Synthesis of N-succinyl-L,L-diaminopimelic acid mimetics via selective protection.

The search for potential inhibitors that target so far unexplored bacterial enzyme mono-N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) has stimulated a development of methodology for quick and efficient preparation of mono-N-acylated 2,6-diaminopimelic acid (DAP) derivatives bearing the different carboxyl groups or lipophilic moieties on their amino group.

Inhibitors of N(alpha)-acetyl-L-ornithine deacetylase: synthesis, characterization and analysis of their inhibitory potency.

A series of N (alpha)-acyl (alkyl)- and N (alpha)-alkoxycarbonyl-derivatives of L- and D-ornithine were prepared, characterized, and analyzed for their potency toward the bacterial enzyme N (alpha)-acetyl-L-ornithine deacetylase (ArgE). ArgE catalyzes the conversion of N (alpha)-acetyl-L-ornithine to L-ornithine in the fifth step of the biosynthetic pathway for arginine, a necessary step for bacterial growth. Most of the compounds tested provided IC(50) values in the muM range toward ArgE, indicating that they are moderately strong inhibitors. N (alpha)-chloroacetyl-L-ornithine (1g) was the best inhibitor tested toward ArgE providing an IC(50) value of 85 microM while N (alpha)-trifluoroacetyl-L-ornithine (1f), N (alpha)-ethoxycarbonyl-L-ornithine (2b), and N (alpha)-acetyl-D-ornithine (1a) weakly inhibited ArgE activity providing IC(50) values between 200 and 410 microM. Weak inhibitory potency toward Bacillus subtilis-168 for N (alpha)-acetyl-D-ornithine (1a) and N (alpha)-fluoro- (1f), N (alpha)-chloro- (1g), N (alpha)-dichloro- (1h), and N (alpha)-trichloroacetyl-ornithine (1i) was also observed. These data correlate well with the IC(50) values determined for ArgE, suggesting that these compounds might be capable of getting across the cell membrane and that ArgE is likely the bacterial enzymatic target.

Structural evidence that the methionyl aminopeptidase from Escherichia coli is a mononuclear metalloprotease.

The Co and Fe K-edge extended X-ray absorption fine structure (EXAFS) spectra of the methionyl aminopeptidase from Escherichia coli (EcMetAP) have been recorded in the presence of 1 and 2 equiv of either Co(II) or Fe(II) (i.e., [Co(II)_(EcMetAP)], [Co(II)Co(II)(EcMetAP)], [Fe(II)_(EcMetAP)], and [Fe(II)Fe(II)(EcMetAP)]). The Fourier transformed data of both [Co(II)_(EcMetAP)] and [Co(II)Co(II)(EcMetAP)] are dominated by a peak at ca. 2.05 A, which can be fit assuming 5 light atom (N,O) scatterers at 2.04 A. Attempts to include a Co-Co interaction (in the 2.4-4.0 A range) in the curve-fitting parameters were unsuccessful. Inclusion of multiple-scattering contributions from the outer-shell atoms of a histidine-imidazole ring resulted in reasonable Debye-Waller factors for these contributions and a slight reduction in the goodness-of-fit value (f '). These data suggest that a dinuclear Co(II) center does not exist in EcMetAP and that the first Co atom is located in the histidine-ligated side of the active site. The EXAFS data obtained for [Fe(II)_(EcMetAP)] and [Fe(II)Fe(II)(EcMetAP)] indicate that Fe(II) binds to EcMetAP in a similar site to Co(II). Since no X-ray crystallographic data are available for any Fe(II)-substituted EcMetAP enzyme, these data provide the first glimpse at the Fe(II) active site of MetAP enzymes. In addition, the EXAFS data for [Co(II)Co(II)(EcMetAP)] incubated with the antiangiogenesis drug fumagillin are also presented.

Inhibition of the aminopeptidase from Aeromonas proteolytica by L-leucinephosphonic acid. Spectroscopic and crystallographic characterization of the transition state of peptide hydrolysis.

The nature of the interaction of the transition-state analogue inhibitor L-leucinephosphonic acid (LPA) with the leucine aminopeptidase from Aeromonas proteolytica (AAP) was investigated. LPA was shown to be a competitive inhibitor at pH 8.0 with a K(i) of 6.6 microM. Electronic absorption spectra, recorded at pH 7.5 of [CoCo(AAP)], [CoZn(AAP)], and [ZnCo(AAP)] upon addition of LPA suggest that LPA interacts with both metal ions in the dinuclear active site. EPR studies on the Co(II)-substituted forms of AAP revealed that the environments of the Co(II) ions in both [CoZn(AAP)] and [ZnCo(AAP)] become highly asymmetric and constrained upon the addition of LPA and clearly indicate that LPA interacts with both metal ions. The X-ray crystal structure of AAP complexed with LPA was determined at 2.1 A resolution. The X-ray crystallographic data indicate that LPA interacts with both metal centers in the dinuclear active site of AAP and a single oxygen atom bridge is absent. Thus, LPA binds to the dinuclear active site of AAP as an eta-1,2-mu-phosphonate with one ligand to the second metal ion provided by the N-terminal amine. A structural comparison of the binding of phosphonate-containing transition-state analogues to the mono- and bimetallic peptidases provides insight into the requirement for the second metal ion in bridged bimetallic peptidases. On the basis of the results obtained from the spectroscopic and X-ray crystallographic data presented herein along with previously reported mechanistic data for AAP, a new catalytic mechanism for the hydrolysis reaction catalyzed by AAP is proposed.

Divalent metal binding properties of the methionyl aminopeptidase from Escherichia coli.

The metal-binding properties of the methionyl aminopeptidase from Escherichia coli (MetAP) were investigated. Measurements of catalytic activity as a function of added Co(II) and Fe(II) revealed that maximal enzymatic activity is observed after the addition of only 1 equiv of divalent metal ion. Based on these studies, metal binding constants for the first metal binding event were found to be 0.3 +/- 0.2 microM and 0.2 +/- 0.2 microM for Co(II)- and Fe(II)-substituted MetAP, respectively. Binding of excess metal ions (>50 equiv) resulted in the loss of approximately 50% of the catalytic activity. Electronic absorption spectral titration of a 1 mM sample of MetAP with Co(II) provided a binding constant of 2.5 +/- 0.5 mM for the second metal binding site. Furthermore, the electronic absorption spectra of Co(II)-loaded MetAP indicated that both metal ions reside in a pentacoordinate geometry. Consistent with the absorption data, electron paramagnetic resonance (EPR) spectra of [CoCo(MetAP)] also indicated that the Co(II) geometries are not highly constrained, suggesting that each Co(II) ion in MetAP resides in a pentacoordinate geometry. EPR studies on [CoCo(MetAP)] also revealed that at pH 7.5 there is no significant spin-coupling between the two Co(II) ions, though a small proportion ( approximately 5%) of the sample exhibited detectable spin-spin interactions at pH values > 9.6. EPR studies on [Fe(III)_(MetAP)] and [Fe(III)Fe(III)(MetAP)] also suggested no spin-coupling between the two metal ions. (1)H nuclear magnetic resonance (NMR) spectra of [Co(II)_(MetAP)] in both H(2)O and D(2)O buffer indicated that the first metal binding site contains the only active-site histidine residue, His171. Mechanistic implications of the observed binding properties of divalent metal ions to the MetAP from E. coli are discussed.

Inhibition of the aminopeptidase from Aeromonas proteolytica by L-leucinethiol: kinetic and spectroscopic characterization of a slow, tight-binding inhibitor-enzyme complex.

The peptide inhibitor L-leucinethiol (LeuSH) was found to be a potent, slow-binding inhibitor of the aminopeptidase from Aeromonas proteolytica (AAP). The overall potency (K(I)*) of LeuSH was 7 nM while the corresponding alcohol L-leucinol (LeuOH) was a simple competitive inhibitor of much lower potency (K(I) = 17 microM). These data suggest that the free thiol is likely involved in the formation of the E x I and E x I* complexes, presumably providing a metal ligand. In order to probe the nature of the interaction of LeuSH and LeuOH with the dinuclear active site of AAP, we have recorded both the electronic absorption and EPR spectra of [CoCo(AAP)], [CoZn(AAP)], and [ZnCo(AAP)] in the presence of both inhibitors. In the presence of LeuSH, all three Co(II)-substituted AAP enzymes exhibited an absorption band centered at 295 nm, characteristic of a S --> Co(II) ligand-metal charge-transfer band. In addition, absorption spectra recorded in the 450 to 700 nm region all showed changes characteristic of LeuSH and LeuOH interacting with both metal ions. EPR spectra recorded at high temperature (19 K) and low power (2.5 mW) indicated that, in a given enzyme molecule, LeuSH interacts weakly with one of the metal ions in the dinuclear site and that the crystallographically identified mu-OH(H) bridge, which has been shown to mediate electronic interaction of the Co(II) ions, is likely broken upon binding LeuSH. EPR spectra of [CoCo(AAP)]-LeuSH, [ZnCo(AAP)]-LeuSH, and [Co_(AAP)]-LeuSH were also recorded at lower temperature (3.5-4.0 K) and high microwave power (50-553 mW). These signals were unusual and appeared to contain, in addition to the incompletely saturated contributions from the signals characterized at 19 K, a very sharp feature at g(eff) approximately 6.5 that is characteristic of thiolate-Co(II) interactions. Combination of the electronic absorption and EPR data indicates that LeuSH perturbs the electronic structure of both metal ions in the dinuclear active site of AAP. Since the spin-spin interaction seen in resting [CoCo(AAP)] is abolished upon the addition of LeuSH, it is unlikely that a mu-S(R) bridge is established.

Slow-binding inhibition of the aminopeptidase from Aeromonas proteolytica by peptide thiols: synthesis and spectroscopic characterization.

Peptide-derived thiols of the general structure N-mercaptoacyl-leucyl-p-nitroanilide (1a-c) were synthesized and found to be potent, slow-binding inhibitors of the aminopeptidase from Aeromonas proteolytica (AAP). The overall potencies (K(I)) of these inhibitors against AAP range from 2.5 to 57 nM exceeding that of the natural product bestatin and approaching that of amastatin. The corresponding alcohols (2a-b) are simple competitive inhibitors of much lower potencies (K(I) = 23 and 360 microM). These data suggest that the free thiols are involved in the formation of the E. I and E.I complexes, presumably serving as a metal ligand. To investigate the nature of the interaction of the thiol-based inhibitors with the dinuclear active site of AAP, we have recorded electronic absorption and EPR spectra of Co(II)Co(II)-, Co(II)Zn(II)-, and Zn(II)Co(II)-AAP in the presence of the strongest binding inhibitor, 1c. Both [CoZn(AAP)] and [ZnCo(AAP)], in the presence of 1c, exhibited an absorption band centered at 320 nm characteristic of an S --> Co(II) ligand-metal charge-transfer band. In addition, absorption spectra recorded between 400 and 700 nm showed changes characteristic of 1c interacting with each active-site metal ion. EPR spectra recorded at high temperature (19 K) and low power (2.5 mW) indicated that in a given enzyme molecule, 1c interacts weakly with one of the metal ions in the dinuclear site and that the crystallographically identified micro-OH(H) bridge, which has been shown to mediate electronic interaction of the Co(II) ions, is likely broken upon 1c binding. EPR spectra of [CoCo(AAP)]-1c, [ZnCo(AAP)]-1c, and [CoZn(AAP)]-1c were also recorded at lower temperature (3.5-4.0 K) and high microwave power (50-553 mW). The observed signals were unusual and appeared to contain, in addition to the incompletely saturated contributions from the signals characterized at 19 K, a very sharp feature at g(eff) approximately 6.8 that is characteristic of thiolate-Co(II) interactions. These data suggest that the thiolate moiety can bind to either of the metal ions in the dinuclear active site of AAP but does not bridge the dinuclear cluster. Compounds 1a-c are readily accessible by synthesis and thus provide a novel class of potent aminopeptidase inhibitors.

Inhibition of the aminopeptidase from Aeromonas proteolytica by aliphatic alcohols. Characterization of the hydrophobic substrate recognition site.

Seven aliphatic and two aromatic alcohols were tested as reporters of the substrate selectivity of the aminopeptidase from Aeromonas proteolytica (AAP). This series of alcohols was chosen to systematically probe the effect of carbon chain length, steric bulk, and inhibitor shape on the inhibition of AAP. Initially, however, the question of whether AAP is denatured in the presence of aliphatic alcohols was addressed. On the basis of circular dichroism (CD), electronic absorption, and fluorescence spectra, the secondary structure of AAP, with and without added aliphatic alcohols, was unchanged. These data clearly indicate that AAP is not denatured in aliphatic alcohols, even up to concentrations of 20% (v/v). All of the alcohols studied were competitive inhibitors of AAP with K(i) values between 860 and 0.98 mM. The clear trend in the data was that as the carbon chain length increases from one to four, the K(i) values increase. Branching of the carbon chains also increases the K(i) values, but large bulky groups, such as that found in tert-butyl alcohol, do not inhibit AAP as well as leucine analogues, such as 3-methyl-1-butanol. The competitive nature of the inhibition indicates that the substrate and each alcohol studied are mutually exclusive due to binding at the same site on the enzyme. On the basis of EPR and electronic absorption data for Co(II)-substituted AAP, none of the alcohols studied binds to the dinuclear metallo-active site of AAP. Thus, reaction of the inhibitory alcohols with the catalytic metal ions cannot constitute the mechanism of inhibition. Combination of these data suggests that each of these inhibitors bind only to the hydrophobic pocket of AAP and, consequently, block the binding of substrate. Thus, the first step in peptide hydrolysis is the recognition of the N-terminal amino acid side chain by the hydrophobic pocket adjacent to the dinuclear active site of AAP.

The methionyl aminopeptidase from Escherichia coli can function as an iron(II) enzyme.

The identity of the physiologically relevant metal ions for the methionyl aminopeptidase (MetAP) from Escherichia coli was investigated and is suggested to be Fe(II). The metal content of whole cells in the absence and presence of expression of the type I MetAP from E. coli was determined by inductively coupled plasma (ICP) emission analysis. The observed change in whole cell concentrations of cobalt, cadmium, copper, nickel, strontium, titanium, and vanadium upon expression of MetAP was negligible. On the other hand, significant increases in the cellular metal ion concentrations of chromium, zinc, manganese, and iron were observed with the increase in iron concentration being 4.4 and 6.2 times greater than that of manganese and zinc, respectively. Activity assays of freshly lysed BL21(DE3) cells containing the pMetAAP plasmid revealed detectable levels (>2 units/mg) of MetAP activity. Control experiments with BL21(DE3) without the MetAP plasmid showed no detectable enzymatic activity. Since MetAP is active upon expression, these data strongly suggest that cobalt is not the in vivo metal ion for the MetAP from E. coli. The MetAP from E. coli as purified was found to be catalytically inactive (

1H NMR studies on the CuA center of nitrous oxide reductase from Pseudomonas stutzeri.

1H NMR spectra of the CuA center of N2OR from Pseudomonas stutzeri, and a mutant enzyme that contains only CuA, were recorded in both H2O- and D2O-buffered solution at pH 7.5. Several sharp, well-resolved hyperfine-shifted 1H NMR signals were observed in the 60 to -10 ppm chemical shift range. Comparison of the native and mutant N2OR spectra recorded in H2O-buffered solutions indicated that several additional signals are present in the native protein spectrum. These signals are attributed to a dinuclear copperII center. At least two of the observed hyperfine-shifted signals associated with the dinuclear center, those at 23.0 and 13.2 ppm, are lost upon replacement of H2O buffer with D2O buffer. These data indicate that at least two histidine residues are ligands of a dinuclear CuII center. Comparison of the mutant N2OR 1H NMR spectra recorded in H2O and D2O indicates that three signals, c (27.5 ppm), e (23.6 ppm), and i (12.4 ppm), are solvent exchangeable. The two most strongly downfield-shifted signals (c and e) are assigned to the two N epsilon 2H (N-H) protons of the coordinated histidine residues, while the remaining exchangeable signal is assigned to a backbone N-H proton in close proximity to the CuA cluster. Signal e was found to decrease in intensity as the temperature was increased, indicating that proton e resides on a more solvent-exposed histidine residue. One-dimensional nOe studies at pH 7.5 allowed the histidine ring protons to be definitively assigned, while the remaining signals were assigned by comparison to previously reported spectra from CuA centers. The temperature dependence of the observed hyperfine-shifted 1H NMR signals of mutant N2OR were recorded over the temperature range of 276-315 K. Both Curie and anti-Curie temperature dependencies are observed for sets of hyperfine-shifted protons. Signals a and h (cysteine protons) follow anti-Curie behavior (contact shift increases with increasing temperatures), while signals b-g, i, and j (histidine protons) follow Curie behavior (contact shift decreases with increasing temperatures). Fits of the temperature dependence of the observed hyperfine-shifted signals provided the energy separation (Delta EL) between the ground (2B3u) and excited (2B2u) states. The temperature data obtained for all of the observed hyperfine-shifted histidine ligand protons provided a Delta EL value of 62 +/- 35 cm-1. The temperature dependence of the observed cysteine C beta H and C alpha H protons (a and h) were fit in a separate experiment providing a Delta EL value of 585 +/- 125 cm-1. The differences between the Delta EL values determined by 1H NMR spectroscopy and those determined by EPR or MCD likely arise from coupling between relatively low-frequency vibrational states and the ground and excited electronic states.

1-Butaneboronic acid binding to Aeromonas proteolytica aminopeptidase: a case of arrested development.

Hydrolases containing two metal ions connected by a bridging ligand catalyze reactions important in carcinogensis, tissue repair, post-translational modification, control and regulation of biochemical pathways, and protein degradation. The aminopeptidase from Aeromonas proteolytica serves as a paradigm for the study of such bridged bimetallic proteases since its three-dimensional structure is known to very high resolution and its catalytic reaction is amenable to spectroscopic examination. Herein, we report the X-ray crystal structure at 1.9 A resolution of AAP complexed with 1-butaneboronic acid (BuBA). This structure suggests that this complex represents a snapshot of the proteolytic reaction in an arrested form between the Michaelis complex and the transition state. Comparison of the structure with spectroscopic and other data allows us to conclude that the apparently structurally symmetrical dizinc site is actually asymmetric electrostatically.

Proton nuclear magnetic resonance investigation of the [2Fe-2S](1-)-containing "Rieske-type" protein from Xanthobacter strain Py2.

Proton NMR spectra of the Rieske-type ferredoxin from Xanthobacter strain Py2 were recorded in both H2O and D2O buffered solutions at pH 7.2. Several well-resolved hyperfine-shifted 1H NMR signals were observed in the 90 to -20 ppm chemical shift range. Comparison of spectra recorded in H2O and D2O buffered solutions indicated that the signals at -11.4 (L) and -15.5 (M) ppm were solvent-exchangeable and thus were assigned to the two histidine N epsilon 2H protons. The remaining observed signals were assigned based upon chemical shift, T1 values, and one-dimensional nuclear Overhauser effect (nOe) saturation transfer experiments to either C beta H or C alpha H protons of cluster cysteinyl or histidine ligands. Upon oxidation of the [2Fe-2S] cluster, only two broad resonances were observed, indicating that the two Fe(III) ions are strongly antiferromagnetically coupled. In addition, the temperature dependence of each observed hyperfine-shifted signal in the reduced state was determined, providing information about the magnetic properties of the [2Fe-2S]1- cluster. Fits of the temperature data observed for each resonance to equations describing the hyperfine shift with their Boltzmann weighting factors provided a delta EL value of 185 +/- 26 cm-1 which, in turn, gives -2J as 124 cm-1. These data indicate that the two iron centers in the reduced [2Fe-2S] Rieske-type center are moderately antiferromagnetically coupled. The combination of these data with the available spectroscopic and crystallographic results for Rieske-type proteins has provided new insights into the role of the Rieske-type protein from Xanthobacter strain Py2 in alkene oxidation.

Spectroscopically distinct cobalt(II) sites in heterodimetallic forms of the aminopeptidase from Aeromonas proteolytica: characterization of substrate binding.

The Co(II)Zn(II)- and Zn(II)Co(II)-substituted derivatives of the aminopeptidase from Aeromonas proteolytica (AAP) were probed by EPR spectroscopy. EPR spectra of the high-spin S = 3/2 Co(II) ions in [CoZn(AAP)] and [ZnCo(AAP)] indicated that each metal binding site provides a spectroscopically distinct signature. For [CoZn(AAP)], subtraction of EPR spectra recorded at pH 7.5 and 10 revealed that two species were present and that the relative contributions to each of the experimental spectra were pH-dependent. The first EPR species, predominant at lower pH values, was simulated as a relatively featureless axial signal with geff values of 2.20, 3.92, and 5.23 which correspond to an Ms = |+/-1/2> ground state transition with a greal of 2.29 and an E/D of 0.1. The second species, predominant at high pH, was simulated with geff values of 1.80, 2.75, and 6.88 and exhibited a characteristic eight-line 59Co hyperfine pattern with an Az(59Co) of 7.0 mT. These parameters correspond to an Ms = |+/-1/2> ground state transition with a greal of 2.54; however, the signal exhibited marked rhombicity (E/D = 0.32) indicative of an asymmetric tetrahedral or five-coordinate Co(II) ion. Summation of these two species provided an excellent simulation of the observed [CoZn(AAP)] EPR spectrum. The EPR spectrum of [ZnCo(AAP)] also contained two species, at least one of which also exhibited 59Co hyperfine features. However, this signal exhibited little pH dependence, and individual species could not be isolated. The addition of the competitive inhibitor 1-butaneboronic acid (BuBA) to [CoZn(AAP)] resulted in a distinct change in the EPR spectrum; however, addition of BuBA to [ZnCo(AAP)] left the EPR spectrum completely unperturbed. These data indicate that BuBA binds only to the first metal binding site in AAP and does not interact with the second site. On the basis of the X-ray crystallographic data for the transition state analog-inhibited complexes of AAP and the aminopeptidase from bovine lens, BuBA was reclassified as a substrate analog inhibitor rather than a transition state analog inhibitor as previously suggested [Baker, J. O., & Prescott, J. M. (1983) Biochemistry 22, 5322-5331]. From difference spectroscopy and from the simulation of the [CoZn(AAP)] EPR spectrum, a third signal appearing upon BuBA binding was isolated. This signal was simulated with geff values of 2.08, 3. 15, and 6.15 which correspond to an Ms = |+/-1/2> ground state transition with a greal of 2.41 and an E/D of 0.22. This simulation also invoked an eight-line unresolved 59Co hyperfine pattern with an Az(59Co) value of 4.0 mT. Summation of the these three species provided an excellent simulation of the observed [CoZn(AAP)] + BuBA EPR spectrum at both pH values. This work establishes that substrate binds only to the first metal binding site in AAP and thus substantiates the first step in catalysis in the recently proposed mechanism of action for AAP [Bennett, B., & Holz, R. C. (1997) J. Am. Chem. Soc. 119, 1923-1933; Chen, G., et al. (1997) Biochemistry 36, 4278-4286].

Mechanistic studies on the aminopeptidase from Aeromonas proteolytica: a two-metal ion mechanism for peptide hydrolysis.

The aminopeptidase from Aeromonas proteolytica (AAP) is uncompetitively inhibited by fluoride ion at pH 8.0 with an inhibition constant (Ki) of 30 mM. Thus, fluoride inactivates AAP only after substrate binding, and only a single fluoride ion binds to AAP. On the other hand, chloride ion does not inhibit AAP up to concentrations of 2 M at pH 8.0. The pH dependence of fluoride inhibition of AAP was measured over the pH range 6.0-9.5. Between pH values of 6.0 and 9.0, fluoride ion acts as a pure uncompetitive inhibitor of AAP, and the Ki increases from 1.2 to 370 mM. From a plot of pKi vs pH, a pKa value of 7.0 +/- 0.3 was extracted which corresponds to a single deprotonation process. At pH values higher than 9.0, the fluoride inhibition pattern changes to competitive. This change in inhibition pattern was attributed to a change in ionic strength or perhaps pH of the solution since fluoride ion was also found to become a competitive inhibitor of AAP at pH 8.0 in the presence of 2 M NaCl. These data, taken together with previous kinetic studies of mono- and dinuclear hydrolases with fluoride ion, suggest that a Zn(II)-bound water/hydroxide exists at the dimetal active site of AAP with a pKa of 7.0 and that this water/hydroxide acts as the active site nucleophile. The hydrolysis of L-leucine-p-nitroanilide was measured spectrophotometrically in triplicate between 25 and 85 degrees C at eight substrate concentrations ranging from 5 to 800 microM. From these data, Km values were derived at each temperature studied and were found to increase exponentially with increasing temperature. Moreover, the calculated Vmax values were also found to increase over this temperature range, mimicking the Km values. An Arrhenius plot was constructed from k(cat) values and was found to be linear over the temperature range 25-85 degrees C, indicating that the rate-limiting step in AAP peptide hydrolysis is product formation and does not change as a function of temperature. From the slope of the line, the activation energy (Ea) was calculated to be 36.5 kJ/mol. The enthalpy and entropy of activation at 25 degrees C calculated over the temperature range 298-358 K were found to be 34.0 kJ/mol and -94.2 J/(mol x K), respectively. The free energy of activation at 25 degrees C was found to be 62.1 kJ/mol. Combination of the available X-ray crystallographic data with the present kinetic and thermodynamic results, as well as the previously reported kinetic and spectroscopic data, has allowed a detailed catalytic mechanism for AAP to be proposed.

Proton NMR investigation of the [4Fe--4S]1+ cluster environment of nitrogenase iron protein from Azotobacter vinelandii: defining nucleotide-induced conformational changes.

This work presents the complete assignment of the isotropically shifted 1H NMR resonances of Azotobacter vinelandii nitrogenase iron protein (Fe protein) to beta-CH2 and alpha-CH protons of the [4Fe--4S]1+ cluster cysteinyl ligands. Four resonances were observed for the reduced Fe protein with chemical shifts of 49, 23, 17, and 13 ppm. T1 measurements and analysis of relative peak areas coupled with one-dimensional nuclear Overhauser effect (NOE) difference spectra were used to assign the two most downfield-shifted resonances (49 and 23 ppm) to cysteinyl ligand beta-CH2 protons and the 17 and 14 ppm resonances to cysteinyl ligand alpha-CH protons. Temperature dependent studies of the isotropically shifted protons revealed both Curie and anti-Curie behavior. These results, along with previous Mossbauer studies of the Fe protein, allowed the assignment of signal A (49 ppm) to four beta-CH2 protons and signal C (17 ppm) to 2 alpha-CH protons of two cysteinyl ligands bound to a mixed-valence iron pair (Fe3(+)--Fe2+) of the [4Fe--4S]1+ cluster. Signal B (23 ppm) was assigned to four beta-CH2 protons, and signal C (17 ppm) and D (13 ppm) were assigned to two alpha-CH protons of two cysteinyl ligands bound to a ferrous pair of irons (2Fe2+). The effects of MgATP, MgADP, and Mg-adenosine-beta, gamma-methylene-5'-triphosphate binding to the Fe protein on the assigned resonances were established and are discussed in the context of nucleotide-induced changes in the protein environment of the [4Fe--4S] cluster.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of porcine uterine fluid purple acid phosphatase with vanadate and vanadyl cation.

Uteroferrin, the purple acid phosphatase from porcine uterine fluid, is noncompetitively inhibited by vanadate in a time-dependent manner under both aerobic and anaerobic conditions. This time-dependent inhibition is observed only with the diiron enzyme and is absent when the FeZn enzyme is used. The observations are attributed to the sequential formation of two uteroferrin-vanadium complexes. The first complex forms rapidly and reversibly, while the second complex forms slowly and results in the production of catalytically inactive oxidized uteroferrin and V(IV), which is observed by EPR. The redox reaction can be reversed by treatment of the oxidized enzyme first with (V(IV)) and then EDTA to generate a catalytically active uteroferrin. Multiple inhibition kinetics suggests that vanadate is mutually exclusive with molybdate, tungstate, and vanadyl cation. The binding site for each of these anions is distinct from the site to which the competitive inhibitors phosphate and arsenate bind. The time-dependent inhibition by vanadate of uteroferrin containing the diiron core represents a new type of mechanism by which vanadium can interact with proteins and gives additional insight into the binding of anions to uteroferrin.

Electrochemical properties of the diiron core of uteroferrin and its anion complexes.

The reduction potentials (Em) of the purple acid phosphatase from porcine uterus, uteroferrin (Uf), and its phosphate, arsenate, and molybdate complexes were determined by coulometric methods at various pH values. The midpoint potential of Uf at the pH value for optimal enzyme activity (pH 5) was found to be +367 mV versus a normal hydrogen electrode (NHE), while at pH 6.01 Uf exhibits a reduction potential of +306 mV. At pH 6.01 molybdate was found to shift the potential of Uf more positive by 192 mV, while phosphate and arsenate shift the potential of Uf more negative by 193 and 89 mV, respectively. These shifts are consistent with the different susceptibilities of Uf to aerobic oxidation in the presence of these anions. Comparison of the reduction potential of Uf at pH 7.0 with those reported for other dinuclear non-heme iron enzymes and various (mu-oxo)diiron model complexes suggest that the potential of Uf is too positive to be consistent with a mu-oxo-bridge in Ufo. The pH dependence of the reduction potentials of Uf (60 mV/pH unit) and the fact that the electron transfer rate increases with decreasing pH indicate a concomitant participation of a proton during the oxidation-reduction process. This process was assigned to the protonation of a terminally bound hydroxide ligand at the Fe(II) center upon reduction of Ufo. Structural implications provided by the electrochemical data indicate that molybdate affects the dinuclear core in a manner that differs from that of phosphate and arsenate. This observation is consistent with previous spectroscopic and biochemical studies.(ABSTRACT TRUNCATED AT 250 WORDS)