Log Kcat Research Articles

Identification of quasi-stable water molecules near the Thr73-Lys13 catalytic diad of Bacillus sp. TB-90 urate oxidase by X-ray crystallography with controlled humidity.

Urate oxidases (UOs) catalyze the cofactor-independent oxidation of uric acid, and an extensive water network in the active site has been suggested to play an essential role in the catalysis. For our present analysis of the structure and function of the water network, the crystal qualities of Bacillus sp. TB-90 urate oxidase were improved by controlled dehydration using the humid air and glue-coating method. After the dehydration, the P21212 crystals were transformed into the I222 space group, leading to an extension of the maximum resolution to 1.42 Å. The dehydration of the crystals revealed a significant change in the five-water-molecules' binding mode in the vicinity of the catalytic diad, indicating that these molecules are quasi-stable. The pH profile analysis of log(kcat) gave two pKa values: pKa1 at 6.07 ± 0.07 and pKa2 at 7.98 ± 0.13. The site-directed mutagenesis of K13, T73 and N276 involved in the formation of the active-site water network revealed that the activities of these mutant variants were significantly reduced. These structural and kinetic data suggest that the five quasi-stable water molecules play an essential role in the catalysis of the cofactor-independent urate oxidation by reducing the energy penalty for the substrate-binding or an on-off switching for the proton-relay rectification.

Altering the Y137-K164-K166 triad of mandelate racemase and its effect on the observed pKa of the Brønsted base catalysts

Mandelate racemase (MR) catalyzes the interconversion of the enantiomers of mandelate using a two-base mechanism with Lys 166 acting as the Brønsted base to abstract the α-proton from (S)-mandelate. The resulting intermediate is subsequently re-protonated by the conjugate acid of His 297 to yield (R)-mandelate. The roles of these amino acids are reversed when (R)-mandelate is the substrate. The side chains of Tyr 137, Lys 164, and Lys 166 form a H-bonding network and the proximity of the two ε-NH3+ groups is believed to lower the pKa of Lys 166. We used site-directed mutagenesis, kinetics, and pH-rate studies to explore the roles of Lys 164 (K164 C/M) and Tyr 137 (Y137 L/F/S/T) in catalysis. The efficiency (kcat/Km) was reduced ∼3.5 × 105-fold for K164C MR, relative to wild-type MR, indicating a major role for this residue in catalysis. The efficiency of Y137F MR, however, was reduced only 25–30-fold. pH-Rate profiles (log kcat vs. pH) revealed that substitution of Tyr 137 by Phe increased the kinetic pKa of Lys 166 from 5.88 ± 0.02 to 7.3 ± 0.2. Hence, Tyr 137 plays an important role in facilitating the reduction of the pKa of the Brønsted base Lys 166 by ∼1.4 units. Interestingly, the Phe substitution also increased the kinetic pKa of His 297 from 5.97 ± 0.04 to 7.1 ± 0.1. Thus, the Tyr 137-Lys 164-Lys 166 H-bonding network plays a broader role in modulating the pKa of catalytic residues by influencing the electrostatic character of the entire active site, not only by decreasing the observed pKa value of Lys 166, but also by decreasing the pKa of His 297 by 1.1 units.

33 - Novel fluorinated Mn porphyrin as a powerful SOD mimic and catalyst for ascorbate-coupled anticancer therapy

Cationic Mn(III) porphyrins (MnPs) are among the most efficacious SOD mimics. A lead MnP, BMX-001 is now in two Clinical Trials as a radioprotector of normal tissues with glioma and head&neck cancer patients. Due to the optimized catalytic properties, MnPs couple efficiently with ascorbate (Asc) thereby inflicting significant anticancer effects. The anticancer activity is due to the intratumoral H2O2 production arising from the MnP-driven catalysis of Asc oxidation. We have recently developed a new class of fluorinated MnPs. Herein we report the synthesis, characterization and redox activity of one member of a new class of fluorinated MnPs, Mn(III) meso-tetrakis(2-fluoroethylpyridinium-2-yl) por-phyrin, MnFE. It is a more potent SOD mimic (log kcat(O2•‒) = 7.91, E1/2 = +242 mV vs NHE) and more powerful catalyst for Asc oxidation (v0(Asc)ox = 543 nM s-1) than the previous lead analogs, MnTE-2-PyP5+ and BMX-001. MnFE accumulates more in tumor vs normal tissue, which is essential for the differential effects of MnP in tumor vs normal tissues. Further, MnFE does not cause dose-limiting hypotension-based acute toxicity seen with non-fluorinated MnPs. Finally, MnFE has excellent safety/toxicity profile. In combination with Asc, MnFE exerts stronger cytotoxicity to various cancer cell lines than the earlier lead MnPs. MnFE/Asc treatment significantly suppressed tumor growth in BalbC mice bearing triple negative 4T1 mammary flank tumor. Furthermore, MnFE/Asc system was also tested as an adjuvant to radiotherapy (RT) in a same tumor model. While single (MnFE, Asc, RT) and double (MnFE/Asc, MnFE/RT, Asc/RT) treatments exhibited strong anticancer effects, the triple combination (MnFE/Asc/RT) was superior in suppressing tumor growth. Our findings demonstrate that the fluorination of MnPs comprises major modification in the development of Mn porphyrin-based therapeutics. In the light of our results and the fact that Asc is already in Clinical Trials, the MnFE alone or in combination with Asc and/or RT carries outstanding therapeutic potential which warrants its clinical development.

32 - Protection of rat prostate and erectile function from radiation-induced damage by novel Mn(III) N- substituted pyridylporphyrin and ascorbate

Prostate cancer is the most common cancer in American men, with approximately 30-50% of men developing loss of erectile function after definitive radiotherapy (RT). Currently, no treatment is available that preserves erectile function. Therefore there is a significant reduction of the quality of life of survivors which incurs considerable healthcare costs. Hence, there is an imminent need to protect normal tissues during prostate cancer RT. We have developed Mn porphyrin-based SOD mimics and demonstrated that they radioprotect normal tissues such as brain, salivary glands and mouth mucosa while radio- and chemosensitizing tumors, such as brain, breast and lymphoma. We have also demonstrated and explained the reasoning behind the significant enhancement of tumor radiosensitization by Mn porphyrins when combined with ascorbate (Asc). A lead drug, BMX-001 is now in two Phase I/II clinical trials as a normal tissue radioprotector with glioma and head&neck cancer patients. Herein we have explored the radioprotection of a lower pelvic region by a member of the new class of powerful Mn porphyrin-based SOD mimics – Mn(III) meso-tetrakis(5',5',5'-trifluoropentylpyridinium-2-yl)porphyrin, MnF3Pen. The SOD-like activity given by log kcat(O2.-) is 7.45, and the reduction potential of the Mn site, E1/2 is +288 mV vs NHE. In this study we also aimed to show that the addition of Asc would not diminish radioprotection of a normal tissue by MnF3Pen. The rat toxicity and pharmacokinetic studies of MnF3Pen/Asc system showed that MnF3Pen reached ~100 to 500 nM levels at targeted sites (penis, prostate, bladder, testes and rectum) within few hours after single subcutaneous (sc) 1 mg/kg injection. We subsequently performed the efficacy study. Single radiation dose of 20 Gy was delivered stereotactically to the prostate of Sprague-Dawley 8 week old rats using image-guided small animal irradiator. MnF3Pen was given sc at 0.5 mg/kg/day for four weeks and continued twice weekly for the next 5 weeks. Ascorbate was given intraperitoneally at 1 g/kg/day for first 3 days and then twice weekly for the rest of 9 weeks. We showed that MnF3Pen/Asc treatment prevented erectile dysfunction observed in RT-treated group. We further showed that MnF3Pen/Asc treatment greatly suppressed RT-induced damage to prostate as evaluated by H&E staining.

Merged Heme and Non-Heme Manganese Cofactors for a Dual Antioxidant Surveillance in Photosynthetic Organisms

The coupling of a polycationic Mn(III)-porphyrin, with a dinuclear Mn2(II,II)L2 core (HL = 2-{[[di(2-pyridyl)methyl](methyl)amino]methyl}phenol), results in a dual Superoxide Dismutase (SOD) and Catalase (CAT) functional analogue, Mn2L2Pn+, enabling a detoxification cascade of the superoxide anion and hydrogen peroxide into benign H2O and O2. The SOD/CAT artificial manifolds, joined in one asset, exhibit a peak catalytic performance under physiological conditions, with log kcat(O2• –) ≥ 7 and kcat(H2O2)/KM = 1890. The dual-enzyme (dizyme) concept allows for a built-in self-protection against the irreversible bleaching of the porphyrin unit (>75% protection), readily induced by H2O2 (200 μM, 20 equiv, in buffer solution, pH 7.8). We show herein that incubation of the photosynthetic green algae, Chlamydomonas reinhardtii, with the synthetic dizyme (as low as 0.1 μM), prevents H2O2 accumulation under high-light illumination conditions, thus providing antioxidant surveillance and photoprotection.

Rational design of superoxide dismutase (SOD) mimics: the evaluation of the therapeutic potential of new cationic Mn porphyrins with linear and cyclic substituents.

Our goal herein has been to gain further insight into the parameters which control porphyrin therapeutic potential. Mn porphyrins (MnTnOct-2-PyP5+, MnTnHexOE-2-PyP5+, MnTE-2-PyPhP5+, and MnTPhE-2-PyP5+) that bear the same positive charge and same number of carbon atoms at meso positions of porphyrin core were explored. The carbon atoms of their meso substituents are organized to form either linear or cyclic structures of vastly different redox properties, bulkiness, and lipophilicities. These Mn porphyrins were compared to frequently studied compounds, MnTE-2-PyP5+, MnTE-3-PyP5+, and MnTBAP3–. All Mn(III) porphyrins (MnPs) have metal-centered reduction potential, E1/2 for MnIIIP/MnIIP redox couple, ranging from −194 to +340 mV versus NHE, log kcat(O2•–) from 3.16 to 7.92, and log kred(ONOO–) from 5.02 to 7.53. The lipophilicity, expressed as partition between n-octanol and water, log POW, was in the range −1.67 to −7.67. The therapeutic potential of MnPs was assessed via: (i) in vitro ability to prevent spontaneous lipid peroxidation in rat brain homogenate as assessed by malondialdehyde levels; (ii) in vivo O2•– specific assay to measure the efficacy in protecting the aerobic growth of SOD-deficient Saccharomyces cerevisiae; and (iii) aqueous solution chemistry to measure the reactivity toward major in vivo endogenous antioxidant, ascorbate. Under the conditions of lipid peroxidation assay, the transport across the cellular membranes, and in turn shape and size of molecule, played no significant role. Those MnPs of E1/2 ∼ +300 mV were the most efficacious, significantly inhibiting lipid peroxidation in 0.5–10 μM range. At up to 200 μM, MnTBAP3– (E1/2 = −194 mV vs NHE) failed to inhibit lipid peroxidation, while MnTE-2-PyPhP5+ with 129 mV more positive E1/2 (−65 mV vs NHE) was fully efficacious at 50 μM. The E1/2 of MnIIIP/MnIIP redox couple is proportional to the log kcat(O2•–), i.e., the SOD-like activity of MnPs. It is further proportional to kred(ONOO–) and the ability of MnPs to prevent lipid peroxidation. In turn, the inhibition of lipid peroxidation by MnPs is also proportional to their SOD-like activity. In an in vivo S. cerevisiae assay, however, while E1/2 predominates, lipophilicity significantly affects the efficacy of MnPs. MnPs of similar log POW and E1/2, that have linear alkyl or alkoxyalkyl pyridyl substituents, distribute more easily within a cell and in turn provide higher protection to S. cerevisiae in comparison to MnP with bulky cyclic substituents. The bell-shape curve, with MnTE-2-PyP5+ exhibiting the highest ability to catalyze ascorbate oxidation, has been established and discussed. Our data support the notion that the SOD-like activity of MnPs parallels their therapeutic potential, though species other than O2•–, such as peroxynitrite, H2O2, lipid reactive species, and cellular reductants, may be involved in their mode(s) of action(s).

SOD therapeutics: latest insights into their structure-activity relationships and impact on the cellular redox-based signaling pathways.

Superoxide dismutase (SOD) enzymes are indispensable and ubiquitous antioxidant defenses maintaining the steady-state levels of O2·(-); no wonder, thus, that their mimics are remarkably efficacious in essentially any animal model of oxidative stress injuries thus far explored. Structure-activity relationship (half-wave reduction potential [E1/2] versus log kcat), originally reported for Mn porphyrins (MnPs), is valid for any other class of SOD mimics, as it is dominated by the superoxide reduction and oxidation potential. The biocompatible E1/2 of ∼+300 mV versus normal hydrogen electrode (NHE) allows powerful SOD mimics as mild oxidants and antioxidants (alike O2·(-)) to readily traffic electrons among reactive species and signaling proteins, serving as fine mediators of redox-based signaling pathways. Based on similar thermodynamics, both SOD enzymes and their mimics undergo similar reactions, however, due to vastly different sterics, with different rate constants. Although log kcat(O2·(-)) is a good measure of therapeutic potential of SOD mimics, discussions of their in vivo mechanisms of actions remain mostly of speculative character. Most recently, the therapeutic and mechanistic relevance of oxidation of ascorbate and glutathionylation and oxidation of protein thiols by MnP-based SOD mimics and subsequent inactivation of nuclear factor κB has been substantiated in rescuing normal and killing cancer cells. Interaction of MnPs with thiols seems to be, at least in part, involved in up-regulation of endogenous antioxidative defenses, leading to the healing of diseased cells. Mechanistic explorations of single and combined therapeutic strategies, along with studies of bioavailability and translational aspects, will comprise future work in optimizing redox-active drugs.

Differential Coordination Demands in Fe versus Mn Water-Soluble Cationic Metalloporphyrins Translate into Remarkably Different Aqueous Redox Chemistry and Biology

The different biological behavior of cationic Fe and Mn pyridylporphyrins in Escherichia coli and mouse studies prompted us to revisit and compare their chemistry. For that purpose, the series of ortho and meta isomers of Fe(III) meso-tetrakis-N-alkylpyridylporphyrins, alkyl being methyl to n-octyl, were synthesized and characterized by elemental analysis, UV/vis spectroscopy, mass spectrometry, lipophilicity, protonation equilibria of axial waters, metal-centered reduction potential, E(1/2) for M(III)P/M(II)P redox couple (M = Fe, Mn, P = porphyrin), kcat for the catalysis of O2(•-) dismutation, stability toward peroxide-driven porphyrin oxidative degradation (produced in the catalysis of ascorbate oxidation by MP), ability to affect growth of SOD-deficient E. coli, and toxicity to mice. Electron-deficiency of the metal site is modulated by the porphyrin ligand, which renders Fe(III) porphyrins ≥5 orders of magnitude more acidic than the analogous Mn(III) porphyrins, as revealed by the pKa1 of axially coordinated waters. The 5 log units difference in the acidity between the Mn and Fe sites in porphyrin translates into the predominance of tetracationic (OH)(H2O)FeP complexes relative to pentacationic (H2O)2MnP species at pH ∼7.8. This is additionally evidenced in large differences in the E(1/2) values of M(III)P/M(II)P redox couples. The presence of hydroxo ligand labilizes trans-axial water which results in higher reactivity of Fe relative to Mn center. The differences in the catalysis of O2(•-) dismutation (log kcat) between Fe and Mn porphyrins is modest, 2.5-5-fold, due to predominantly outer-sphere, with partial inner-sphere character of two reaction steps. However, the rate constant for the inner-sphere H2O2-based porphyrin oxidative degradation is 18-fold larger for (OH)(H2O)FeP than for (H2O)2MnP. The in vivo consequences of the differences between the Fe and Mn porphyrins were best demonstrated in SOD-deficient E. coli growth. On the basis of fairly similar log kcat(O2(•-)) values, a very similar effect on the growth of SOD-deficient E. coli was anticipated by both metalloporphyrins. Yet, while (H2O)2MnTE-2-PyP(5+) was fully efficacious at ≥20 μM, the Fe analogue (OH)(H2O)FeTE-2-PyP(4+) supported SOD-deficient E. coli growth at as much as 200-fold lower doses in the range of 0.1-1 μM. Moreover the pattern of SOD-deficient E. coli growth was different with Mn and Fe porphyrins. Such results suggested a different mode of action of these metalloporphyrins. Further exploration demonstrated that (1) 0.1 μM (OH)(H2O)FeTE-2-PyP(4+) provided similar growth stimulation as the 0.1 μM Fe salt, while the 20 μM Mn salt provides no protection to E. coli; and (2) 1 μM Fe porphyrin is fully degraded by 12 h in E. coli cytosol and growth medium, while Mn porphyrin is not. Stimulation of the aerobic growth of SOD-deficient E. coli by the Fe porphyrin is therefore due to iron acquisition. Our data suggest that in vivo, redox-driven degradation of Fe porphyrins resulting in Fe release plays a major role in their biological action. Possibly, iron reconstitutes enzymes bearing [4Fe-4S] clusters as active sites. Under the same experimental conditions, (OH)(H2O)FePs do not cause mouse arterial hypotension, whereas (H2O)2MnPs do, which greatly limits the application of Mn porphyrins in vivo.

α-Thioglycoligase-based synthesis of O-aryl α-glycosides as chromogenic substrates for α-glycosidases

α-Thioglycoligases are retaining α-glycosidase mutants, with modification of their general acid/base catalytic residue to an inactive amino acid residue, catalyzing the formation of S-glycosidic linkages using a sugar donor with an excellent leaving group and suitable sugar acceptors with a thiol group as the substrate. In this study, we describe the enzymatic synthesis of O-aryl α-glycosides catalyzed by α-thioglycoligases. An α-xylosidase mutant (YicI-D482A) efficiently catalyzed the synthesis of O-aryl α-xylosides in near-quantitative yields (up to 99%) using 4-methylumbelliferone and nitrophenols. Synthesis did not occur with those acceptors having a nitro group at the ortho-position. The conversion yields of 3-nitrophenol markedly increased at pH 8.0, whereas those of other aryl compounds were nearly independent of pH, ranging from pH 6.0 to 8.0. The O-aryl α-xylosides were prepared on a preparative scale with yields of up to 96%. Upon employing the O-aryl α-xylosides as the substrate for the wild-type YicI, Brønsted relationships of log kcat versus pKa and log (kcat/KM) versus pKa both showed a linear monotonic dependence on the leaving group pKa with low βlg values of 0.39 and 0.38, respectively. In addition, synthesis of O-aryl α-glucosides was successfully conducted by an α-glucosidase mutant (MalA-D416A) in the same fashion with high yields. Therefore, this strategy can be used for the synthesis of O-aryl α-glycosides using an acid/base mutant of retaining α-glycosidases that hydrolyze the glycosides.

Understanding the Role of Tyrosine 381 in the Activity of E. coli Aminopeptidase N (PepN)

Aminopeptidase N (PepN) from E. coli is an M1 class, Zn(II)‐dependent peptidase with broad specificity. PepN was mechanistically characterized in an effort to understand the role of tyrosine 381. pH Dependence kinetic studies revealed a log kcat versus pH and kcat/KM versus pH curves with inflection points at a pH of 9.0. Solvent isotope studies suggest at least one rate‐limiting proton transfer in the mechanism. In an effort to characterize the functional roles of binding region components, several active‐site amino acids were mutated. A tyrosine to alanine mutated PepN (Y381A) was successfully over‐expressed, purified, and characterized. Y381A‐PepN maintained Zn(II) binding efficiency but exhibited greater than 900‐fold loss of activity (kcat) when compared to the wild‐type PepN. This suggests that Tyr381 has an essential mechanistic role in the function of PepN beyond the suggested role of hydrogen bond stabilization of substrate and product. This work was funded by Augustana College New Faculty Research Fund, Augustana College Research Foundation, and Miami University.

A new SOD mimic, Mn(III) ortho N-butoxyethylpyridylporphyrin, combines superb potency and lipophilicity with low toxicity

The Mn porphyrins of kcat(O2.-) as high as that of a superoxide dismutase enzyme and of optimized lipophilicity have already been synthesized. Their exceptional in vivo potency is at least in part due to their ability to mimic the site and location of mitochondrial superoxide dismutase, MnSOD. MnTnHex-2-PyP5+ is the most studied among lipophilic Mn porphyrins. It is of remarkable efficacy in animal models of oxidative stress injuries and particularly in central nervous system diseases. However, when used at high single and multiple doses it becomes toxic. The toxicity of MnTnHex-2-PyP5+ has been in part attributed to its micellar properties, i.e., the presence of polar cationic nitrogens and hydrophobic alkyl chains. The replacement of a CH2 group by an oxygen atom in each of the four alkyl chains was meant to disrupt the porphyrin micellar character. When such modification occurs at the end of long alkyl chains, the oxygens become heavily solvated, which leads to a significant drop in the lipophilicity of porphyrin. However, when the oxygen atoms are buried deeper within the long heptyl chains, their excessive solvation is precluded and the lipophilicity preserved. The presence of oxygens and the high lipophilicity bestow the exceptional chemical and physical properties to Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, MnTnBuOE-2-PyP5+. The high SOD-like activity is preserved and even enhanced: log kcat(O2.-)=7.83 vs 7.48 and 7.65 for MnTnHex-2-PyP5+ and MnTnHep-2-PyP5+, respectively. MnTnBuOE-2-PyP5+ was tested in an O2.- -specific in vivo assay, aerobic growth of SOD-deficient yeast, Saccharomyces cerevisiae, where it was fully protective in the range of 5–30μM. MnTnHep-2-PyP5+ was already toxic at 5μM, and MnTnHex-2-PyP5+ became toxic at 30μM. In a mouse toxicity study, MnTnBuOE-2-PyP5+ was several-fold less toxic than either MnTnHex-2-PyP5+ or MnTnHep-2-PyP5+.

2H Kinetic Isotope Effects and pH Dependence of Catalysis as Mechanistic Probes of Rat Monoamine Oxidase A: Comparisons with the Human Enzyme

Monoamine oxidase A (MAO A) is a mitochondrial outer membrane-bound flavoenzyme important in the regulation of serotonin and dopamine levels. Because the rat is extensively used as an animal model in drug studies, it is important to understand how rat MAO A behaves in comparison with the more extensively studied human enzyme. For many reversible inhibitors, rat MAO A exhibits K(i) values similar to those of human MAO A. The pH profile of k(cat) for rat MAO A shows a pK(a) of 8.2 ± 0.1 for the benzylamine ES complex and pK(a) values of 7.5 ± 0.1 and 7.6 ± 0.1 for the ES complexes with p-CF(3)-(1)H- and p-CF(3)-(2)H-benzylamine, respectively. In contrast to the human enzyme, the rat enzyme exhibits a single pK(a) value (8.3 ± 0.1) with k(cat)/K(m) for benzylamine versus pH and pK(a) values of 7.8 ± 0.1 and 8.1 ± 0.2 for the ascending limbs, respectively, of k(cat)/K(m) versus pH profiles for p-CF(3)-(1)H- and p-CF(3)-(2)H-benzylamine and 9.3 ± 0.1 and 9.1 ± 0.2 for the descending limbs, respectively. The oxidation of para-substituted benzylamine substrate analogues by rat MAO A has large deuterium kinetic isotope effects on k(cat) and on k(cat)/K(m). These effects are pH-independent and range from 7 to 14, demonstrating a rate-limiting α-C-H bond cleavage step in catalysis. Quantitative structure-activity correlations of log k(cat) with the electronic substituent parameter (σ) at pH 7.5 and 9.0 show a dominant contribution with positive ρ values (1.2-1.3) and a pH-independent negative contribution from the steric term. Quantitative structure-activity relationship analysis of the binding affinities of the para-substituted benzylamine analogues for rat MAO A shows an increased van der Waals volume (V(w)) increases the affinity of the deprotonated amine for the enzyme. These results demonstrate that rat MAO A exhibits functional properties similar but not identical with those of the human enzyme and provide additional support for C-H bond cleavage via a polar nucleophilic mechanism.

Methoxy-derivatization of alkyl chains increases the in vivo efficacy of cationic Mn porphyrins. Synthesis, characterization, SOD-like activity, and SOD-deficient E. coli study of meta Mn(iii) N-methoxyalkylpyridylporphyrins

Cationic Mn(III) N-alkylpyridylporphyrins (MnPs) are potent SOD mimics and peroxynitrite scavengers and diminish oxidative stress in a variety of animal models of central nervous system (CNS) injuries, cancer, radiation, diabetes, etc. Recently, properties other than antioxidant potency, such as lipophilicity, size, shape, and bulkiness, which influence the bioavailability and the toxicity of MnPs, have been addressed as they affect their in vivo efficacy and therapeutic utility. Porphyrin bearing longer alkyl substituents at pyridyl ring, MnTnHex-2-PyP(5+), is more lipophilic, thus more efficacious in vivo, particularly in CNS injuries, than the shorter alkyl-chained analog, MnTE-2-PyP(5+). Its enhanced lipophilicity allows it to accumulate in mitochondria (relative to cytosol) and to cross the blood-brain barrier to a much higher extent than MnTE-2-PyP(5+). Mn(III) N-alkylpyridylporphyrins of longer alkyl chains, however, bear micellar character, and when used at higher levels, become toxic. Recently we showed that meta isomers are ∼10-fold more lipophilic than ortho species, which enhances their cellular accumulation, and thus reportedly compensates for their somewhat inferior SOD-like activity. Herein, we modified the alkyl chains of the lipophilic meta compound, MnTnHex-3-PyP(5+) via introduction of a methoxy group, to diminish its toxicity (and/or enhance its efficacy), while maintaining high SOD-like activity and lipophilicity. We compared the lipophilic Mn(III) meso-tetrakis(N-(6'-methoxyhexyl)pyridinium-3-yl)porphyrin, MnTMOHex-3-PyP(5+), to a hydrophilic Mn(III) meso-tetrakis(N-(2'-methoxyethyl)pyridinium-3-yl)porphyrin, MnTMOE-3-PyP(5+). The compounds were characterized by uv-vis spectroscopy, mass spectrometry, elemental analysis, electrochemistry, and ability to dismute O(2)˙(-). Also, the lipophilicity was characterized by thin-layer chromatographic retention factor, R(f). The SOD-like activities and metal-centered reduction potentials for the Mn(III)P/Mn(II)P redox couple were similar-to-identical to those of N-alkylpyridyl analogs: log k(cat) = 6.78, and E(1/2) = +68 mV vs. NHE (MnTMOHex-3-PyP(5+)), and log k(cat) = 6.72, and E(1/2) = +64 mV vs. NHE (MnTMOE-3-PyP(5+)). The compounds were tested in a superoxide-specific in vivo model: aerobic growth of SOD-deficient E. coli, JI132. Both MnTMOHex-3-PyP(5+) and MnTMOE-3-PyP(5+) were more efficacious than their alkyl analogs. MnTMOE-3-PyP(5+) is further significantly more efficacious than the most explored compound in vivo, MnTE-2-PyP(5+). Such a beneficial effect of MnTMOE-3-PyP(5+) on diminished toxicity, improved efficacy and transport across the cell wall may originate from the favorable interplay of the size, length of pyridyl substituents, rotational flexibility (the ortho isomer, MnTE-2-PyP(5+), is more rigid, while MnTMOE-3-PyP(5+) is a more flexible meta isomer), bulkiness and presence of oxygen.

On the question of stepwise vs. concerted cleavage of RNA models promoted by a synthetic dinuclear Zn(ii) complex in methanol: implementation of a noncleavable phosphonate probe

To address the question of concerted versus a stepwise reaction mechanisms for the cyclization of the 2-hydroxypropyl aryl and alkyl RNA models (1a-k) promoted by dinuclear Zn(II) complex (4) at (s)spH 9.8 and 25 degrees C, the non-cleavable O-hydroxypropyl phenylphosphonate analogues 6a and 6b were subjected to the catalytic reaction in methanol. These phosphonates did not undergo isomerization in the study, the only observable methanolysis reaction being release of 1,2-propanediol and the formation of O-methyl phenylphosphonate. The observed first order rate constants for methanolysis promoted by 4 are k(obs)(6a) = (1.47 +/- 0.09) x 10(-4) s(-1) and k(obs)(6b) = (2.08 +/- 0.09) x 10(-6) s(-1), respectively. The rates of methanolysis of a series of O-aryl phenylphosphonates (8a-f) in the presence of increasing [4] were analyzed to provide binding constants, Kb, and the catalytic rate constant, kcat(max), for the unimolecular decomposition of the 8:4 Michaelis complex. A Brønsted plot of the log (k(cat)(max)) vs. sspKa(phenol) (acidity constant of the conjugate acid of the leaving group in methanol) was fitted to a linear regression of log kcat(max) = (-0.80 +/- 0.07)(s)spKa + (10.2 +/- 1.0) which includes the datum for 6a. The datum for 6b, which reacts approximately 70-fold slower, falls significantly below the linear correlation. The data provide additional evidence consistent with a concerted cyclization of RNA models 1a-k promoted by 4.

The Electrostatic Driving Force for Nucleophilic Catalysis inl-Arginine Deiminase: A Combined Experimental and Theoretical Study

L-arginine deiminase (ADI) catalyzes the hydrolysis of L-arginine to form L-citrulline and ammonia via two partial reactions. A working model of the ADI catalytic mechanism assumes nucleophilic catalysis by a stringently conserved active site Cys and general acid-general base catalysis by a stringently conserved active site His. Accordingly, in the first partial reaction, the Cys attacks the substrate guanidino C zeta atom to form a tetrahedral covalent adduct, which is protonated by the His at the departing ammonia group to facilitate the formation of the Cys- S-alkylthiouronium intermediate. In the second partial reaction, the His activates a water molecule for nucleophilic addition at the thiouronium C zeta atom to form the second tetrahedral intermediate, which eliminates the Cys in formation of the L-citrulline product. The absence of a basic residue near the Cys thiol suggested that the electrostatic environment of the Cys thiol, in the enzyme-substrate complex, stabilizes the Cys thiolate anion. The studies described in this paper explore the mechanism of stabilization of the Cys thiolate. First, the log(k(cat)/K(m)) and log k(cat) pH rate profiles were measured for several structurally divergent ADIs to establish the pH range for ADI catalysis. All ADIs were optimally active at pH 5, which suggested that the Cys pKa is strongly perturbed by the prevailing electrostatics of the ADI active site. The p K a of the Bacillus cereus ADI (BcADI) was determined by UV-pH titration to be 9.6. In contrast, the pKa determined by iodoacetamide Cys alkylation is 6.9. These results suggest that the negative electrostatic field from the two opposing Asp carboxylates perturbs the Cys pKa upward in the apoenzyme and that the binding of the iodoacetamide (a truncated analogue of the citrulline product) between the Cys thiol and the two Asp carboxylates shields the Cys thiol, thereby reducing its pKa. It is hypothesized that the bound positively charged guanidinium group of the L-arginine substrate further stabilizes the Cys thiolate. The so-called "substrate-assisted" Cys ionization, first reported by Fast and co-workers to operate in the related enzyme dimethylarginine dimethylaminohydrolase [Stone, E. M., Costello, A. L., Tierney, D. L., and Fast, W. (2006) Biochemistry 45, 5618-5630], was further explored computationally in ADI by using an ab initio quantum mechanics/molecular mechanics method. The energy profiles for formation of the tetrahedral intermediate in the first partial reaction were calculated for three different reaction scenarios. From these results, we conclude that catalytic turnover commences from the active configuration of the ADI(L-arginine) complex which consists of the Cys thiolate (nucleophile) and His imidazolium ion (general acid) and that the energy barriers for the nucleophilic addition of Cys thiolate to the thiouronium C zeta atom and His imidazolium ion-assisted elimination from the tetrahedral intermediate are small.

New PEG-ylated Mn(iii) porphyrins approaching catalytic activity of SOD enzyme

Two new tri(ethyleneglycol)-derivatized Mn(III) porphyrins were synthesized with the aim of increasing their bioavailability, and blood-circulating half-life. These are Mn(III) tetrakis(N-(1-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)pyridinium-2-yl)porphyrin, MnTTEG-2-PyP5+ and Mn(III) tetrakis(N,N'-di(1-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)imidazolium-2-yl)porphyrin, MnTDTEG-2-ImP5+. Both porphyrins have ortho pyridyl or di-ortho imidazolyl electron-withdrawing substituents at meso positions of the porphyrin ring that assure highly positive metal centered redox potentials, E1/2 = +250 mV vs. NHE for MnTTEG-2-PyP5+ and E1/2 = + 412 mV vs. NHE for MnTDTEG-2-ImP5+. As expected, from established E1/2 vs. log kcat(O2 *-) structure-activity relationships for metalloporphyrins (Batinic-Haberle et al., Inorg. Chem., 1999, 38, 4011), both compounds exhibit higher SOD-like activity than any meso-substituted Mn(III) porphyrins-based SOD mimic thus far, log kcat = 8.11 (MnTTEG-2-PyP5+) and log kcat = 8.55 (MnTDTEG-2-ImP5+), the former being only a few-fold less potent in disproportionating O2*- than the SOD enzyme itself. The new porphyrins are stable to both acid and EDTA, and non toxic to E. coli. Despite elongated substituents, which could potentially lower their ability to cross the cell wall, MnTTEG-2-PyP5+ and MnTDTEG-2-ImP5+ exhibit similar protection of SOD-deficient E. coli as their much smaller ethyl analogues MnTE-2-PyP5+ and MnTDE-2-ImP5+, respectively. Consequently, with anticipated increased blood-circulating half-life, these new Mn(III) porphyrins may be more effective in ameliorating oxidative stress injuries than ethyl analogues that have been already successfully explored in vivo.

Mechanistic Roles of Ser-114, Tyr-155, and Lys-159 in 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase from Comamonas testosteroni

3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) from Comamonas testosteroni, a short chain dehydrogenase/reductase, catalyzes the oxidation of androsterone with NAD+ to form androstanedione and NADH. A catalytic triad of Ser-114, Tyr-155, and Lys-159 in 3alpha-HSD/CR has been proposed based on structural analysis and sequence alignment of the short chain dehydrogenase/reductase family. The 3alpha-HSD/CR-catalyzed reaction has not been kinetically analyzed in detail, however. In this study, we combined steady-state kinetics, site-directed mutagenesis, and pH profile to explore the function of Ser-114, Tyr-155, and Lys-159 in 3alpha-HSD/CR-catalyzed reaction. The catalytic efficiency of wild-type and mutants S114A, Y155F, K159A, and Y155F/K159A is 4.3 x 10(7), 7.3 x 10(4), 1.7 x 10(4), 2.4 x 10(5), and 71 m(-1)s(-1), respectively. The values of pKa on kcat/Km for the wild-type, S114A, Y155F, K159A, and Y155F/K159A are 7.2, 7.4, 8.4, 9.1, and 10.2, respectively. Mutant S114A/Y155F exhibits a pH-independent profile with 10(-5) times of wild-type activity at pH 10.5. The activity decreases as the pH lowers, which indicates that a functional group with an apparent pKa of 7.2 is involved in the general base catalysis for wild-type 3alpha-HSD/CR. The pKa shift to 9.1 for mutant K159A suggests the role of Lys-159 is to lower the pKa of the residues involved in the general base catalysis. Because pH dependence is observed for both S114A and Y155F mutants and pH independence is observed in S114A/Y155F, Tyr-155 may be important as a general base catalysis in the wild-type, whereas Ser-114 may act as a general base on mutant Y155F to catalyze the reaction.

Metal ion promoted transesterifications of carboxylate esters. A structure/activity study of the efficacy of Zn2+ and La3+ to catalyze the methanolysis of some aryl and aliphatic esters

The methanolysis of various aryl and aliphatic carboxylate esters promoted by methoxide, 1,5,9-triazacyclododecane : Zn2+(-OCH3) and La3+(-OCH3), were studied and the derived rate constants (kOCH3, kcat3:Zn(OCH3) and kcatLa(OCH3)) correlated in various ways. The metal ion catalyzed reactions are very much faster than the background reactions in some cases reaching up to 7 x 10(6)-fold acceleration when present at concentrations of 5 mmol dm(-3). The data for both metals exhibit non-linear Bronsted correlations with the pKa of the leaving group which are analyzed in terms of a change in rate limiting step from formation to breakdown of a metal-coordinated tetrahedral intermediate as the pKa increases above values of approximately 14.7. Plots of the log kOCH3 reaction vs. the log kcat values for each metal ion indicate low sensitivity for aryl esters and a higher sensitivity for the aliphatic esters. A mechanistic rationale for the observations is presented.

New class of potent catalysts of O2.-dismutation. Mn(III) ortho-methoxyethylpyridyl- and di-ortho-methoxyethylimidazolylporphyrins.

Three new Mn(III) porphyrin catalysts of O2.-dismutation (superoxide dismutase mimics), bearing ether oxygen atoms within their side chains, were synthesized and characterized: Mn(III) 5,10,15,20-tetrakis[N-(2-methoxyethyl)pyridinium-2-yl]porphyrin (MnTMOE-2-PyP(5+)), Mn(III)5,10,15,20-tetrakis[N-methyl-N'-(2-methoxyethyl)imidazolium-2-yl]porphyrin (MnTM,MOE-2-ImP(5+)) and Mn(III) 5,10,15,20-tetrakis[N,N'-di(2-methoxyethyl)imidazolium-2-yl]porphyrin (MnTDMOE-2-ImP(5+)). Their catalytic rate constants for O2.-dismutation (disproportionation) and the related metal-centered redox potentials vs. NHE are: log k(cat)= 8.04 (E(1/2)=+251 mV) for MnTMOE-2-PyP(5+), log k(cat)= 7.98 (E(1/2)=+356 mV) for MnTM,MOE-2-ImP(5+) and log k(cat)= 7.59 (E(1/2)=+365 mV) for MnTDMOE-2-ImP(5+). The new porphyrins were compared to the previously described SOD mimics Mn(III) 5,10,15,20-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP(5+)), Mn(III) 5,10,15,20-tetrakis(N-n-butylpyridinium-2-yl)porphyrin (MnTnBu-2-PyP(5+)) and Mn(III) 5,10,15,20-tetrakis(N,N'-diethylimidazolium-2-yl)porphyrin (MnTDE-2-ImP(5+)). MnTMOE-2-PyP(5+) has side chains of the same length and the same E(1/2), as MnTnBu-2-PyP(5+)(k(cat)= 7.25, E(1/2)=+ 254 mV), yet it is 6-fold more potent a catalyst of O2.-dismutation , presumably due to the presence of the ether oxygen. The log k(cat)vs. E(1/2) relationship for all Mn porphyrin-based SOD mimics thus far studied is discussed. None of the new compounds were toxic to Escherichia coli in the concentration range studied (up to 30 microM), and protected SOD-deficient E. coli in a concentration-dependent manner. At 3 microM levels, the MnTDMOE-2-ImP(5+), bearing an oxygen atom within each of the eight side chains, was the most effective and offered much higher protection than MnTE-2-PyP(5+), while MnTDE-2-ImP(5+) was of very low efficacy.

Examining the relative timing of hydrogen abstraction steps during NAD(+)-dependent oxidation of secondary alcohols catalyzed by long-chain D-mannitol dehydrogenase from Pseudomonas fluorescens using pH and kinetic isotope effects.

Mannitol dehydrogenases (MDH) are a family of Zn(2+)-independent long-chain alcohol dehydrogenases that catalyze the regiospecific NAD(+)-dependent oxidation of a secondary alcohol group in polyol substrates. pH and primary deuterium kinetic isotope effects on kinetic parameters for reaction of recombinant MDH from Pseudomonas fluorescens with D-mannitol have been measured in H(2)O and D(2)O at 25 degrees C and used to determine the relative timing of C-H and O-H bond cleavage steps during alcohol conversion. The enzymatic rates decreased at low pH; apparent pK values for log(k(cat)/K(mannitol)) and log k(cat) were 9.2 and 7.7 in H(2)O, respectively, and both were shifted by +0.4 pH units in D(2)O. Proton inventory plots for k(cat) and k(cat)/K(mannitol) were determined at pL 10.0 using protio or deuterio alcohol and were linear at the 95% confidence level. They revealed the independence of primary deuterium isotope effects on the atom fraction of deuterium in a mixed H(2)O-D(2)O solvent and yielded single-site transition-state fractionation factors of 0.43 +/- 0.05 and 0.47 +/- 0.01 for k(cat)/K(mannitol) and k(cat), respectively. (D)(k(cat)/K(mannitol)) was constant (1.80 +/- 0.20) in the pH range 6.0-9.5 and decreased at high pH to a limiting value of approximately 1. Measurement of (D)(k(cat)/K(fructose)) at pH 10.0 and 10.5 using NADH deuterium-labeled in the 4-pro-S position gave a value of 0.83, the equilibrium isotope effect on carbonyl group reduction. A mechanism of D-mannitol oxidation by MDH is supported by the data in which the partly rate-limiting transition state of hydride transfer is stabilized by a single solvation catalytic proton bridge. The chemical reaction involves a pH-dependent internal equilibrium which takes place prior to C-H bond cleavage and in which proton transfer from the reactive OH to the enzyme catalytic base may occur. Loss of a proton from the enzyme at high pH irreversibly locks the ternary complex with either alcohol or alkoxide bound in a conformation committed of undergoing NAD(+) reduction at a rate about 2.3-fold slower than the corresponding reaction rate of the protonated complex. Transient kinetic studies for D-mannitol oxidation at pH(D) 10.0 showed that the solvent isotope effect on steady-state turnover originates from a net rate constant of NADH release that is approximately 85% rate-limiting for k(cat) and 2-fold smaller in D(2)O than in H(2)O.