Inulavosin and its benzo-derivatives, melanogenesis inhibitors, target the copper loading mechanism to the active site of tyrosinase.

Tyrosinase, a melanosomal membrane protein containing copper, is a key enzyme for melanin synthesis in melanocytes. Inulavosin inhibits melanogenesis by enhancing a degradation of tyrosinase in lysosomes. However, the mechanism by which inulavosin redirects tyrosinase to lysosomes is yet unknown. The analyses of structure-activity relationship of inulavosin and its benzo-derivatives reveal that the hydroxyl and the methyl groups play a critical role in their inhibitory activity. Intriguingly, the docking studies to tyrosinase suggest that the compounds showing inhibitory activity bind through hydrophobic interactions to the cavity of tyrosinase below which the copper-binding sites are located. This cavity is proposed to be required for the association with a chaperon that assists in copper loading to tyrosinase in Streptomyces antibioticus. Inulavosin and its benzo-derivatives may compete with the copper chaperon and result in a lysosomal mistargeting of apo-tyrosinase that has a conformational defect.

Physiologically relevant metal cofactor for methionine aminopeptidase-2 is manganese.

The identity of the physiological metal cofactor for human methionine aminopeptidase-2 (MetAP2) has not been established. To examine this question, we first investigated the effect of eight divalent metal ions, including Ca(2+), Co(2+), Cu(2+), Fe(2+), Mg(2+), Mn(2+), Ni(2+), and Zn(2+), on recombinant human methionine aminopeptidase apoenzymes in releasing N-terminal methionine from three peptide substrates: MAS, MGAQFSKT, and (3)H-MASK(biotin)G. The activity of MetAP2 on either MAS or MGAQFSKT was enhanced 15-25-fold by Co(2+) or Mn(2+) metal ions in a broad concentration range (1-1000 microM). In the presence of reduced glutathione to mimic the cellular environment, Co(2+) and Mn(2+) were also the best stimulators (approximately 30-fold) for MetAP2 enzyme activity. To determine which metal ion is physiologically relevant, we then tested inhibition of intracellular MetAP2 with synthetic inhibitors selective for MetAP2 with different metal cofactors. A-310840 below 10 microM did not inhibit the activity of MetAP2-Mn(2+) but was very potent against MetAP2 with other metal ions including Co(2+), Fe(2+), Ni(2+), and Zn(2+) in the in vitro enzyme assays. In contrast, A-311263 inhibited MetAP2 with Mn(2+), as well as Co(2+), Fe(2+), Ni(2+), and Zn(2+). In cell culture assays, A-310840 did not inhibit intracellular MetAP2 enzyme activity and did not inhibit cell proliferation despite its ability to permeate and accumulate in cytosol, while A-311263 inhibited both intracellular MetAP2 and proliferation in a similar concentration range, indicating cellular MetAP2 is functioning as a manganese enzyme but not as a cobalt, zinc, iron, or nickel enzyme. We conclude that MetAP2 is a manganese enzyme and that therapeutic MetAP2 inhibitors should inhibit MetAP2-Mn(2+).

Mechanism-based inactivation of cytochrome P-450-3A4 by mifepristone (RU486).

Mifepristone (RU486), an 11beta-substituted nor-steroid containing a 17alpha-1-propynyl group used clinically as an antiprogestin agent for medical abortions, was demonstrated to be a selective mechanism-based inactivator of human cytochrome P-450-3A4 (CYP-3A4). The loss of testosterone 6beta-hydroxylation activity was time- and concentration-dependent as well as requiring metabolism of mifepristone in a purified CYP-3A4 reconstituted system. The inactivation exhibited pseudofirst-order kinetics. The values for KI and kinactivation were 4.7 microM and 0.089 min-1, respectively. The reduced-CO spectrum of CYP-3A4 was decreased by 76%, whereas approximately 81% of the activity was lost following incubation with mifepristone in the reconstituted system in the presence of NADPH. However, the Soret peak of the inactivated CYP-3A4 was slightly increased. High-performance liquid chromatography analysis of the incubation mixture showed that the peak containing the heme dissociated from the inactivated CYP3A4 was almost identical with that seen for the -NADPH control. Covalent binding of [3H]mifepristone to apoCYP3A4 was demonstrated by SDS-PAGE and high-pressure liquid chromatography analyses of the reconstituted system containing CYP-3A4, NADPH-CYP reductase, cytochrome b5 and lipids in the presence of NADPH. The stoichiometry was determined to be approximately 1 mol of mifepristone bound per 1 mol of CYP-3A4 inactivated. Therefore, the mechanism of inactivation of CYP-3A4 by mifepristone involves irreversible modification of the apoprotein at the enzyme active site instead of being the result of heme adduct formation or heme fragmentation. Mifepristone exhibits selectivity for CYP-3A4 as evidenced by the fact that it did not show mechanism-based inactivation of CYPs 1A, 2B, 2D6, and 2E1, although a competitive inhibition of CYP 2B1 and 2D6 was observed.

PIP: This study demonstrates mifepristone (RU-486) as a potent and selective mechanism-based inactivator of cytochrome P-450-3A4 (CYP-3A4) via irreversible modification of the apoprotein. The results of this clinical research indicate that loss of testosterone 6-beta-hydroxylation activity was time- and concentration-dependent, as well as requiring metabolism of mifepristone in purified CYP-3A4 reconstituted system. Inactivation using several different concentrations of mifepristone exhibited pseudo-first-order kinetics. Reduced-CO difference spectrum of CYP-3A4 decreased by 76%, whereas approximately 81% of CYP-3A4 activity was lost following incubation with mifepristone, indicating the occurrence of N-heme adduct formation detected by HPLC and UV-visible spectroscopy. The peak containing the heme dissociated from the mifepristone-inactivated CYP-3A4 was almost identical with that of the -NADPH control when the incubation mixtures were analyzed by HPLC. 3H-mifepristone proved to be covalently bound to the apoCYP-3A4 by HPLC and SDC-PAGE. The stoichiometry for the binding of the mifepristone was determined to be 1.02 +or- 0.15, approximately 1 mol of mifepristone bound per mole of inactivated CYP-3A4. In summary, mifepristone has been shown to be a potent mechanism-based inactivator of human CYP-3A4. The mechanism of the inactivation showed to involve irreversible modification of the apoprotein at the enzyme active site instead of heme adduct formation or heme fragmentation.

Zn(II) dependence of the Aeromonas hydrophila AE036 metallo-beta-lactamase activity and stability.

Two Zn2+ binding sites were found in the Aeromonas hydrophila AE036 metallo-beta-lactamase. The affinity of the first binding site for Zn2+ ions is so high that the dissociation constant could not be determined, but it is significantly lower than 20 nM. The mono-Zn2+ form of the enzyme exhibits a maximum activity against its carbapenem substrates. The presence of a Zn2+ ion in the second lower affinity binding site results in a loss of enzymatic activity with a Ki value of 46 microM at pH 6.5. The kinetic analysis is in agreement with a noncompetitive inhibition mechanism. The Zn content of the A. hydrophila enzyme is also strongly pH-dependent. With an external Zn2+ ion concentration of 0.4 microM, occupancy of the higher affinity site by metal ions is lower than 10% at pH 5 and 10. The affinity for the second binding site seems to increase from pH 6 to 7.5. Fluorescence emission and circular dichroism spectra revealed slight conformational changes upon titration of the apoenzyme by Zn2+ ions, resulting in the successive saturation of the first and second binding sites. Differential scanning calorimetry transitions and intrinsic fluorescence emission spectra in the presence of increasing concentrations of urea demonstrate that the catalytic zinc strongly stabilizes the conformation of the enzyme whereas the di-Zn enzyme is even more resistant to thermal and urea denaturation than the mono-Zn enzyme. The Zn2+ dependency of the activity of this metallo-beta-lactamase thus appears to be very different from that of the homologous Bacteroides fragilis enzyme for which the presence of two Zn2+ ions per molecule of protein appears to result in maximum activity.

Reconstitution of the holoenzyme form of Escherichia coli porphobilinogen deaminase from apoenzyme with porphobilinogen and preuroporphyrinogen: a study using circular dichroism spectroscopy.

Porphobilinogen deaminase (PBG-D), an early enzyme of the tetrapyrrole biosynthetic pathway, catalyzes the formation of a tetrapyrrole chain, preuroporphyrinogen, from four molecules of porphobilinogen (PBG). The PBG-D apoenzyme is responsible for the autocatalytic synthesis and covalent attachment of a dipyrromethane cofactor at its active site. In this paper an efficient method for the purification of Escherichia coli PBG-D apoenzyme using an affinity chromatography resin is reported. Circular dichroism (CD) spectra of apoenzyme and holoenzyme were recorded and significant differences in both the backbone and aromatic region of the spectra were observed. The differences in the spectra allowed the reconstitution of holoenzyme from purified apoenzyme with PBG and preuroporphyrinogen in solution to be monitored separately by CD. Apoenzyme incubated with preuroporhyrinogen gave a CD spectrum that was much more like the CD spectrum of holoenzyme than apoenzyme incubated with PBG. The results showed clearly that the cofactor was generated much more rapidly from preuroporphyrinogen than from PBG. Changes in the CD spectrum associated with the aromatic side-chain region, in particular the contribution assigned to phenylalanine-62, were found to correlate well with the activity of the reconstituted enzyme. Phenylalanine-62 is located in close proximity to the cofactor and acts as a sensitive probe to active-site changes. The stability of the holoenzyme and apoenzyme were compared with respect to both heat and susceptibility to proteolysis. The results were consistent with a model for the apoenzyme in which, in the absence of the cofactor, the three domains of the protein are held less rigidly together, thereby making the protein more susceptible to heat denaturation and proteolysis. The CD spectrum of the holoenzyme was found to be similar at both pH 5.1 and 7.4, suggesting that the crystal structure, determined at pH 5.1, is likely to be similar at physiological pH values.