N-alkyl urea hydroxamic acids as a new class of peptide deformylase inhibitors with antibacterial activity.

Peptide deformylase (PDF) is a prokaryotic metalloenzyme that is essential for bacterial growth and is a new target for the development of antibacterial agents. All previously reported PDF inhibitors with sufficient antibacterial activity share the structural feature of a 2-substituted alkanoyl at the P(1)' site. Using a combination of iterative parallel synthesis and traditional medicinal chemistry, we have identified a new class of PDF inhibitors with N-alkyl urea at the P(1)' site. Compounds with MICs of 200 micro M for matrilysin and other mammalian metalloproteases. Structure-activity relationship analysis identified preferred substitutions resulting in improved potency and decreased cytotoxity. One of the compounds (VRC4307) was cocrystallized with PDF, and the enzyme-inhibitor structure was determined at a resolution of 1.7 A. This structural information indicated that the urea compounds adopt a binding position similar to that previously determined for succinate hydroxamates. Two compounds, VRC4232 and VRC4307, displayed in vivo efficacy in a mouse protection assay, with 50% protective doses of 30.8 and 17.9 mg/kg of body weight, respectively. These N-alkyl urea hydroxamic acids provide a starting point for identifying new PDF inhibitors that can serve as antimicrobial agents.

Amidation of salicyluric acid and gentisuric acid: a possible role for peptidylglycine alpha-amidating monooxygenase in the metabolism of aspirin.

Bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the copper-, ascorbate-, and O2-dependent cleavage of C-terminal glycine-extended peptides, N-acylglycines, and the bile acid glycine conjugates to the corresponding amides and glyoxylate. Two known metabolites of aspirin, salicyluric acid and gentisuric acid, are also substrates for PAM, leading to the formation of salicylamide and gentisamide. The time course for O2 consumption and glyoxylate production indicates that salicylurate amidation is a two-step reaction. Salicylurate is first converted to N-salicyl-alpha-hydroxyglycine, which is ultimately dealkylated to salicylamide and glyoxylate. The enzymatically generated salicylamide and N-salicyl-alpha-hydroxyglycine were characterized by mass spectrometry and two-dimensional 1H-13C heteronuclear multiple quantum coherence NMR.

Structural and functional investigations on the role of zinc in bifunctional rat peptidylglycine alpha-amidating enzyme.

Bifunctional peptidylglycine alpha-amidating enzyme (alpha-AE) catalyzes the two-step conversion of C-terminal glycine-extended peptides to C-terminal alpha-amidated peptides and glyoxylate. The first step is the ascorbate-, O2-, and copper-dependent hydroxylation of the alpha-carbon of the glycyl residue, producing an alpha-hydroxyglycine-extended peptide. The second step is the ascorbate-, O2-, and copper-independent dealkylation of the carbinolamide intermediate. We show that alpha-AE requires 1.1 +/- 0. 2 mol of zinc/mol of enzyme for maximal (S)-N-dansyl-Tyr-Val-alpha-hydroxyglycine dealkylation activity. Treatment of the enzyme with EDTA abolishes both the peptide hydroxylation and the carbinolamide dealkylation activities. Addition of Zn(II), Co(II), Cd(II), and Mn(II) partially restores carbinolamide dealkylation activity to the EDTA-treated enzyme. Addition of Co(II) produces the greatest restoration of dealkylation activity, 32% relative to a control not treated with EDTA, while Mn(II) addition results in the smallest restoration of dealkylation activity, only 3% relative to an untreated control. The structure and coordination of the zinc center has been investigated by X-ray absorption spectroscopy. EXAFS data are best interpreted by an average coordination of 2-3 histidine ligands and 1-2 non-histidine O/N ligands. Since catalytic zinc centers in other zinc metalloenzymes generally exhibit only O/N ligands to the zinc atom, a zinc-bound water or hydroxide may serve as a general base for the abstraction of the hydroxyl proton from the carbinolamide intermediate. Alternatively, the zinc may function in a structural role.

Structural investigations on the coordination environment of the active-site copper centers of recombinant bifunctional peptidylglycine alpha-amidating enzyme.

The structure and coordination chemistry of the copper centers in the bifunctional peptidylglycine alpha-amidating enzyme (alpha-AE) have been investigated by EPR, EXAFS, and FTIR spectroscopy of a carbonyl derivative. The enzyme contains 2 coppers per 75 kDa protein molecule. Double integration of the EPR spectrum of the oxidized enzyme indicates that 98 +/- 13% of the copper is EPR detectable, indicating that the copper centers are located in mononuclear coordination environments. The Cu(II) coordination of the oxidized enzyme is typical of type 2 copper proteins. EXAFS data are best interpreted by an average coordination of 2-3 histidines and 1-2 O/N (probably O from solvent, Asp or Glu) as equatorial ligands. Reduction causes a major structural change. The Cu(I) centers are shown to be structurally inequivalent since only one of them binds CO. EXAFS analysis of the reduced enzyme data indicates that the nonhistidine O/N shell is displaced, and the Cu(I) coordination involves a maximum of 2.5 His ligands together with 0.5 S/CI ligand per copper. The value of v(CO) (2093 cm-1) derived from FTIR spectroscopy suggests coordination of a weak donor such as methionine, which is supported by a previous observation that the delta Pro-PHM382s mutant M314I is totally inactive. Binding of the peptide substrate N-Ac-Tyr-Val-Gly causes minimum structural perturbation at the Cu(I) centers but appears to induce a more rigid conformation in the vicinity of the S-Met ligand. The unusually intense 8983 eV Cu K-absorption edge feature in reduced and substrate-bound-reduced enzymes is suggestive of a trigonal or digonal coordination environment for Cu(I). A structural model is proposed for the copper centers involving 3 histidines as ligands to CuIA and 2 histidines and 1 methionine as ligands to CuIB. However, in view of the intense 8934 eV edge feature and the lack of CO-binding ability, a 2-coordinate structure for CuA is also entirely consistent with the data.

The irreversible inactivation of two copper-dependent monooxygenases by sulfite: peptidylglycine alpha-amidating enzyme and dopamine beta-monooxygenase.

Peptidylglycine alpha-amidating enzyme (alpha-AE) and dopamine beta-monooxygenase (D beta M), two copper-dependent monooxygenases that have catalytic and structural similarities, are irreversibly inactivated by sodium sulfite in a time- and concentration-dependent manner. Studies with alpha-AE show that the sulfite-mediated inactivation is dependent on the presence of redox active transition metals free in solution, with Cu(II) being the most effective in supporting the inactivation reaction. Sulfite inactivation of alpha-AE is specific for the monooxygenase reaction of this bifunctional enzyme and amidated peptides provide protection against the inactivation. Consequently, the sulfite-mediated inactivation of alpha-AE and D beta M most likely results from the transition metal-catalyzed oxidation of sulfite to the sulfite radical, SO3-.

The inactivation of bifunctional peptidylglycine alpha-amidating enzyme by benzylhydrazine: evidence that the two enzyme-bound copper atoms are nonequivalent.

Peptidylglycine alpha-amidating enzyme catalyzes the two-step conversion of C-terminal glycine-extended peptides to C-terminal alpha-amidated peptides and glyoxylate in a reaction that requires O2, ascorbate and 2 mol of copper per mole of enzyme [Kulathila et al. (1994) Arch. Biochem. Biophys. 311, 191-195]. Peptides with a C-terminal alpha-hydroxyglycine residue are intermediates in the amidation reaction. Benzylhydrazine inactivates the enzymatic conversion of dansyl-Tyr-Val-Gly to dansyl-Tyr-Val-NH2 in a time- and concentration-dependent manner. In contrast, the enzymatic conversion of dansyl-Tyr-Val-alpha-hydroxyglycine to dansyl-Tyr-Val-NH2 is unaffected by benzylhydrazine. The plot of 1/(inactivation rate) vs 1/[benzylhydrazine] is parabolic, indicating that the inactivation results from the interaction of 2 mol of benzylhydrazine per mole of enzyme. EPR spectra obtained from benzylhydrazine inactivation reactions carried out in the presence of a radical trap, alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone, show the formation of a carbon-centered benzyl radical. The benzyl radical most likely results from redox chemistry between benzylhydrazine and the enzyme-bound Cu(II) ions because EPR studies show that enzyme-bound Cu(II) is reduced to Cu(I) in the presence of benzylhydrazine. The kinetic constants for benzylhydrazine as a reductant in the amidation reaction were determined at benzylhydrazine concentrations too low to cause significant enzyme inactivation. Mimosine exhibits mixed inhibition vs benzylhydrazine; however, previous results have shown that benzylhydrazine is competitive vs ascorbate [Miller et al. (1992) Arch. Biochem. Biophys. 298, 380-388]. This change in kinetic mechanism coupled with the nonlinear inactivation kinetics have lead to a proposal that the two enzyme-bound Cu(II) atoms are nonequivalent with respect to their reduction by benzylhydrazine.

Bifunctional peptidylglcine alpha-amidating enzyme requires two copper atoms for maximum activity.

The conversion of C-terminal glycine-extended peptides to C-terminal alpha-amidated peptides occurs in two distinct reactions, both of which are catalyzed by bifunctional peptidylglycine alpha-amidating enzyme. The first step is the alpha-hydroxylation of the C-terminal glycine residue and the second step is the dealkylation of the alpha-hydroxyglycine-extended peptide to the alpha-amidated peptide and glyoxylate. We show that the bifunctional enzyme requires 1.9 +/- 0.2 mol of copper/mol of enzyme for maximal dansyl-Tyr-Lys-Gly amidation activity under the conditions of high enzyme concentration (approximately 80 microM) required to measure initial rates for this poor substrate. The enzyme, as purified, contains a substoichiometric amount of copper and has only trace levels of amidation activity. Addition of exogenous Cu(II) ions stimulates amidation activity approximately 3000-fold at the optimum copper stoichiometry and the enzyme is then inhibited by excess Cu(II). No stimulation of amidation activity is observed upon the addition of the following divalent metal ions: Mn(II), Fe(II), Ni(II), Cd(II), and the oxovanadium cation, VO(II). The enzyme-catalyzed dealkylation of alpha-hydroxyhippuric acid to benzamide shows no dependence on copper, indicating that the copper dependence of the amidation reaction must be attributed to a copper dependence in peptide alpha-hydroxylation.

Characterization of a bifunctional peptidylglycine alpha-amidating enzyme expressed in Chinese hamster ovary cells.

Peptidylglycine alpha-amidating enzyme (alpha-AE) catalyzes the conversion of glycine-extended prohormones to their biologically active alpha-amidated forms. We have derived a clonal Chinese hamster ovary cell line that secretes significant quantities of active alpha-AE. Enzyme production was increased by selection for methotrexate-resistant cells expressing a dicistronic message. Amplification of the alpha-AE gene was monitored by Southern blot analysis, enzyme activity, and immunoreactive protein throughout the selection process. The soluble enzyme is bifunctional as determined by the ability to convert either the glycine-extended substrate, dansyl-Tyr--Val--Gly, or the intermediate, dansyl-Tyr--Val--alpha-hydroxyglycine, to the dansyl-Tyr--Val--NH2 product. The recombinant alpha-AE was purified by a simple two-step chromatographic process. The purified enzyme is partially glycosylated and the glycosylated and nonglycosylated forms of the enzyme were separated on a Con A-Sepharose column. The kinetic constants for dansyl-Tyr--Val--Gly, dansyl-Tyr--Val--alpha-hydroxyglycine, ascorbate, and catechol were the same for both forms of alpha-AE. In addition, mimosine is competitive vs ascorbate with K(is) = 3.5 microM for the nonglycosylated alpha-AE and K(is) = 4.2 microM for the glycosylated alpha-AE. Therefore, the presence or absence of asparagine-linked oligosaccharide does not affect the catalytic efficiency of the enzyme. Overexpression of the recombinant enzyme in CHO cells greatly enhances expression of the endogenous gene, implicating a feedback mechanism on the alpha-AE gene.

18O isotopic 13C NMR shift as proof that bifunctional peptidylglycine alpha-amidating enzyme is a monooxygenase.

The biosynthesis of C-terminal alpha-amidated peptides from their corresponding C-terminal glycine-extended precursors is catalyzed by peptidylglycine alpha-amidating enzyme (alpha-AE) in a reaction that requires copper, ascorbate, and molecular oxygen. Using bifunctional type A rat alpha-AE, we have shown that O2 is the source of the alpha-carbonyl oxygen of pyruvate produced during the amidation of dansyl-Tyr-Val-[alpha-13C]-D-Ala, as demonstrated by the 18O isotopic shift in the 13C NMR spectrum of [alpha-13C]lactate generated from [alpha-13C]pyruvate in the presence of lactate dehydrogenase and NADH. In addition, one-to-one stoichiometries have been determined for glyoxylate formed/dansyl-Tyr-Val-Gly consumed, pyruvate formed/dansyl-Tyr-Val-D-Ala consumed, dansyl-Tyr-Val-NH2 formed/ascorbate oxidized, and dansyl-Tyr-Val-NH2 formed/O2 consumed. Quantitative coupling of NADH oxidation to dansyl-Tyr-Val-NH2 production using Neurospora crassa semidehydroascorbate reductase showed that two one-electron reductions by ascorbate occurred per alpha-AE turnover. The stoichiometry of approximately 1.0 dansyl-Tyr-Val-NH2 produced/ascorbate oxidized observed in the absence of a semidehydroascorbate trap resulted from the disproportionation of two semidehydroascorbate molecules to ascorbate and dehydroascorbate.

Selective inactivation of the hydroxylase activity of bifunctional rat peptidylglycine alpha-amidating enzyme.

Conversion of dansyl-Tyr-Val-Gly to dansyl-Tyr-Val-NH2 by recombinant type A rat 75-kDa peptidylglycine alpha-amidating enzyme (alpha-AE) is inactivated by ascorbate, dehydroascorbate, and hydrogen peroxide in a time- and concentration-dependent manner. Both ascorbate- and dehydroascorbate-mediated inactivation are saturable with apparent kinact/Kinact values of 1.7 and 0.23 s-1 M-1, respectively. Hydrogen peroxide-mediated inactivation is not saturable with a second-order rate constant of 50 s-1 M-1. Peptidyl-Gly substrates, EDTA, and H2O2 scavengers protect against ascorbate-mediated inactivation while EDTA and semidehydroascorbate scavengers protect against dehydroascorbate-mediated inactivation. Under similar conditions, ascorbate, dehydroascorbate, and H2O2 have no effect on the alpha-AE-catalyzed conversion of dansyl-Tyr-Val-alpha-hydroxyglycine to dansyl-Tyr-Val-NH2 which is consistent with the hypothesis that the 75-kDa enzyme consists of distinct peptidyl-Gly hydroxylase and peptidyl-alpha-hydroxyglycine lyase active sites.