb, J. W., Misono, K. S., and Berkner, K. L. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 13033-13038 reported that Cys-99 and Cys-450 are the carboxylase active site residues. We determined the form of all cysteines in carboxylase using in-gel protease digestion and matrix-assisted laser desorption/ionization mass spectrometry. The spectrum of non-reduced, trypsin-digested carboxylase revealed a peak at m/z 1991.9. Only this peak disappeared in the spectrum of the reduced sample. This peak's m/z is consistent with the mass of peptide 92-100 (Cys-99) disulfide-linked with peptide 446-453 (Cys-450). To confirm its identity, the m/z 1991.9 peak was isolated by a timed ion selector as the precursor ion for further MS analysis. The fragmentation pattern exhibited two groups of triplet ions characteristic of the symmetric and asymmetric cleavage of disulfide-linked tryptic peptides containing Cys-99 and Cys-450. Mutation of either Cys-99 or Cys-450 caused loss of enzymatic activity. We created a carboxylase variant with both C598A and C700A, leaving Cys-450 as the only remaining cysteine residue in the 60-kDa fragment created by limited trypsin digestion. Analysis of this fully active mutant enzyme showed a 30- and the 60-kDa fragment were joined under non-reducing conditions, thus confirming Cys-450 participates in a disulfide bond. Our results indicate that Cys-99 and Cys-450 form the only disulfide bond in carboxylase.     

Probing the CYP3A4 active site by cysteine scanning mutagenesis and photoaffinity labeling

The mechanism of CYP3A4-substrate interactions has been investigated using a battery of techniques including cysteine scanning mutagenesis, photoaffinity labeling, and structural modeling. In this study, cysteine scanning mutagenesis was performed at seven sites within CYP3A4 proposed to be involved in substrate interaction and/or cooperativity. Photolabeled CYP3A4 peptide adducts were further characterized by mass spectrometric analysis for each mutant after proteolytic digestion and isolation of fluorescent photolabeled peptides. Among the tryptic peptides of seven tested mutants, three photolabeled peptides of the F108C mutant, ECYSVFTNR (positions 97-105), VLQNFSFKPCK (positions 459-469), and RPCGPVGFMK (positions 106-115) were identified by MALDI-TOF-MS and nano-LC/ESI QTOF MS. The site of modification was further localized to the substituted Cys-108 residue in the mutant peptide adduct RPCGPVGFMK (positions 106-115) by nano-LC/ESI QTOF MS/MS. In summary, we described a potentially useful method to study P450 active sites using a combination of cysteine scanning mutagenesis and photoaffinity labeling.     

Mechanism of inactivation of rat liver microsomal cytochrome P-450c by 2-bromo-4'-nitroacetophenone

The mechanism by which 2-bromo-4'-nitroacetophenone (BrNAP) inactivates cytochrome P-450c, which involves alkylation primarily at Cys-292, is shown in the present study to involve an uncoupling of NADPH utilization and oxygen consumption from product formation. Alkylation of cytochrome P-450c with BrNAP markedly stimulated (approximately 30-fold) its rate of anaerobic reduction by NADPH-cytochrome P-450 reductase, as determined by stopped flow spectroscopy. This marked stimulation in reduction rate is highly unusual in that Cys-292 is apparently not part of the heme- or substrate-binding site, and its alkylation by BrNAP does not cause a low spin to high spin state transition in cytochrome P-450c. Under aerobic conditions the rapid oxidation of NADPH catalyzed by alkylated cytochrome P-450c was associated with rapid reduction of molecular oxygen to hydrogen peroxide via superoxide anion. The intermediacy of superoxide anion, formed by the one-electron reduction of molecular oxygen, established that alkylation of cytochrome P-450c with BrNAP uncouples the catalytic cycle prior to introduction of the second electron. The generation of superoxide anion by decomposition of the Fe2+ X O2 complex was consistent with the observations that, in contrast to native cytochrome P-450c, alkylated cytochrome P-450c failed to form a 430 nm absorbing chromophore during the metabolism of 7-ethoxycoumarin. Alkylation of cytochrome P-450c with BrNAP did not completely uncouple the catalytic cycle such that 5-20% of the catalytic activity remained for the alkylated cytochrome compared to the native protein depending on the substrate assayed. The uncoupling effect was, however, highly specific for cytochrome P-450c. Alkylation of nine other rat liver microsomal cytochrome P-450 isozymes with BrNAP caused little or no increase in hydrogen peroxide formation in the presence of NADPH-cytochrome P-450 reductase and NADPH.     

Ketoconazole-induced conformational changes in the active site of cytochrome P450eryF

The azole-based P450 inhibitor ketoconazole is used to treat fungal infections and functions by blocking ergosterol biosynthesis in yeast. Ketoconazole binds to mammalian P450 enzymes and this can result in drug-drug interactions and lead to liver damage. To identify protein-drug interactions that contribute to binding specificity and affinity, we determined the crystal structure of ketoconazole complexed with P450eryF. In the P450eryF/ketoconazole structure, the azole moiety and nearby rings of ketoconzole are positioned in the active site similar to the substrate, 6-deoxyerythronolide B, with the azole nitrogen atom coordinated to the heme iron atom. The remainder of the ketoconazole molecule extends into the active-site pocket, which is occupied by water in the substrate complex. Binding of ketoconazole led to unexpected conformational changes in the I-helix. The I-helix cleft near the active site has collapsed with a helical pitch of 5.4 A compared to 6.6 A in the substrate complex. P450eryF/ketoconazole crystals soaked in 6-deoxyerythronolide B to exchange ligands exhibit a structure identical with that of the original P450eryF/substrate complex, with the I-helix cleft restored to a pitch of 6.6 A. These findings indicate that the I-helix region of P450eryF is flexible and can adopt multiple conformations. An improved understanding of the flexibility of the active-site region of cytochrome P450 enzymes is important to gain insight into determinants of ligand binding/specificity as well as to evaluate models for catalytic mechanism based on static crystal structures.     

Identification of the cysteine residues in the amino-terminal extracellular domain of the human Ca(2+) receptor critical for dimerization. Implications for function of monomeric Ca(2+) receptor.

We analyzed the effect of substituting serine for each of the 19 cysteine residues within the amino-terminal extracellular domain of the human Ca(2+) receptor on cell surface expression and receptor dimerization. C129S, C131S, C437S, C449S, and C482S were similar to wild type receptor; the other 14 cysteine to serine mutants were retained intracellularly. Four of these, C60S, C101S, C358S and C395S, were unable to dimerize. A C129S/C131S double mutant failed to dimerize but was unique in that the monomeric form expressed at the cell surface. Substitution of a cysteine for serine 132 within the C129S/C131S mutant restored receptor dimerization. Mutation of residues Cys-129, Cys-131, and Ser-132, singly and in various combinations caused a left shift in Ca(2+) response compared with wild type receptor. These results identify cysteines 129 and 131 as critical in formation of intermolecular disulfide bond(s) responsible for receptor dimerization. In a "venus flytrap" model of the receptor extracellular domain, Cys-129 and Cys-131 are located within a region protruding from one lobe of the flytrap. We suggest that this region represents a dimer interface for the receptor and that mutation of residues within the interface causes important changes in Ca(2+) response of the receptor.