Homotropic cooperativity of monomeric cytochrome P450 3A4 in a nanoscale native bilayer environment

Mechanistic studies of mammalian cytochrome P450s are often obscured by the phase heterogeneity of solubilized preparations of membrane enzymes. The various protein-protein aggregation states of microsomes, detergent solubilized cytochrome or a family of aqueous multimeric complexes can effect measured substrate binding events as well as subsequent steps in the reaction cycle. In addition, these P450 monooxygenases are normally found in a membrane environment and the bilayer composition and dynamics can also effect these catalytic steps. Here, we describe the structural and functional characterization of a homogeneous monomeric population of cytochrome P450 3A4 (CYP 3A4) in a soluble nanoscale membrane bilayer, or Nanodisc [Nano Lett. 2 (2002) 853]. Cytochrome P450 3A4:Nanodisc assemblies were formed and purified to yield a 1:1 ratio of CYP 3A4 to Nanodisc. Solution small angle X-ray scattering was used to structurally characterize this monomeric CYP 3A4 in the membrane bilayer. The purified CYP 3A4:Nanodiscs showed a heretofore undescribed high level of homotropic cooperativity in the binding of testosterone. Soluble CYP 3A4:Nanodisc retains its known function and shows prototypic hydroxylation of testosterone when driven by hydrogen peroxide. This represents the first functional characterization of a true monomeric preparation of cytochrome P450 monooxygenase in a phospholipid bilayer and elucidates new properties of the monomeric form.     

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.     

Indole hydroxylation by bacterial cytochrome P450 BM-3 and modulation of activity by cumene hydroperoxide

Cytochrome P450 BM-3 from Bacillus megaterium catalyzed NADPH-supported indole hydroxylation under alkaline conditions with homotropic cooperativity toward indole. The activity was also found with the support of H2O2, tert-butyl hydroperoxide (tBuOOH), or cumene hydroperoxide (CuOOH). Enhanced activity and heterotropic cooperativity were observed in CuOOH-supported hydroxylation, and both the Hill coefficient and substrate concentration required for half-maximal activity in the CuOOH-supported reaction were much lower than those in the H2O2-, tBuOOH-, or NADPH-supported reactions. CuOOH greatly enhanced NADPH consumption and indole hydroxylation in the NADPH-supported reaction. However, when CuOOH was replaced by tBuOOH or H2O2, heterotropic cooperativity was not observed. Spectral studies also confirmed that CuOOH stimulated indole binding to P450 BM-3. Interestingly, a mutant enzyme with enhanced indole-hydroxylation activity, F87V (Phe87 was replaced by Val), lost homotropic cooperativity towards indole and heterotropic cooperativity towards CuOOH, indicating that the active-site structure affects the cooperativities.     

Methionine-321 in the C-terminal α-helix of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa is important for positive homotropic cooperativity

Abstract Pseudomonas aeruginosa has a pair of distinct ornithine carbamoyltransferases. The anabolic ornithine carbamoyltransferase encoded by the argF gene catalyzes the formation of citrulline from ornithine and carbamoylphosphate. The catabolic ornithine carbamoyltransferase encoded by the arcB gene promotes the reverse reaction in vivo; although this enzyme can be assayed in vitro for citrulline synthesis, its unidirectionality in vivo is determined by its high concentration at half maximum velocity for carbamoylphosphate ([S]_0.5) and high cooperativity toward this substrate. We have mutant forms of catabolic ornithine carbamoyltransferase catalyzing the anabolic reaction in vivo. The corresponding arcB mutant alleles on a multicopy plasmid specifically suppressed an argF mutation of P. aeruginosa . Two new mutant enzymes were obtained. When methionine 321 was replaced by isoleucine, the mutant enzyme showed loss of homotropic cooperativity at physiological carbamoylphosphate concentrations. Substitution of glutamate 105 by lysine resulted in a partial loss of the sigmoidal response to increasing carbamoylphosphate concentrations. However, both mutant enzymes were still sensitive to the allosteric activator AMP and to the inhibitor spermidine. These results indicate that at least two residues of catabolic ornithine carbamoyltransferase are critically involved in positive carbamoylphisphate cooperativity: glutamate 105 (previously known to be important) and methionine 321. Mutational changes in either amino acid will affect the geometry of helix H2, which contains several residues required for carbamoylphosphate binding.     

The structural basis for homotropic and heterotropic cooperativity of midazolam metabolism by human cytochrome P450 3A4

Human cytochrome P450 3A4 (CYP3A4) metabolizes a significant portion of clinically relevant drugs and often exhibits complex steady-state kinetics that can involve homotropic and heterotropic cooperativity between bound ligands. In previous studies, the hydroxylation of the sedative midazolam (MDZ) exhibited homotropic cooperativity via a decrease in the ratio of 1'-OH-MDZ to 4-OH-MDZ at higher drug concentrations. In this study, MDZ exhibited heterotropic cooperativity with the antiepileptic drug carbamazepine (CBZ) with characteristic decreases in the 1'-OH-MDZ to 4-OH-MDZ ratios. To unravel the structural basis of MDZ cooperativity, we probed MDZ and CBZ bound to CYP3A4 using longitudinal T(1) nuclear magnetic resonance (NMR) relaxation and molecular docking with AutoDock 4.2. The distances calculated from longitudinal T(1) NMR relaxation were used during simulated annealing to constrain the molecules to the substrate-free X-ray crystal structure of CYP3A4. These simulations revealed that either two MDZ molecules or an MDZ molecule and a CBZ molecule assume a stacked configuration within the CYP3A4 active site. In either case, the proton at position 4 of the MDZ molecule was closer to the heme than the protons of the 1'-CH(3) group. In contrast, molecular docking of a single molecule of MDZ revealed that the molecule was preferentially oriented with the 1'-CH(3) position closer to the heme than position 4. This study provides the first detailed molecular analysis of heterotropic and homotropic cooperativity of a human cytochrome P450 from an NMR-based model. Cooperativity of ligand binding through direct interaction between stacked molecules may represent a common motif for homotropic and heterotropic cooperativity.     

Elucidation of distinct ligand binding sites for cytochrome P450 3A4

Cytochrome P450 (P450) 3A4 is the most abundant human P450 enzyme and has broad selectivity for substrates. The enzyme can show marked catalytic regioselectivity and unusual patterns of homotropic and heterotropic cooperativity, for which several models have been proposed. Spectral titration studies indicated one binding site for the drug indinavir (M(r) 614), a known substrate and inhibitor. Several C-terminal aminated peptides, including the model morphiceptin (YPFP-NH(2)), bind with spectral changes indicative of Fe-NH(2) bonding. The binding of the YPFP-NH(2) N-terminal amine and the influence of C-terminal modification on binding argue that the entire molecule (M(r) 521) fits within P450 3A4. YPFP-NH(2) was not oxidized by P450 3A4 but blocked binding of the substrates testosterone and midazolam, with K(i) values similar to the spectral binding constant (K(s)) for YPFP-NH(2). YPFP-NH(2) inhibited the oxidations of several typical P450 substrates with K(i) values 10-fold greater than the K(s) for binding YPFP-NH(2) and its K(i) for inhibiting substrate binding. The n values for cooperativity of these oxidations were not altered by YPFP-NH(2). YPFP-NH(2) inhibited the oxidations of midazolam at two different positions (1'- and 4-) with 20-fold different K(i) values. The differences in the K(i) values for blocking the binding to ferric P450 3A4 and the oxidation of several substrates may be attributed to weaker binding of YPFP-NH(2) to ferrous P450 3A4 than to the ferric form. The ferrous protein can be considered a distinct form of the enzyme in binding and catalysis because many substrates (but not YPFP-NH(2)) facilitate reduction of the ferric to ferrous enzyme. Our results with these peptides are considered in the context of several proposed models. A P450 3A4 model based on these peptide studies contains at least two and probably three distinct ligand sites, with testosterone and alpha-naphthoflavone occupying distinct sites. Midazolam appears to be able to bind to P450 3A4 in two modes, one corresponding to the testosterone binding mode and one postulated to reflect binding in a third site, distinct from both testosterone and alpha-naphthoflavone. The work with indinavir and YPFP-NH(2) also argues that room should be present in P450 3A4 to bind more than one smaller ligand in the "testosterone" site, although no direct evidence for such binding exists. Although this work with peptides provides evidence for the existence of multiple ligand binding sites, the results cannot be used to indicate their juxtaposition, which may vary through the catalytic cycle.     

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.     

Structural characterization of the homotropic cooperative binding of azamulin to human cytochrome P450 3A5

Cytochrome P450 3A4 and 3A5 catalyze the metabolic clearance of a large portion of therapeutic drugs. Azamulin is used as a selective inhibitor for 3A4 and 3A5 to define their roles in metabolism of new chemical entities during drug development. In contrast to 3A4, 3A5 exhibits homotropic cooperativity for the sequential binding of two azamulin molecules at concentrations used for inhibition. To define the underlying sites and mechanisms for cooperativity, an X-ray crystal structure of 3A5 was determined with two azamulin molecules in the active site that are stacked in an antiparallel orientation. One azamulin resides proximal to the heme in a pose similar to the 3A4–azamulin complex. Comparison to the 3A5 apo structure indicates that the distal azamulin in 3A5 ternary complex causes a significant induced fit that excludes water from the hydrophobic surfaces of binding cavity and the distal azamulin, which is augmented by the stacking interaction with the proximal azamulin. Homotropic cooperativity was not observed for the binding of related pleuromutilin antibiotics, tiamulin, retapamulin, and lefamulin, to 3A5, which are larger and unlikely to bind in the distal site in a stacked orientation. Formation of the 3A5 complex with two azamulin molecules may prevent time-dependent inhibition that is seen for 3A4 by restricting alternate product formation and/or access of reactive intermediates to vulnerable protein sites. These results also contribute to a better understanding of sites for cooperative binding and the differential structural plasticity of 3A5 and 3A4 that contribute to differential substrate and inhibitor binding.     

The role of cytochrome P450 BM3 phenylalanine-87 and threonine-268 in binding organic hydroperoxides

BackgroundCytochrome P450 (P450) BM3, from Bacillus megaterium, catalyzes a wide range of chemical reactions and is routinely used as a model system to study mammalian P450 reactions and structure.MethodsThe metabolism of 2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (BHTOOH) and 2-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadien-1-one (BMPOOH) was examined with P450 BM3 and with the conserved T268 and F87 residues mutated to investigate their effects on organic hydroperoxide metabolism. To determine the effects of the mutations on the active site volume and architecture, the X-ray crystal structure of the F87A/T268A P450 BM3 heme domain (BMP) was determined and compared to previous structures. To investigate the interactions of the substrates with the F87 and T268 residues, BHTOOH and BMPOOH were docked into the BMP X-ray crystal structures.ResultsLower metabolism of BHTOOH and BMPOOH was observed in the WT P450 BM3 and the T268A P450 BM3 mutant than in the F87A and F87A/T268A P450 BM3 mutants. Large differences were found in the F–G loop regions and active site cavity volumes for the F87A mutated structures.ConclusionsAnalysis of the metabolism, X-ray crystal structures, and molecular docking simulations suggests that P450 BM3 activity toward BHTOOH and BMPOOH is mediated through substrate recognition by T268 and F87, and the active site cavity volume. Based on this information, a simplified representation is presented with the relative orientation of organic hydroperoxides in the P450 BM3 active site.General significanceThe metabolism results and structural analysis of this model P450 allowed us to rationalize the structural factors that influence organic hydroperoxide metabolism.     

Evaluation of alkoxyresorufins as fluorescent substrates for cytochrome P450 BM3 and site-directed mutants

In this study, the first fluorescent assay for bacterial cytochrome P450 BM3 (BM3) and mutants is described. BM3 mutants are potentially very versatile biocatalysts for the production of fine chemicals. A fluorescent assay would be very useful for the identification of nonnatural ligands in high-throughput inhibition assays. Because of the ease and sensitivity of alkoxyresorufin O-dealkylation assays, four different alkoxyresorufins were evaluated as substrates. Wild-type BM3 showed extremely low activity toward all four alkoxyresorufins tested. Five different BM3 mutants were constructed, carrying different combinations of mutations R47L, F87V, and L188Q, which were previously shown to increase activity toward nonnatural substrates. For all mutants, a high benzyloxyresorufin O-dealkylation (BROD) activity was found. The triple mutant of BM3, R47L/F87V/L188Q, showed the highest activity, increasing 900-fold compared to wild-type BM3. The BROD assay could also be applied in whole Escherichia coli cells; permeabilization by lipopolysaccharide deficiency strongly increased activity. To demonstrate the applicability of the BROD assay to screening for novel ligands of BM3 R47L/F87V/L188Q, a library of 45 drug-like compounds was tested for inhibition. Of these compounds, 8 showed strong inhibition of the BROD activity, demonstrating for the first time that drug-like molecules also can bind with high affinity to BM3 mutants.     

Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1)

Fatty acid binding and oxidation kinetics for wild type P450_BM3 (CYP102A1) from Bacillus megaterium have been found to display chain length-dependent homotropic behavior. Laurate and 13-methyl-myristate display Michaelis-Menten behavior while there are slight deviations with myristate at low ionic strengths. Palmitate shows Michaelis-Menten kinetics and hyperbolic binding behavior in 100 mmol/L phosphate, pH 7.4, but sigmoidal kinetics (with an apparent intercept) in low ionic strength buffers and at physiological phosphate concentrations. In low ionic strength buffers both the heme domain and the full-length enzyme show complex palmitate binding behavior that indicates a minimum of four fatty acid binding sites, with high cooperativity for the binding of the fourth palmitate molecule, and the full-length enzyme showing tighter palmitate binding than the heme domain. The first flavin-to-heme electron transfer is faster for laurate, myristate and palmitate in 100 mmol/L phosphate than in 50 mmol/L Tris (pH 7.4), yet each substrate induces similar high-spin heme content. For palmitate in low phosphate buffer concentrations, the rate constant of the first electron transfer is much larger than k_cat. The results suggest that phosphate has a specific effect in promoting the first electron transfer step, and that P450_BM3 could modulate Bacillus membrane morphology and fluidity via palmitate oxidation in response to the external phosphate concentration.     

Catalytic turnover of pyrene by CYP3A4: evidence that cytochrome b5 directly induces positive cooperativity

The metabolism of pyrene to hydroxypyrene by CYP3A4 was investigated to determine the effect of cytochrome b5 (b5) on turnover kinetics. In the absence of b5, formation of hydroxypyrene in in vitro incubations showed a biphasic substrate-velocity curve where K(m1) and V(max1) were 1.3 microM and 0.5 pmol/min/pmol P450, respectively. The addition of testosterone to the incubation mixture completely abolished the second phase to yield a typical, hyperbolic curve, presumably through the disruption in the formation of a pi-pi stacked pyrene complex within the CYP3A4 active site. Finally, the addition of b5 yielded an increase hydroxypyrene formation that resulted in a sigmoidal substrate velocity curve. The V(max) was 15.7 pmol/min/pmol P450, the K(m) was 7.5 microM, and the Hill coefficient was greater than two. This demonstrated that b5 could directly induce positive cooperativity on CYP3A4 and that this biological factor needs to be carefully considered when included in in vitro P450 reactions.     

Active site substitution A82W improves the regioselectivity of steroid hydroxylation by cytochrome P450 BM3 mutants as rationalized by spin relaxation nuclear magnetic resonance studies

Cytochrome P450 BM3 from Bacillus megaterium is a monooxygenase with great potential for biotechnological applications. In this paper, we present engineered drug-metabolizing P450 BM3 mutants as a novel tool for regioselective hydroxylation of steroids at position 16β. In particular, we show that by replacing alanine at position 82 with a tryptophan in P450 BM3 mutants M01 and M11, the selectivity toward 16β-hydroxylation for both testosterone and norethisterone was strongly increased. The A82W mutation led to a ≤42-fold increase in V(max) for 16β-hydroxylation of these steroids. Moreover, this mutation improves the coupling efficiency of the enzyme, which might be explained by a more efficient exclusion of water from the active site. The substrate affinity for testosterone increased at least 9-fold in M11 with tryptophan at position 82. A change in the orientation of testosterone in the M11 A82W mutant as compared to the orientation in M11 was observed by T(1) paramagnetic relaxation nuclear magnetic resonance. Testosterone is oriented in M11 with both the A- and D-ring protons closest to the heme iron. Substituting alanine at position 82 with tryptophan results in increased A-ring proton-iron distances, consistent with the relative decrease in the level of A-ring hydroxylation at position 2β.     

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.     

A novel type of allosteric regulation: functional cooperativity in monomeric proteins

Cooperative functional properties and allosteric regulation in cytochromes P450 play an important role in xenobiotic metabolism and define one of the main mechanisms of drug-drug interactions. Recent experimental results suggest that ability to bind simultaneously two or more small organic molecules can be the essential feature of cytochrome P450 fold, and often results in rich and complex pattern of allosteric behavior. Manifestations of non-Michaelis kinetics include homotropic and heterotropic activation and inhibition effects depending on the stoichiometric ratios of substrate and effector, changes in the regio- and stereospecificity of catalytic transformations, and often give rise to the clinically important drug-drug interactions. In addition, functional response of P450 systems is modulated by the presence of specific and non-specific effector molecules, metal ions, membrane incorporation, formation of homo- and hetero-oligomers, and interactions with the protein redox partners. In this article we briefly overview the main factors contributing to the allosteric effects in cytochromes P450 with the main focus on the sources of cooperative behavior in xenobiotic metabolizing monomeric heme enzymes with their conformational flexibility and extremely broad substrate specificity. The novel mechanism of functional cooperativity in P450 enzymes does not require substantial binding cooperativity, rather it implies the presence of one or more binding sites with higher affinity than the single catalytically active site in the vicinity of the heme iron.     

Multiple sequential steps involved in the binding of inhibitors to cytochrome P450 3A4

Cytochrome P450 (P450) 3A4 is an extensively studied human enzyme involved in the metabolism of >50% of drugs. The mechanism of the observed homotropic and heterotropic cooperativity in P450 3A4-catalyzed oxidations is not well understood, and together with the cooperative behavior, a detailed understanding of interaction of drug inhibitors with P450 3A4 is important in predicting clinical drug-drug interactions. The interactions of P450 3A4 with several structurally diverse inhibitors were investigated using both kinetic and thermodynamic approaches to resolve the steps involved in binding of these ligands. The results of pre-steady-state absorbance and fluorescence experiments demonstrate that inhibitor binding is clearly a multistep process, even more complex than the binding of substrates. Based on spectrophotometric equilibrium binding titrations as well as isothermal titration calorimetry experiments, the stoichiometry of binding appears to be 1:1 in the concentration ranges studied. Using a sequential-mixing stopped-flow approach, we were also able to show that the observed multiphasic binding kinetics is the result of sequential events as opposed to the existence of multiple enzyme populations in dynamic equilibrium that interact with ligands at different rates. We propose a three-step minimal model for inhibitor binding, developed with kinetic simulations, consistent with our previously reported model for the binding of substrates, although it is possible that even more steps are involved.     

Is there a toxicological advantage for non-hyperbolic kinetics in cytochrome P450 catalysis? Functional allostery from "distributive catalysis"

The cytochrome P450s (CYPs) are the major enzymatic detoxification and drug metabolism system. Recently, it has become clear that several CYP isoforms exhibit positive and negative homotropic cooperativity. However, the toxicological implications of allosteric kinetics have not been considered, nor understood. The allosteric kinetics are particularly enigmatic in several respects. In many cases, CYPs bioactivate substrates to more toxic products, thus making it difficult to rationalize a functional advantage for positive cooperativity. Also, CYPs exhibit cooperativity with many structurally diverse ligands, in marked contrast to the specificity observed with other allosteric systems. Here, kinetic simulations are used to compare the probabilistic time- and concentration-dependent integrated toxicity function during conversion of substrate to product for CYP models exhibiting Michaelis-Menten (non-cooperative) kinetics, positive cooperativity, or negative cooperativity. The results demonstrate that, at low substrate concentrations, the slower substrate turnover afforded by cooperative CYPs compared with Michaelis-Menten enzymes can be a significant toxicological advantage, when toxic thresholds exist. When present, the advantage results from enhanced "distribution" of toxin in two pools, substrate and product, for an extended period, thus minimizing the chance that either exceeds its toxic threshold. At intermediate concentrations, the allosteric kinetics can be a modest advantage or modest disadvantage, depending on the kinetic parameters. However, at high substrate concentrations associated with a high probability of toxicity, fast turnover is desirable, and this advantage is provided also by the cooperative enzymes. For the positive homotropic cooperativity, the allosteric kinetics minimize the probability of toxicity over the widest range of system parameters. Furthermore, this apparent functional cooperativity is achieved without specific molecular recognition that is the hallmark of "traditional" allostery.     

The one-electron autoxidation of human cytochrome P450 3A4

Monomeric cytochrome P450 3A4 (CYP3A4), the most prevalent cytochrome P450 in human liver, can simultaneously bind one, two, or three molecules of substrates and effectors. The difference in the functional properties of such binding intermediates gives rise to homotropic and heterotropic cooperative kinetics of this enzyme. To understand the overall kinetic processes operating in CYP3A4, we documented the kinetics of autoxidation of the oxy-ferrous intermediate of CYP3A4 as a function of testosterone concentration. The rate of autoxidation in the presence of testosterone was significantly lower than that observed with no substrate present. Stability of the oxy-ferrous complex in CYP3A4 and the amplitude of the geminate CO rebinding increased significantly as a result of binding of just one testosterone molecule. In contrast, the slow phase in the kinetics of cyanide binding to the ferric CYP3A4 correlated with a shift of the heme iron spin state, which is only caused by the association of a second molecule of testosterone. Our results show that the first substrate binding event prevents the escape of diatomic ligands from the distal heme binding pocket, stabilizes the oxy-ferrous complex, and thus serves as an important modulator of the uncoupling channel in the cytochromes P450.     

Site-directed mutagenesis identifies catalytic residues in the active site of Escherichia coli phosphofructokinase.

Six active site mutants of Escherichia coli phosphofructokinase have been constructed and characterized using steady-state kinetics. All but one of the mutants (ES222) have significantly lower maximal activity, implicating these residues in the catalytic process. Replacement of Asp127, the key catalytic residue in the forward reaction with Glu, results in an enzyme with wild-type cooperative and allosteric behavior but severely decreased Fru6P binding. Replacement of the same residue with Tyr abolishes cooperativity while retaining sensitivity to allosteric inhibition and activation. Thus, this mutant has uncoupled homotropic from heterotropic allostery. Mutation of Asp103 to Ala results in an enzyme which retains wild-type Fru6P-binding characteristics with reduced activity. GDP, which allosterically activates the wild-type enzyme, acts as a mixed inhibitor for this mutant. Mutation of Thr125 to Ala and Asp129 to Ser produces mutants with impaired Fru6P binding and decreased cooperativity. In the presence of the activator GDP, both these mutants display apparent negative cooperativity. In addition, ATP binding is now allosterically altered by GDP. These results extend the number of active site residues known to participate in the catalytic process and help to define the mechanisms behind catalysis and homotropic and heterotropic allostery.     

Co-expression of P450 BM3 and glucose dehydrogenase by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> and its application in an NADPH-dependent indigo production system

P450 BM3 mutant can catalyze indole to indoxyl, and indoxyl can dimerize to form indigo. But the reaction catalyzed by P450 BM3 requires NADPH, as coenzyme regeneration is very important in this system. As we know, when glucose dehydrogenase oxidizes glucose to glucolactone, NADH or NADPH can be formed, which can contribute to NADPH regeneration in the reaction catalyzed by P450 BM3. In this paper, a recombinant Escherichia coli BL21 (pET28a (+)-P450 BM3-gdh0310) was constructed to co-express both P450 BM3 gene and glucose dehydrogenase (GDH) gene. To improve the expression level of P450 BM3 and GDH in E. coli and to avoid the complex and low-efficiency refolding operation in the purification procedure, the expression conditions were optimized. Under the optimized conditions, the maximum P450 BM3 and GDH activities amounted to 8173.13 and 0.045 U/mg protein, respectively. Then bioconversion of indole to indigo was carried out by adding indole and glucose to the culture after improved expression level was obtained under optimized conditions, and 2.9 mM (760.6 mg/L) indigo was formed with an initial indole concentration of 5 mM.