Characterization of the Active Site and Insight into the Binding Mode of the Anti-angiogenesis Agent Fumagillin to the Manganese(II)-Loaded Methionyl Aminopeptidase from \u3cem\u3eEscherichia coli\u3c/em\u3e

EPR spectra were recorded for methionine aminopeptidase from Escherichia coli (EcMetAP-I) samples (~2.5 mM) to which one and two equivalents of Mn(II) were added (the latter is referred to as [MnMn(EcMetAP-I)]). The spectra for each sample were indistinguishable except that the spectrum of [MnMn(EcMetAP-I)] was twice as intense. The EPR spectrum of [MnMn(EcMetAP-I)] exhibited the characteristic six-line g≈2 EPR signal of mononuclear Mn(II) with A av(55Mn)=9.3 mT (93 G) and exhibited Curie-law temperature dependence. This signal is typical of Mn(II) in a ligand sphere comprising oxygen and/or nitrogen atoms. Other features in the spectrum were observed only as the temperature was raised from that of liquid helium. The temperature dependences of these features are consistent with their assignment to excited state transitions in the S=1, 2 ... 5 non-Kramer’s doublets, due to two antiferromagnetically coupled Mn(II) ions with an S=0 ground state. This assignment is supported by the observation of a characteristic 4.5 mT hyperfine pattern, and by the presence of signals in the parallel mode consistent with a non-Kramers’ spin ladder. Upon the addition of the anti-angiogenesis agent fumagillin to [MnMn(EcMetAP-I)], very small changes were observed in the EPR spectrum. MALDI-TOF mass spectrometry indicated that fumagillin was, however, covalently coordinated to EcMetAP-I. Therefore, the inhibitory action of this anti-angiogenesis agent on EcMetAP-I appears to involve covalent binding to a polypeptide component at or near the active site rather than direct binding to the metal ions

Divalent Metal Binding Properties of the Methionyl Aminopeptidase from \u3cem\u3eEscherichia coli\u3c/em\u3e

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 μM and 0.2 ± 0.2 μM for Co(II)- and Fe(II)-substituted MetAP, respectively. Binding of excess metal ions (\u3e50 equiv) resulted in the loss of ∼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 (∼5%) of the sample exhibited detectable spin−spin interactions at pH values \u3e 9.6. EPR studies on [Fe(III)_(MetAP)] and [Fe(III)Fe(III)(MetAP)] also suggested no spin-coupling between the two metal ions. 1H nuclear magnetic resonance (NMR) spectra of [Co(II)_(MetAP)] in both H2O and D2O 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

The Methionyl Aminopeptidase from \u3cem\u3eEscherichia coli\u3c/em\u3e 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 (\u3e2 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 (≤2 units/mg). ICP emission analysis of the as-purified enzyme revealed no catalytically relevant metal ions. Both the Co(II)− and Fe(II)−MetAP enzymes are susceptible to autoxidation, so strict care must be taken to remove all dissolved oxygen. Enzymatic assays performed under anaerobic conditions indicated that of the di- and trivalent metal cations tested to date, only Co(II) (37.3 units/mg), Fe(II) (29.7 units/mg), Mn(II) (7.0 units/mg), and Zn(II) (3.3 units/mg) provided detectable levels of enzymatic activity. In each case, excess metal ions were found to be inhibitory. The observed specific activity of Co(II)−MetAP is more than 3 times greater than that previously reported for the MetAP from E. coli [Ben-Bassat, A., et al. (1987) J. Bacteriol. 169, 751−757]. This increase in activity is likely due to the strict exclusion of air from reaction samples. Oxidation of either the Fe(II) or Co(II) form of the enzyme resulted in the complete loss of catalytic activity. The substrate binding constants (Km) for Met-Gly-Met-Met binding to the Co(II)- or Fe(II)-substituted MetAP enzymes, under anaerobic conditions, were found to be 3.16 and 1.95 mM, respectively. The combination of these data suggests that the in vivo metal ions for the MetAP enzyme from E. coli are likely Fe(II) ions

Function of the Signal Peptide and N- and C-terminal Propeptides in the Leucine Aminopeptidase from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e

The leucine aminopeptidase from Aeromonas proteolytica (also known as Vibrio proteolyticus) (AAP) is a metalloenzyme with broad substrate specificity. The open reading frame (ORF) for AAP encodes a 54 kDa enzyme, however, the extracellular enzyme has a molecular weight of 43 kDa. This form of AAP is further processed to a mature, thermostable 32 kDa form but the exact nature of this process is unknown. Over-expression of different forms of AAP in Escherichia coli (with AAP\u27s native leader sequence, with and without the N- and/or C-terminal propeptides, and as fusion protein) has allowed a model for the processing of wild-type AAP to be proposed. The role of the A. proteolytica signal peptide in protein secretion as well as comparison to other known signal peptides reveals a close resemblance of the A. proteolytica signal peptide to the outer membrane protein (OmpA) signal peptide. Over-expression of the full 54 kDa AAP enzyme provides an enzyme that is significantly less active, due to a cooperative inhibitory interaction between both propeptides. Over-expression of AAP lacking its C-terminal propeptide provided an enzyme with an identical kcat value to wild-type AAP but exhibited a larger Km value, suggesting competitive inhibition of AAP by the N-terminal propeptide (Ki∼0.13 nM). The recombinant 32 kDa form of AAP was characterized by kinetic and spectroscopic methods and was shown to be identical to mature, wild-type AAP. Therefore, the ease of purification and processing of rAAP along with the fact that large quantities can be obtained now allow new detailed mechanistic studies to be performed on AAP through site-directed mutagenesis

Structurally Distinct Active Sites in the Copper(II)-Substituted Aminopeptidases from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e and \u3cem\u3eEscherichia coli\u3c/em\u3e

The aminopeptidase from Aeromonas proteolytica (AAP) was titrated with copper, which bound sequentially at two distinct sites. Both the mono- and disubstituted forms of AAP exhibited catalytic hyperactivity relative to the native dizinc enzyme. Monosubstituted AAP exhibited an axial Cu(II) EPR spectrum with slight pH dependence:  at pH 6.0 g|| = 2.249, g⊥ = 2.055, and A||(63/65Cu) = 1.77 × 10-2 cm-1, whereas at pH 9.65 g|| = 2.245, g⊥ = 2.056, and A||(63/65Cu) = 1.77 × 10-2 cm-1. These data indicate oxygen and nitrogen ligation of Cu. AAP further substituted with copper exhibited a complex signal with features around g ∼ 2 and 4. The features at g ∼ 4 were relatively weak in the B0 ⊥ B1 (perpendicular) mode EPR spectrum but were intense in the B0 || B1 (parallel) mode spectrum. The g ∼ 2 region of the perpendicular mode spectrum exhibited two components, one corresponding to mononuclear Cu(II) with g|| = 2.218, g⊥ = 2.023, and A||(63/65Cu) = 1.55 × 10-2 cm-1 and likely due to adventitious binding of Cu(II) to a site distant from the active site. Excellent simulations were obtained for the second component of the spectrum assuming that two Cu(II) ions experience dipolar coupling corresponding to an inter-copper distance of 5 Å with the two Cu(II) gz directions parallel to each other and at an angle of ∼17° to the inter-copper vector (ℋ = βB·gCuA·SCuA + βB·gCuB·SCuB + [S·A·I]CuA + [S·A·I]CuB + [SCuA·J·SCuB]; g||(CuA,CuB) = 2.218, g⊥(CuA,CuB) = 2.060; A||(CuA,CuB)(63/65Cu) = 1.59 × 10-2 cm-1, Jisotropic = 50 cm-1, rCu-Cu = 4.93 Å, and χ = 17°). The exchange coupling between the two copper ions was found to be ferromagnetic as the signals exhibited Curie law temperature dependence. The Cu−Cu distance of ∼5 Å indicated by EPR was significantly higher than the inter-zinc distance of 3.5 Å in the native enzyme, and the dicopper species therefore represents a novel dinuclear site capable of catalysis of hydrolysis. In contrast to AAP, the related methionyl aminopeptidase from Escherichia coli (EcMetAP) was found to bind only one Cu(II) ion despite possessing a dinuclear binding site motif. A further difference was the marked pH dependence of the signal in EcMetAP, suggestive of a change in ligation. The structural motifs of these two Cu(II)-substituted aminopeptidases provide important insight into the observed catalytic activity

Mechanistic Studies on the Aminopeptidase from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e: 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 °C at eight substrate concentrations ranging from 5 to 800 μM. 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 kcat values and was found to be linear over the temperature range 25−85 °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 °C calculated over the temperature range 298−358 K were found to be 34.0 kJ/mol and −94.2 J/(mol·K), respectively. The free energy of activation at 25 °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

Kinetic and Spectroscopic Characterization of the H178A Methionyl Aminopeptidase from \u3cem\u3eEscherichia coli\u3c/em\u3e

To gain insight into the role of the strictly conserved histidine residue, H178, in the reaction mechanism of the methionyl aminopeptidase from Escherichia coli (EcMetAP-I), the H178A mutant enzyme was prepared. Metal-reconstituted H178A binds only one equivalent of Co(II) or Fe(II) tightly with affinities that are identical to the WT enzyme based on kinetic and isothermal titration calorimetry (ITC) data. Electronic absorption spectra of Co(II)-loaded H178A EcMetAP-I indicate that the active site divalent metal ion is pentacoordinate, identical to the WT enzyme. These data indicate that the metal binding site has not been affected by altering H178. The effect of altering H178 on activity is, in general, due to a decrease in kcat. The kcat value for Co(II)-loaded H178A decreased 70-fold toward MGMM and 290-fold toward MP-p-NA compared to the WT enzyme, while kcat decreased 50-fold toward MGMM for the Fe(II)-loaded H178A enzyme and 140-fold toward MP-p-NA. The Km values for MGMM remained unaffected, while those for MP-p-NA increased approximately 2-fold for Co(II)- and Fe(II)-loaded H178A. The kcat/Km values for both Co(II)- and Fe(II)-loaded H178A toward both substrates ranged from ∼50- to 580-fold reduction. The pH dependence of log Km, log kcat, and log(kcat/Km) of both WT and H178A EcMetAP-I were also obtained and are identical, within error, for H178A and WT EcMetAP-I. Therefore, H178A is catalytically important but is not required for catalysis. Assignment of one of the observed pKa values at 8.1 for WT EcMetAP-I was obtained from plots of molar absorptivity at λmax(640) vs pH for both WT and H178A EcMetAP-I. Apparent pKa values of 8.1 and 7.6 were obtained for WT and H178A EcMetAP-I, respectively, and were assigned to the deprotonation of a metal-bound water molecule. The data reported herein provide support for the key elements of the previously proposed mechanism and suggest that a similar mechanism can apply to the enzyme with a single metal in the active site

Structural Evidence That the Methionyl Aminopeptidase from \u3cem\u3eEscherichia coli\u3c/em\u3e 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 Å, which can be fit assuming 5 light atom (N,O) scatterers at 2.04 Å. Attempts to include a Co−Co interaction (in the 2.4−4.0 Å 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

Anesthesiology Posters - 2019

Anesthesiology Postershttps://scholarlycommons.libraryinfo.bhs.org/research_education/1000/thumbnail.jp

Postoperative Non-steroidal Anti-inflammatory Drugs and Risk of Bleeding in Pediatric Intracapsular Tonsillectomy

Tonsillectomy with or without adenoidectomy is one of the most frequently performed surgeries in the United States, with over 500,000 performed annually. Post-tonsillectomy hemorrhage is one of the most feared complications; thus, medications that could increase the risk of postoperative bleeding traditionally have been avoided. With recent FDA guidelines encouraging a departure from codeine-based medications in pediatric patients undergoing tonsillectomy, we examined the use of ibuprofen for post-tonsillectomy pain control. The records of 449 children who underwent tonsillectomy and received ibuprofen for postoperative pain control were reviewed and compared to a cohort of 1731 children who received codeine for pain postoperatively. Outcomes measured included rates of secondary post-tonsillectomy hemorrhage (PTH), secondary PTH requiring operative control, and emergency room evaluation for dehydration. Use of ibuprofen after pediatric intracapsular tonsillectomy was found to be associated with a statistically significant increase in secondary PTH and secondary PTH requiring operative control; however, ibuprofen was found to provide pain control that is at least equivalent to narcotic. Rates of secondary PTH with postoperative ibuprofen use remain within the national average. We propose that, despite the increased risk of bleeding, the use of ibuprofen is appropriate for use postoperatively in pediatric tonsillectomy patients, given its ability to control pain and lack of respiratory depression effects