Effector-mediated alteration of substrate orientation in cytochrome P450 2C9

Cytochrome P450 2C9 (CYP2C9)-mediated flurbiprofen 4'-hydroxylation is activated by the presence of dapsone resulting in reduction of the K(m) for flurbiprofen hydroxylation and an increase in V(m). Previous spectral binding studies have demonstrated that the binding of flurbiprofen with CYP2C9 is increased (decrease in K(S)) by the presence of dapsone. We hypothesized that the two compounds are simultaneously in the active site with the presence of dapsone causing flurbiprofen to be oriented more closely to the heme. T(1) relaxation rates determined by NMR were used to estimate the distances of protons on these compounds from the paramagnetic heme-iron center. Samples contained 0.014 microM CYP2C9 and 145 microM flurbiprofen in the presence and absence of 100 microM dapsone. Estimated distances of various flurbiprofen protons from the heme ranged from 4.2 to 4.5 A in the absence of dapsone and from 3.2 to 3.8 A in the presence of dapsone. The 4' proton of flurbiprofen, the site of metabolism, showed one of the greatest differences in distance from the heme in the presence of dapsone, 3.50 A, as compared to the absence of dapsone, 4.41 A. Dapsone protons were less affected, being 4.40 A from the heme in the absence of flurbiprofen and 4.00-4.01 A from the heme in the presence of flurbiprofen. Molecular modeling studies were also performed to corroborate the relative orientations of flurbiprofen and dapsone in the active site of CYP2C9. Shift of the 4' proton of flurbiprofen closer to the heme iron of CYP2C9 in the presence of dapsone may play a role in activation.     

Activation of cytochrome P450 2C9-mediated metabolism: mechanistic evidence in support of kinetic observations

Studies were designed to investigate the possible mechanisms associated with the kinetic observation of CYP2C9 activation by dapsone and its phase I metabolite, N-hydroxydapsone. Kinetic studies suggested that dapsone activated CYP2C9-mediated flurbiprofen 4(')-hydroxylation by decreasing the K(m) (alpha=0.2) and increasing the V(max) (beta=1.9). Interestingly, N-hydroxydapsone also activated flurbiprofen 4(')-hydroxylation by increasing V(max) (beta=1.5) but had no effect on K(m) (alpha=0.98). To study the effects of these modulators on the binding affinity of flurbiprofen, spectral binding studies were performed. In the presence of dapsone, the spectral binding constant (K(s)) for flurbiprofen was reduced from 14.1 to 2.1 microM, while in the presence of N-hydroxydapsone, the K(s) remained unchanged (14.0 microM), which suggests that dapsone causes an increase in the affinity of flurbiprofen for CYP2C9, whereas N-hydroxydapsone does not. Additionally, stoichiometry measurements under activation conditions in the presence of dapsone resulted in a doubling of both NADPH and oxygen consumption for flurbiprofen 4(')-hydroxylation, with an overall increase in metabolite formation and a decrease in formation of peroxide and excess water. Interestingly, the presence of N-hydroxydapsone generally caused the same effects on stoichiometry as those of flurbiprofen 4(')-hydroxylation but failed to reduce excess water formation, which suggests that, while N-hydroxydapsone activates CYP2C9, it does so less efficiently and possibly through a mechanism different from that of dapsone.     

Activation of microsomal epoxide hydrolase by interaction with cytochromes P450: kinetic analysis of the association and substrate-specific activation of epoxide hydrolase function

The kinetics of the association between cytochrome P450 (P450) and microsomal epoxide hydrolase (mEH) was studied by means of resonant mirror based on the principle of surface plasmon resonance. The dissociation equilibrium constants (K(D)) for the affinity of P450 enzymes for mEH were estimated by resonant mirror using an optical biosensor cell covalently bound to rat mEH. Comparable K(D) values were obtained for CYP1A1 and 2B1, and these were greater by one order of magnitude than that for the CYP2C11. To clarify the influences of P450 enzymes on the catalytic activity of mEH, the hydrolyzing activity for styrene oxide and benzo(a)pyrene-7,8-oxide [B(a)P-oxide] was analyzed in the presence or absence of P450s. Styrene oxide hydrolysis was activated by all P450s including the CYP1A, 2B, 2C, and 3A subfamilies. In agreement with the association affinity determined by resonant mirror, CYP2C11 tends to have enhanced activity for styrene oxide hydrolysis. On the other hand, B(a)P-oxide hydrolysis was enhanced by only CYP2C11 while CYP1A1 and CYP2B1 had no effect. These results suggest that (1) many P450 enzymes associate nonspecifically with mEH, (2) the CYP2C11 plays a greater role in the association/activation of mEH and (3) the P450-mediated activation of mEH depends upon the substrate of mEH.     

Efficient catalytic turnover of cytochrome P450(cam) is supported by a T252N mutation

A Thr (or Ser) residue on the I-helix is a highly conserved structural feature of cytochrome P450 enzymes. It is believed to be indispensable as a proton delivery shuttle in the oxygen activation process. Previous work showed that P450_cin (CYP176A1), which contains an Asn instead of the conserved Thr, is fully functional in the catalytic oxidation of cineole [D.B. Hawkes, G.W. Adams, A.L. Burlingame, P.R. Ortiz de Montellano, J.J. De Voss, J. Biol. Chem. 277 (2002) 27725-27732]. To determine whether the substitution of Asn for Thr is specific or general, the conserved Thr252 in P450_cam (CYP101) was mutated to generate the T252N, T252N/V253T, and T252A mutants. Steady-state kinetic analysis of the oxidation of camphor by these mutants indicated that the T252N and T252N/V253T mutants have comparable turnover numbers but higher K_m values relative to the wild-type enzyme. Spectroscopic binding assays indicate that the higher K_m values reflect a decrease in the camphor binding affinity. Non-productive H₂O₂ generation was negligible with the T252N and T252N/V253T mutants, but, as previously observed, was dominant in the T252A mutant. Our results, and a structure model based on the crystal structures of the ferrous dioxygen complexes of P450_cam and its T252A mutant, suggest that Asn252 can stabilize the ferric hydroperoxy intermediate, preventing premature release of H₂O₂ and enabling addition of the second proton to the distal oxygen to generate the catalytic ferryl species.     

Allene oxide synthase from Arabidopsis thaliana (CYP74A1) exhibits dual specificity that is regulated by monomer-micelle association

We investigate the effects of detergent on the kinetics and oligomeric state of allene oxide synthase (AOS) from Arabidopsis thaliana (CYP74A1). We show that detergent-free CYP74A1 is monomeric and highly water soluble with dual specificity, but has relatively low activity. Detergent micelles promote a 48-fold increase in k(cat)/K(m) (to 5.9 x 10(7)M(-1)s(-1)) with concomitant changes in the spin state equilibrium of the haem-iron due to the binding of a single detergent micelle to the protein monomer, which is atypical of P450 enzymes. This mechanism is shown to be an important determinant of the substrate specificity of CYP74A1. CYP74A1 may be suited for structural resolution of the first plant cytochrome P450 and its 9-AOS activity and behaviour in vitro has implications for its role in planta.     

P450 enzymes from the bacterium Novosphingobium aromaticivorans

Twelve of the fifteen potential P450 enzymes from the bacterium Novosphingobium aromaticivorans, which is known to degrade a wide range of aromatic hydrocarbons, have been produced via heterologous expression in Escherichia coli. The enzymes were tested for their ability to bind a range of substrates including polyaromatic hydrocarbons. While two of the enzymes were found to bind aromatic compounds (CYP108D1 and CYP203A2), the others show binding with a variety of compounds including linear alkanes (CYP153C1) and mono- and sesqui-terpenoid compounds (CYP101B1, CYP101C1, CYP101D1, CYP101D2, CYP111A1, and CYP219A1). A 2Fe-2S ferredoxin (Arx-A), which is associated with CYP101D2, was also produced. The activity of five of the P450 enzymes (CYP101B1, CYP101C1, CYP101D1, CYP101D2, and CYP111A2) was reconstituted with Arx-A and putidaredoxin reductase (of the P450cam system from Pseudomonas putida) in a Class I type electron transfer system. Preliminary characterisation of the majority of the substrate oxidation products is reported.     

Suppression of fetal testicular cytochrome P450 17 by maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin: a mechanism involving an initial effect on gonadotropin synthesis in the pituitary

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the fetal expression of testicular cytochrome P450 17 (CYP17), one of the enzymes necessary for sex steroid synthesis, was studied in Wistar rats. Fetal testicular CYP17 exhibited reduced mRNA and protein levels following exposure of the dams at gestational day 15 to 1 microg/kg TCDD. In support of this, CYP17 activity catalyzed by fetal testis homogenate was also reduced by maternal exposure to TCDD. The reduction in CYP17 expression seemed to be specific for fetal stages, because 7 day-old pups born from TCDD-treated dams did not exhibit any reduction in CYP17. In sharp contrast to the in vivo observations, TCDD failed to reduce CYP17 expression in cultured fetal testis, although CYP17 could be induced by activating cAMP-dependent signaling. To assess the role of pituitary luteinizing hormone (LH) on TCDD-induced reduction in fetal testicular CYP17, a further investigation was performed to examine whether the direct injection of LH into fetuses restores the altered CYP17 expression. The results showed that in utero injection of equine chorionic gonadotropin, an LH-mimicking hormone, completely abolishes the TCDD-produced reduction in fetal CYP17. However, neither the alpha- nor beta-subunits of LH in cultured fetal pituitary was reduced by TCDD. These results suggest that 1) maternal exposure to TCDD impairs the expression of testicular CYP17 in a fetal stage-specific manner; 2) this effect is due, at least partially, to a TCDD-produced reduction in circulating LH; and 3) TCDD exerts such an effect by affecting the upstream mechanism regulating the pituitary synthesis of LH.     

The crystal structure of MCAT from Mycobacterium tuberculosis reveals three new catalytic models

The malonyl coenzyme A (CoA)-acyl carrier protein (ACP) transacylase (MCAT) plays a key role in cell wall biosynthesis in Mycobacterium tuberculosis and other bacteria. The M. tuberculosis MCAT (MtMCAT) is encoded by the FabD gene and catalyzes the transacylation of malonate from malonyl-CoA to holo-ACP. Malonyl-ACP is the substrate in fatty acid biosynthesis and is a by-product of the transacylation reaction. This ability for fatty acid biosynthesis enables M. tuberculosis to survive in hostile environments, and thus understanding the mechanism of biosynthesis is important for the design of new anti-tuberculosis drugs. The 2.3 A crystal structure of MtMCAT reported here shows that its catalytic mechanism differs from those of ScMCAT and EcMCAT, whose structures have previously been determined. In MtMCAT, the C(beta)-O(gamma) bond of Ser91 turns upwards, resulting in a different orientation and thus an overall change of the active pocket compared to other known MCAT enzymes. We identify three new nucleophilic attack chains from the MtMCAT structure: His90-Ser91, Asn155-Wat6-Ser91 and Asn155-His90-Ser91. Enzyme activity assays show that His90A, Asn155A and His90A-Asn155A mutants all have substantially reduced MCAT activity, indicating that M. tuberculosis MCAT supports a unique means of proton transfer. Furthermore, His194 cannot form part of a His-Ser catalytic dyad and only stabilizes the substrate. This new discovery should provide a deeper insight into the catalytic mechanisms of MCATs.     

Estimation of binding constants for the substrate and activator of Rhodobacter sphaeroides adenosine 5'-diphosphate-glucose pyrophosphorylase using affinity capillary electrophoresis

Binding constants were determined for the activator fructose-6-phosphate (F6P) and substrate adenosine 5'-triphosphate (ATP) (in the presence and absence of F6P) to the recombinant wild-type (WT) Rhodobacter sphaeroides adenosine 5'-diphosphate-(ADP)-glucose pyrophosphorylase (ADPGlc PPase) using affinity capillary electrophoresis (ACE). In these binding studies, the capillary is initially injected with a plug of sample containing ADPGlc PPase and noninteracting standards. The sample is then subjected to increasing concentrations of F6P or ATP in the running buffer and electrophoresed. Analysis of the change in the migration times of ADPGlc PPase, relative to those of the noninteracting standards, as a function of the varying concentration of F6P or ATP yields a binding constant. The values obtained were in good agreement with kinetic parameters obtained from steady state activity assays. The method was extended to examine the F6P binding constants for the R33A and R22A enzymes and the ATP binding constants for the R8A enzyme in the presence and absence of F6P. The R33A enzyme has been shown by activity assays to be insensitive to F6P activation, indicating a defect in binding or in downstream transmission of the allosteric signal required for full activation. ACE indicated no apparent binding of F6P, supporting the former hypothesis. The R22A enzyme was shown by activity assays to have a approximately 15-fold decrease in apparent affinity for F6P compared to that of WT while ACE indicated an affinity comparable to that of WT; potential reasons for this discrepancy are discussed. The R8A enzyme as measured by activity assays exhibits reduced fold-activation by F6P compared to that of WT but increased apparent affinity for ATP in the presence of F6P. The ACE results were in good agreement with the activity assay data, confirming the increased affinity for ATP in the presence of F6P. This method demonstrates the quantitative ability of ACE to study different binding sites/ligand interactions in allosteric enzymes.     

Beta sheet 2–alpha helix C loop of cytochrome P450 reductase serves as a docking site for redox partners

As a promiscuous redox partner, the biological role of cytochrome P450 reductase (CPR) depends significantly on protein–protein interactions. We tested a hypothesized CPR docking site by mutating D113, E115, and E116 to alanine and assaying activity toward various electron acceptors as a function of ionic strength. Steady-state cytochrome c studies demonstrated the mutations improved catalytic efficiency and decreased the impact of ionic strength on catalytic parameters when compared to wild type. Based on activity toward 7-ethoxy-4-trifluoro-methylcoumarin, CYP2B1 and CPR favored formation of an active CYP2B1•CPR complex and inactive (CYP2B1)₂•CPR complex until higher ionic strength whereby only the binary complex was observed. The mutations increased dissociation constants only for the binary complex and suppressed the ionic strength effect. Studies with a non-binding substrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) suggest changes in activity toward cytochrome c and CYP2B1 reflect alterations in the route of electron transfer caused by the mutations. Electrostatic modeling of catalytic and binding parameters confirmed the importance of D113 and especially the double mutant E115 and E116 as mediators in forming charge–charge interactions between CPR and complex partners.     

The effect of individual N-glycans on enzyme activity

In a series of investigations, N-glycosylation has proven to be a key determinant of enzyme secretion, activity, binding affinity and substrate specificity, enabling a protein to fine-tune its activity. In the majority of cases elimination of all putative N-glycosylation sites of an enzyme results in significantly reduced protein secretion levels, while removal of individual N-glycosylation sites often leads to the expression of active enzymes showing markedly reduced catalytic activity, with the decreased activity often commensurate with the number of glycosylation sites available, and the fully deglycosylated enzymes showing only minimal activity relative to their glycosylated counterparts. On the other hand, several cases have also recently emerged where deglycosylation of an enzyme results in significantly increased catalytic activity, binding affinity and altered substrate specificity, highlighting the very unique and diverse roles that individual N-glycans play in regulating enzyme function.     

Cytochrome b5 reductase and cytochrome b5 support the CYP2E1-mediated activation of nitrosamines in a recombinant Ames test

With CYP2E1 in vitro both the first and the second electron of the catalytic cycle can come from cytochrome b(5) via either NADPH-cytochrome P450 reductase or NADH-cytochrome b(5) reductase, and the presence of cytochrome b(5) stimulates CYP2E1 turnover both in vitro and in vivo. To determine whether electron input via the NADH-dependent pathway was similarly functional in whole cells and necessary for the stimulation by cytochrome b(5), we constructed five plasmids designed to express human CYP2E1 in various combinations with cytochrome b(5) reductase, cytochrome b(5), and cytochrome P450 reductase. CYP2E1 activity in Salmonella typhimurium cells transformed with each plasmid was assessed by mutagenic reversion frequency in the presence of dimethylnitrosamine. A fivefold increase in reversion frequency when cytochrome b(5) was coexpressed with P450 reductase was abolished by disruption of heme-binding in cytochrome b(5) by site-directed mutagenesis (His68Ala), suggesting that electron transfer to cytochrome b(5) was necessary for the stimulation. Addition of cytochrome b(5) reductase to the cytochrome b(5)/P450 reductase coexpression plasmid did not further increase the stimulation by cytochrome b(5), but b(5) reductase could support CYP2E1 activity in the absence of P450 reductase at a level equivalent to that obtained with just CYP2E1 and P450 reductase. Neither cytochrome b(5) reductase nor cytochrome b(5) alone could support CYP2E1 activity. These results demonstrate that the cytochrome b(5) reductase/cytochrome b(5) pathway can support CYP2E1 activity in bacterial cells.     

Mechanistic study of CMP-Neu5Ac hydrolysis by α2,3-sialyltransferase from Pasteurella dagmatis

Bacterial sialyltransferases of the glycosyltransferase family GT-80 exhibit pronounced hydrolase activity toward CMP-activated sialyl donor substrates. Using in situ proton NMR, we show that hydrolysis of CMP-Neu5Ac by Pasteurella dagmatis α2,3-sialyltransferase (PdST) occurs with axial-to-equatorial inversion of the configuration at the anomeric center to release the α-Neu5Ac product. We propose a catalytic reaction through a single displacement-like mechanism where water replaces the sugar substrate as a sialyl group acceptor. PdST variants having His²⁸⁴ in the active site replaced by Asn, Asp or Tyr showed up to 10⁴-fold reduced activity, but catalyzed CMP-Neu5Ac hydrolysis with analogous inverting stereochemistry. The proposed catalytic role of His²⁸⁴ in the PdST hydrolase mechanism is to facilitate the departure of the CMP leaving group.     

Reduced catalytic activity of human CYP2C9 natural alleles for gliclazide: molecular dynamics simulation and docking studies

Amongst sulfonylureas, gliclazide is one of the mostly prescribed drugs to diabetic patients and is metabolized extensively by P450 CYP2C9. Among 24-CYP2C9 alleles, the *2/*2 and *3/*3 genotypes showed significantly lower gliclazide clearances with reductions of 25 and 57%, respectively. However, the reason for the change in drug-metabolizing activity induced by these natural alleles is unknown. In the present study, we used molecular dynamics simulation and autodocking studies to provide models for gliclazide-bound complexes of CYP2C9*2, *3 and *2/*3 mutants, which give insight into CYP2C9-gliclazide interactions and explain the reduced enzymatic activity seen in these variants. Our data shows that the size of the substrate-access entry site is significantly reduced in mutants, which limits the access of gliclazide to heme and the active site. The distance from the substrate oxidation site and heme is >5 Å in *3 and *2/*3. Therefore, the addition of an active oxygen molecule by heme-Fe is hindered. The absence of F100, F114 and F476 in the interacting amino acid pocket in *3 reduces catalytic efficiency toward gliclazide. In *1, gliclazide is stabilized by the formation of two hydrogen bonds with R108 while it is absent in mutants. Further in *3 and *2/*3, the key heme-stabilizing residue, R97 stabilization is greatly reduced. Therefore, the decreased catalytic activity of these variants can be explained from the reduced access of the gliclazide to heme, and the interaction between heme and substrate is affected due to their instability in the active site.     

Catalysis by N-Acetyl-d-glucosaminylphosphatidylinositol De-N-acetylase (PIG-L) from Entamoeba histolytica: NEW ROLES FOR CONSERVED RESIDUES*

We showed previously that Entamoeba histolytica PIG-L exhibits a novel metal-independent albeit metal-stimulated activity. Using mutational and biochemical analysis, here we identify Asp-46 and His-140 of the enzyme as being important for catalysis. We show that these mutations neither affect the global conformational of the enzyme nor alter its metal binding affinity. The defect in catalysis, due to the mutations, is specifically due to an effect on V_max and not due to altered substrate affinity (or K_m). We propose a general acid-base pair mechanism to explain our results.     

Inhibition of human drug metabolizing cytochrome P450 enzymes by plant isoquinoline alkaloids

The human cytochrome P450 (CYP) enzymes play a major role in the metabolism of endobiotics and numerous xenobiotics including drugs. Therefore it is the standard procedure to test new drug candidates for interactions with CYP enzymes during the preclinical development phase. The purpose of this study was to determine in vitro CYP inhibition potencies of a set of isoquinoline alkaloids to gain insight into interactions of novel chemical structures with CYP enzymes. These alkaloids (n = 36) consist of compounds isolated from the Papaveraceae family (n = 20), synthetic analogs (n = 15), and one commercial compound. Their inhibitory activity was determined towards all principal human drug metabolizing CYP enzymes: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4. All alkaloids were assayed in vitro in a 96-well plate format using pro-fluorescent probe substrates and recombinant human CYP enzymes. Many of these alkaloids inhibited the CYP3A4 form, with 30/36 alkaloids inhibiting CYP3A4 with at least moderate potency (IC₅₀ < 10 μM) and 15/36 inhibiting CYP3A4 potently (IC₅₀ < 1 μM). Among them corydine, parfumine and 8-methyl-2,3,10,11-tetraethoxyberbine were potent and selective inhibitors for CYP3A4. CYP2D6 was inhibited with at least moderate potency by 26/34 alkaloids. CYP2C19 was inhibited by 15/36 alkaloids at least moderate potently, whereas CYP1A2, CYP2B6, CYP2C8, and CYP2C9 were inhibited to a lesser degree. CYP2A6 was not significantly inhibited by any of the alkaloids. The results provide initial structure-activity information about the interaction of isoquinoline alkaloids with major human xenobiotic-metabolizing CYP enzymes, and illustrate potential novel structures as CYP form-selective inhibitors.     

Essential requirements for substrate binding affinity and selectivity toward human CYP2 family enzymes

A detailed analysis of substrate selectivity within the cytochrome P450 2 (CYP2) family is reported. From a consideration of specific interactions between drug substrates for human CYP2 family enzymes and the putative active sites of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1, it is likely that the number and disposition of hydrogen bond donor/acceptors and aromatic rings within the various P450 substrate molecules determines their enzyme selectivity and binding affinity, together with directing their preferred routes of metabolism by the CYP2 enzymes concerned. Although many aliphatic residues are present in most P450 active sites, it would appear that their main contribution centers around hydrophobic interactions and desolvation processes accompanying substrate binding. Molecular modeling studies based on the recent CYP2C5 crystal structure appear to show close agreement with site-directed mutagenesis experiments and with information on substrate metabolism and selectivity within the CYP2 family.     

The crystal structure of pectate lyase Pel9A from Erwinia chrysanthemi

The "family 9 polysaccharide lyase" pectate lyase L (Pel9A) from Erwinia chrysanthemi comprises a 10-coil parallel beta-helix domain with distinct structural features including an asparagine ladder and aromatic stack at novel positions within the superhelical structure. Pel9A has a single high affinity calcium-binding site strikingly similar to the "primary" calcium-binding site described previously for the family Pel1A pectate lyases, and there is strong evidence for a common second calcium ion that binds between enzyme and substrate in the "Michaelis" complex. Although the primary calcium ion binds substrate in subsite -1, it is the second calcium ion, whose binding site is formed by the coming together of enzyme and substrate, that facilitates abstraction of the C5 proton from the sacharride in subsite +1. The role of the second calcium is to withdraw electrons from the C6 carboxylate of the substrate, thereby acidifying the C5 proton facilitating its abstraction and resulting in an E1cb-like anti-beta-elimination mechanism. The active site geometries and mechanism of Pel1A and Pel9A are closely similar, but the catalytic base is a lysine in the Pel9A enzymes as opposed to an arginine in the Pel1A enzymes.     

Influence of various polymorphic variants of cytochrome P450 oxidoreductase (POR) on drug metabolic activity of CYP3A4 and CYP2B6

Cytochrome P450 oxidoreductase (POR) is known as the sole electron donor in the metabolism of drugs by cytochrome P450 (CYP) enzymes in human. However, little is known about the effect of polymorphic variants of POR on drug metabolic activities of CYP3A4 and CYP2B6. In order to better understand the mechanism of the activity of CYPs affected by polymorphic variants of POR, six full-length mutants of POR (e.g., Y181D, A287P, K49N, A115V, S244C and G413S) were designed and then co-expressed with CYP3A4 and CYP2B6 in the baculovirus-Sf9 insect cells to determine their kinetic parameters. Surprisingly, both mutants, Y181D and A287P in POR completely inhibited the CYP3A4 activity with testosterone, while the catalytic activity of CYP2B6 with bupropion was reduced to approximately ~70% of wild-type activity by Y181D and A287P mutations. In addition, the mutant K49N of POR increased the CLint (Vmax/Km) of CYP3A4 up to more than 31% of wild-type, while it reduced the catalytic efficiency of CYP2B6 to 74% of wild-type. Moreover, CLint values of CYP3A4-POR (A115V, G413S) were increased up to 36% and 65% of wild-type respectively. However, there were no appreciable effects observed by the remaining two mutants of POR (i.e., A115V and G413S) on activities of CYP2B6. In conclusion, the extent to which the catalytic activities of CYP were altered did not only depend on the specific POR mutations but also on the isoforms of different CYP redox partners. Thereby, we proposed that the POR-mutant patients should be carefully monitored for the activity of CYP3A4 and CYP2B6 on the prescribed medication.     

Kinetic and thermodynamic characterization of the functional properties of a hybrid versatile peroxidase using isothermal titration calorimetry: Insight into manganese peroxidase activation and lignin peroxidase inhibition

Isothermal titration calorimetry (ITC) was developed for measuring lignin peroxidase (LiP) and manganese peroxidase (MnP) activities of versatile peroxidase (VP) from Bjerkandera adusta. Developing an ITC approach provided an alternative to colorimetric methods that enabled reaction kinetics to be accurately determined. Although VP from Bjerkandera adjusta is a hybrid enzyme, specific conditions of [Mn+2] and pH were defined that limited activity to either LiP or MnP activities, or enabled both to be active simultaneously. MnP activity was found to be more efficient than LiP activity, with activity increasing with increasing concentrations of Mn+2. These properties of MnP were explained by a second metal binding site involved in homotropic substrate (Mn+2) activation. The activation of MnP was also accompanied by a decrease in both activation energy and substrate (Mn) affinity, reflecting a flexible enzyme structure. In contrast to MnP activity, LiP activity was inhibited by high dye (substrate) concentrations arising from uncompetitive substrate inhibition caused by substrate binding to a site distinct from the catalytic site. Our study provides a new level of understanding about the mechanism of substrate regulation of catalysis in VP from B. adjusta, providing insight into a class of enzyme, hybrid class II peroxidases, for which little experimental data is available.