Discovery of halopyridines as quiescent affinity labels: inactivation of dimethylarginine dimethylaminohydrolase

In an effort to develop novel covalent modifiers of dimethylarginine dimethylaminohydrolase (DDAH) that are useful for biological applications, a set of "fragment"-sized inhibitors that were identified using a high-throughput screen are tested for time-dependent inhibition. One structural class of inactivators, 4-halopyridines, show time- and concentration-dependent inactivation of DDAH, and the inactivation mechanism of one example, 4-bromo-2-methylpyridine (1), is characterized in detail. The neutral form of halopyridines is not very reactive with excess glutathione. However, 1 readily reacts, with loss of its halide, in a selective, covalent, and irreversible manner with the active-site Cys249 of DDAH. This active-site Cys is not particularly reactive (pK(a) ca. 8.8), and 1 does not inactivate papain (Cys pK(a) ca. ≤4), suggesting that, unlike many reagents, Cys nucleophilicity is not a predominating factor in selectivity. Rather, binding and stabilization of the more reactive pyridinium form of the inactivator by a second moiety, Asp66, is required for facile reaction. This constraint imparts a unique selectivity profile to these inactivators. To our knowledge, halopyridines have not previously been reported as protein modifiers, and therefore they represent a first-in-class example of a novel type of quiescent affinity label.     

Design of a conformationally restricted analogue of the antiepilepsy drug Vigabatrin that directs its mechanism of inactivation of gamma-aminobutyric acid aminotransferase

The antiepilepsy drug vigabatrin (1, 4-aminohex-5-enoic acid, gamma-vinylGABA) is known to be a mechanism-based inactivator of the pyridoxal phosphate (PLP)-dependent enzyme gamma-aminobutyric acid aminotransferase (GABA-AT). Inactivation has been shown to proceed by two divergent mechanisms (Nanavati, S. M.; Silverman, R. B. J. Am. Chem. Soc. 1991, 113, 9341-9349). The major pathway involves gamma-proton removal, tautomerization into the PLP ring, followed by Michael addition of an active site lysine residue at the conjugated vinyl group to give a stable covalent adduct with the protein (Scheme 2, pathway a). The minor inactivation mechanism also involves gamma-proton removal, but tautomerization occurs through the vinyl group, followed by an enamine rearrangement that leads to attachment of the inactivator to the PLP, which is bound to the protein (Scheme 2, pathway b). The cause for the two different inactivation pathways was hypothesized to be potential overlap of the incipient carbanion with the pi-orbitals of both the PLP and the vinyl group. With use of the crystal structure data for GABA-AT recently reported (Storici, P.; Capitani, C.; De Biase, D.; Moser, M.; John, R. A.; Jansonius, J. N.; Schirmer, T. Biochemistry 1999, 38, 8628-8634) a computer model of vigabatrin bound to the PLP was constructed and energy minimized. This model indicated that the major Michael addition pathway could only occur if the vinyl group were allowed to rotate by 180 degrees. A conformationally rigid analogue of vigabatrin, cis-3-aminocyclohex-4-ene-1-carboxylic acid (9), was designed to prevent bond rotation and block the Michael addition pathway. A detailed study of the mechanism of inactivation of GABA-AT by 9 revealed that it inactivates by a single mechanism, the enamine pathway.     

Native chemical ligation in covalent caspase inhibition by p35

Wide-spectrum caspase inhibition by the baculoviral p35 protein was previously shown to be a consequence of covalent inhibition in which a thioester bond is stably formed between the cleavage residue Asp87 of p35 and the active site Cys360' of caspase-8. Here we show that the N-terminal fragment of cleaved p35 (p35-N) is a circular peptide when dissociated from the caspase. Biochemical and crystallographic data suggest that p35-N circularization results from the trapping of a native chemical ligation intermediate in the p35/caspase complex, in which the N-terminal Cys2 of p35 attacks the Asp87-Cys360' thioester to form an equilibrium between Asp87-Cys2 and Asp87-Cys360'. This provides a crucial covalent interaction for keeping the N terminus of p35 bound in the caspase active site, which explains the absolute requirement of Cys2 for caspase inhibition. Participation of native chemical ligation in caspase inhibition by p35 illustrates an unusual mechanism of protease inhibition.     

Hydroxylase activity of Met471Cys tyramine beta-monooxygenase

A series of mutations was targeted at the methionine residue, Met471, coordinating the Cu(M) site of tyramine beta-monooxygenase (TbetaM). The methionine ligand at Cu(M) is believed to be key to dioxygen activation and the hydroxylation chemistry of the copper monooxygenases. The reactivity and copper binding properties of three TbetaM mutants, Met471Asp, Met471Cys, and Met471His, were examined. All three mutants show similar metal binding affinities to wild type TbetaM in the oxidized enzyme forms. EPR spectroscopy suggests that the Cu(II) coordination geometry is identical to that of the WT enzyme. However, substrate hydroxylation was observed for the reaction of tyramine solely with Met471Cys TbetaM. Met471Cys TbetaM provides the first example of an active mutant directed at the Cu(M) site of this class of hydroxylases. The reactivity and altered kinetics of the Met471Cys mutant further highlight the central role of the methionine residue in the enzyme mechanism. The sole ability of the cysteine residue to support activity among the series of alternate amino acids investigated is relevant to theoretical and biomimetic investigations of dioxygen activation at mononuclear copper centers.     

Kinetic stability of the peroxidase activity of unfolded cytochrome c: heme degradation and catalyst inactivation by hydrogen peroxide

Unfolding converts Paracoccus versutus cytochrome c-550 into a potent peroxidase (Diederix, R. E. M.; Ubbink, M.; Canters, G. W. ChemBioChem 2002, 3, 110-112). The catalytic activity is accompanied by peroxide-driven inactivation that is prevented, in part, by reducing substrate. Here, the kinetics of inactivation are described, and evidence is presented for the occurrence of a labile intermediate on the catalytic peroxidase pathway of unfolded cytochrome c-550. This intermediate represents a branching point, whereby the protein proceeds along either the productive pathway or self-inactivates. Reducing substrate suppresses inactivation by decreasing the steady-state concentration of the labile intermediate. Inactivation is accompanied by heme degradation. Its chemical reactivity, UV-vis, and EPR properties identify the first intermediate as hydroxyheme-cytochrome c-550, i.e. with heme hydroxylated at one of the heme meso positions. The occurrence of this species argues for the peroxo-iron species in the peroxidase mechanism as the labile intermediate leading to inactivated cytochrome c-550.     

A proton-shuttle mechanism mediated by the porphyrin in benzene hydroxylation by cytochrome p450 enzymes

Benzene hydroxylation is a fundamental process in chemical catalysis. In nature, this reaction is catalyzed by the enzyme cytochrome P450 via oxygen transfer in a still debated mechanism of considerable complexity. The paper uses hybrid density functional calculations to elucidate the mechanisms by which benzene is converted to phenol, benzene oxide, and ketone, by the active species of the enzyme, the high-valent iron-oxo porphyrin species. The effects of the protein polarity and hydrogen-bonding donation to the active species are mimicked, as before (Ogliaro, F.; Cohen, S.; de Visser, S. P.; Shaik, S. J. Am. Chem. Soc. 2000, 122, 12892-12893). It is verified that the reaction does not proceed either by hydrogen abstraction or by initial electron transfer (Ortiz de Montellano, P. R. In Cytochrome P450: Structure, Mechanism and Biochemistry, 2nd ed.; Ortiz de Montellano, P. R., Ed.; Plenum Press: New York, 1995; Chapter 8, pp 245-303). In accord with the latest experimental conclusions, the theoretical calculations show that the reactivity is an interplay of electrophilic and radicalar pathways, which involve an initial attack on the pi-system of the benzene to produce sigma-complexes (Korzekwa, K. R.; Swinney, D. C.; Trager, W. T. Biochemistry 1989, 28, 9019-9027). The dominant reaction channel is electrophilic and proceeds via the cationic sigma-complex,( 2)3, that involves an internal ion pair made from a cationic benzene moiety and an anionic iron porphyrin. The minor channel proceeds by intermediacy of the radical sigma-complex, (2)2, in which the benzene moiety is radicalar and the iron-porphyrin moiety is neutral. Ring closure in these intermediates produces the benzene oxide product ((2)4), which does not rearrange to phenol ((2)7) or cyclohexenone ((2)6). While such a rearrangement can occur post-enzymatically under physiological conditions by acid catalysis, the computations reveal a novel mechanism whereby the active species of the enzyme catalyzes directly the production of phenol and cyclohexenone. This enzymatic mechanism involves proton shuttles mediated by the porphyrin ring through the N-protonated intermediate, (2)5, which relays the proton either to the oxygen atom to form phenol ((2)7) or to the ortho-carbon atom to produce cyclohexenone product ((2)6). The formation of the phenol via this proton-shuttle mechanism will be competitive with the nonenzymatic conversion of benzene oxide to phenol by external acid catalysis. With the assumption that (2)5 is not fully thermalized, this novel mechanism would account also for the observation that there is a partial skeletal retention of the original hydrogen of the activated C-H bond, due to migration of the hydrogen from the site of hydroxylation to the adjacent carbon (so-called "NIH shift" (Jerina, D. M.; Daly, J. W. Science 1974, 185, 573-582)). Thus, in general, the computationally discovered mechanism of a porphyrin proton shuttle suggests thatthere is an enzymatic pathway that converts benzene directly to a phenol and ketone, in addition to nonenzymatic production of these species by conversion of arene oxide to phenol and ketone. The potential generality of protonated porphyrin intermediates in P450 chemistry is discussed in the light of the H/D exchange observed during some olefin epoxidation reactions (Groves, J. T.; Avaria-Neisser, G. E.; Fish, K. M.; Imachi, M.; Kuczkowski, R. J. Am. Chem. Soc. 1986, 108, 3837-3838) and the general observation of heme alkylation products (Kunze, K. L.; Mangold, B. L. K.; Wheeler, C.; Beilan, H. S.; Ortiz de Montellano, P. R. J. Biol. Chem. 1983, 258, 4202-4207). The competition, similarities, and differences between benzene oxidation viz. olefin epoxidation and alkanyl C-H hydroxylation are discussed, and comparison is made with relevant experimental and computational data. The dominance of low-spin reactivity in benzene hydroxylation viz. two-state reactivity (Shaik, S.; de Visser, S. P.; Ogliaro, F.; Schwarz, H.; Schr?der, D. Curr. Opin. Chem. Biol. 2002, 6, 556-567) in olefin epoxidation and alkane hydroxylation is traced to the loss of benzene resonance energy during the bond activation step.     

Mechanistic insight into the inactivation of carboxypeptidase A by alpha-benzyl-2-oxo-1,3-oxazolidine-4-acetic acid, a novel type of irreversible inhibitor for carboxypeptidase A with no stereospecificity

On the basis of the active site topology and enzymic catalytic mechanism of carboxypeptidase A (CPA), a prototypical zinc-containing proteolytic enzyme, alpha-benzyl-2-oxo-1,3-oxazolidine-4-acetic acid (1), was designed as a novel type of mechanism-based inactivator of the enzyme. All four possible stereoisomers of the inhibitor were synthesized in an enantiomerically pure form starting with optically active aspartic acid, and their CPA inhibitory activities were evaluated to find that surprisingly all of the four stereoisomers inhibit CPA in a time dependent manner. The inhibited enzyme did not regain its enzymic activity upon dialysis. The inactivations were prevented by 2-benzylsuccinic acid, a competitive inhibitor that is known to bind the active site of the enzyme. These kinetic results strongly support that the inactivators attach covalently to the enzyme at the active site. The analysis of ESI mass spectral data of the inactivated CPA ascertained the conclusion from the kinetic results. The values of second-order inhibitory rate constants (k(obs)/[I](o)) fall in the range of 1.7-3.6 M(-1) min(-1). The lack of stereospecificity shown in the inactivation led us to propose that the ring cleavage occurs by the nucleophilic attack at the 2-position rather than at the 5-position and the ring opening takes place in an addition-elimination mechanism. The tetrahedral transition state that would be generated in this pathway is thought to be stabilized by the active site zinc ion, which was supported by the PM3 semiemprical calculations. In addition, alpha-benzyl-2-oxo-1,3-oxazolidine-5-acetic acid (18), a structural isomer of 1 was also found to inactivate CPA in an irreversible manner, reinforcing the nucleophilic addition-elimination mechanism. The present study demonstrates that the transition state for the inactivation pathway plays a critical role in determining stereochemistry of the inactivation.     

Spectroscopic studies of Pyrococcus furiosus superoxide reductase: implications for active-site structures and the catalytic mechanism

The combination of UV/visible/NIR absorption, CD and variable-temperature magnetic circular dichroism (VTMCD), EPR, and X-ray absorption (XAS) spectroscopies has been used to investigate the electronic and structural properties of the oxidized and reduced forms of Pyrococcus furiosus superoxide reductase (SOR) as a function of pH and exogenous ligand binding. XAS shows that the mononuclear ferric center in the oxidized enzyme is very susceptible to photoreduction in the X-ray beam. This observation facilitates interpretation of ground- and excited-state electronic properties and the EXAFS results for the oxidized enzyme in terms of the published X-ray crystallographic data (Yeh, A. P.; Hu, Y.; Jenney, F. E.; Adams, M. W. W.; Rees, D. C. Biochemistry 2000, 39, 2499-2508). In the oxidized state, the mononuclear ferric active site has octahedral coordination with four equatorial histidyl ligands and axial cysteinate and monodentate glutamate ligands. Fe EXAFS are best fit by one Fe-S at 2.36 A and five Fe-N/O at an average distance of 2.12 A. The EPR-determined spin Hamiltonian parameters for the high-spin (S = (5)/(2)) ferric site in the resting enzyme, D = -0.50 +/- 0.05 cm(-1) and E/D = 0.06, are consistent with tetragonally compressed octahedral coordination geometry. UV/visible absorption and VTMCD studies facilitate resolution and assignment of pi His --> Fe(3+)(t(2g)) and (Cys)S(p) --> Fe(3+)(t(2g)) charge-transfer transitions, and the polarizations deduced from MCD saturation magnetization studies indicate that the zero-field splitting (compression) axis corresponds to one of the axes with trans-histidyl ligands. EPR and VTMCD studies provide evidence of azide, ferrocyanide, hydroxide, and cyanide binding via displacement of the glutamate ligand. For azide, ferrocyanide, and hydroxide, ligand binding occurs with retention of the high-spin (S = 5/2) ground state (E/D = 0.27 and D < 0 for azide and ferrocyanide; E/D = 0.25 and D = +1.1 +/- 0.2 cm(-1) for hydroxide), whereas cyanide binding results in a low-spin (S = 1/2) species (g = 2.29, 2.25, 1.94). The ground-state and charge-transfer/ligand-field excited-state properties of the low-spin cyanide-bound derivative are shown to be consistent with a tetragonally elongated octahedral coordination with the elongation axis corresponding to an axis with trans-histidyl ligands. In the reduced state, the ferrous site of SOR is shown to have square-pyramidal coordination geometry in frozen solution with four equatorial histidines and one axial cysteine on the basis of XAS and UV and NIR VTMCD studies. Fe EXAFS are best fit by one Fe-S at 2.37 A and four Fe-N/O at an average distance of 2.15 A. VTMCD reveals a high-spin (S = 2) ferrous site with (Cys)S(p) --> Fe(2+) charge-transfer transitions in the UV region and (5)T(2g) --> (5)E(g) ligand-field transitions in the NIR region at 12400 and <5000 cm(-1). The ligand-field bands indicate square-pyramidal coordination geometry with 10Dq < 8700 cm(-1) and a large excited-state splitting, Delta (5)E(g) > 7400 cm(-1). Analysis of MCD saturation magnetization data leads to ground-state zero-field splitting parameters for the S = 2 ground state, D approximately +10 cm(-1) and E/D approximately 0.1, and complete assessment of ferrous d-orbital splitting. Azide binds weakly at the vacant coordination site of reduced SOR to give a coordination geometry intermediate between octahedral and square pyramidal with 10Dq = 9700 cm(-1) and Delta (5)E(g) = 4800 cm(-1). Cyanide binding results in an octahedral ferrous site with 10Dq = 10,900 cm(-1) and Delta (5)E(g) = 1750 cm(-1). The ability to bind exogenous ligands to both the ferrous and ferric sites of SOR is consistent with an inner-sphere catalytic mechanism involving superoxide binding at the ferrous site to yield a ferric-(hydro)peroxo intermediate. The structural and electronic properties of the SOR active site are discussed in relation to the role and bonding of the axial cysteine residue and the recent proposals for the catalytic mechanism.     

Fe3+-hydroxide ligation in the superoxide reductase from Desulfoarculus baarsii is associated with pH dependent spectral changes

Superoxide reductase (SOR) catalyzes the reduction of O2*- to H2O2. Its active site consists of a non-heme Fe2+ center in an unusual square-pyramidal [His4 Cys] coordination. Like many SORs, the electronic absorption band corresponding to the oxidized active site of the SOR from Desulfoarculus baarsii exhibits a pH-dependent alkaline transition changing from ca. 644 to 560 nm as the pH increases and with an apparent pKa of 9.0. Variants in which the conserved amino acids glutamate 47 and lysine 48 were replaced by the neutral residues alanine (E47A) and isoleucine (K48I), respectively, exhibited the same alkaline transition but at lower apparent pKa values of 6.7 and 7.6, respectively. Previous work [Nivière, V.; Asso, M.; Weill, C. O.; Lombard, M.; Guigliarelli, B.; Favaudon, V.; Houée-Levin, C. Biochemistry 2004, 43, 808-818] has shown that this alkaline transition is associated with the protonation/deprotonation of an unidentified base, B-, which is neither E47 nor K48. In this work, we show by resonance Raman spectroscopy that at basic pH a high-spin Fe3+-OH species is formed at the active site. The presence of the HO- ligand was directly associated with an absorption band maximum at 560 nm, whereas upon protonation, the band shifts to 644 nm. With respect to our previous work, B- can be identified with this high-spin Fe3+-OH species, which upon protonation results in a water molecule at the active site. Implications for the SOR catalytic cycle are proposed.     

Active site labeling of the gentamicin resistance enzyme AAC(6')-APH(2") by the lipid kinase inhibitor wortmannin

BACKGROUND: Aminoglycoside antibiotic resistance is largely the result of the production of enzymes that covalently modify the drugs including kinases (APHs) with structural and functional similarity to protein and lipid kinases. One of the most important aminoglycoside resistance enzymes is AAC(6')-APH(2"), a bifunctional enzyme with both aminoglycoside acetyltransferase and kinase activities. Knowledge of enzyme active site structure is important in deciphering the molecular mechanism of antibiotic resistance and here we explored active site labeling techniques to study AAC(6')-APH(2") structure and function. RESULTS: AAC(6')-APH(2") was irreversibly inactivated by wortmannin, a potent phosphatidylinositol 3-kinase inhibitor, through the covalent modification of a conserved lysine in the ATP binding pocket. 5'-[p-(Fluorosulfonyl)benzoyl]adenosine, an electrophilic ATP analogue and known inactivator of other APH enzymes such as APH(3')-IIIa, did not inactivate AAC(6')-APH(2"), and reciprocally, wortmannin did not inactivate APH(3')-IIIa. CONCLUSIONS: These distinct active site label sensitivities point to important differences in aminoglycoside kinase active site structures and suggest that design of broad range, ATP binding site-directed inhibitors against APHs will be difficult. Nonetheless, given the sensitivity of APH enzymes to both protein and lipid kinase inhibitors, potent lead inhibitors of this important resistance enzyme are likely to be found among the libraries of compounds directed against other pharmacologically important kinases.     

Synthetic model of the phosphate binding protein: solid-state structure and solution-phase anion binding properties of a large oligopyrrolic macrocycle

The macrocyclic receptors 4-6 were synthesized via the anion-templated condensation of appropriately chosen dialdehyde and diamine building blocks. Whereas all three products could be obtained directly via the appropriate choice of reaction conditions, the larger [3+3] product, 6, which incorporates three of each precursor subunit, could also be obtained conveniently via an indirect procedure involving ring expansion of the smaller [2+2] macrocycle 4. As detailed earlier (Sessler, J. L.; Katayev, E. A.; Pantos, G. D.; Reshetova, M. D.; Khrustalev, V. N.; Lynch, V. M.; Ustynyuk, Y. A. Angew. Chem. 2005, 117, 7552-7556; Angew. Chem., Int. Ed. 2005, 44, 7386-7390), this ring expansion occurs under thermodynamic control in the presence of HSO4- and H2PO4- anions in acetonitrile solution and serves to effect the conversion of 4 to 6. An analysis of the X-ray crystal structure of complex 6H22+.HPO42- revealed a strong resemblance to the active site of the phosphate binding protein (PBP) with similar structural analogies being drawn between the active site of the sulfate binding protein (SBP) and the corresponding hydrogensulfate anion complex. In both cases, the anions are bound in a 1:1 fashion in the solid state through a complementary hydrogen bond network involving both the receptor 6 and the anions. UV-vis spectroscopic titrations provide support for the conclusion that macrocycle 6 binds the hydrogensulfate and dihydrogenphosphate anion (studied as the corresponding tetrabutylammonium salts) with high selectivity and affinity in acetonitrile (log Ka for the first binding interaction approaching 7), albeit with different receptor-to-anion binding stoichiometries (1:1 vs 1:3 for HSO4- and H2PO4-, respectively).     

Coordination chemistry at the molybdenum site of sulfite oxidase: redox-induced structural changes in the cysteine 207 to serine mutant

The redox chemistry of the molybdenum site of the C207S mutant of recombinant human sulfite oxidase has been studied via potentiometric titrations employing both electron paramagnetic resonance (EPR) spectroscopy and X-ray absorption spectroscopy (XAS) as probes of the active site structure. In earlier EXAFS studies, oxidized Cys207Ser enzyme has been shown to possess a novel tri-oxo active site, in which Ser207 does not appear to be a ligand to Mo [George, G. N.; Garrett, R. M.; Prince, R. C.; Rajagopalan, K. V. J. Am. Chem. Soc. 1996, 118, 8588-8592]. Redox titrations show that the active site is modified under reducing conditions to a mono-oxo Mo(IV) species, probably with Ser207 ligated to the metal. The Mo(IV) species can be reoxidized to a mono-oxo Mo(V) species still coordinated to Ser207, which in turn can be further reoxidized to yield the initial tri-oxo Mo(VI) structure with loss of Ser207 ligation.     

Hydrogen‐bonding interactions in the active site of a low molecular weight protein‐tyrosine phosphatase

Molecular dynamics simulation of the Michaelis complex, phospho‐enzyme intermediate, and the wild‐type and C12S mutant have been carried out to examine hydrogen‐bonding interactions in the active site of the bovine low molecular weight protein‐tyrosine phosphatase (BPTP). It was found that the S_γ atom of the nucleophilic residue Cys‐12 is ideally located at a position opposite from the phenylphosphate dianion for an inline nucleophilic substitution reaction. In addition, electrostatic and hydrogen‐bonding interactions from the backbone amide groups of the phosphate‐binding loop strongly stabilize the thiolate anion, making Cys‐12 ionized in the active site. In the phospho‐enzyme intermediate, three water molecules are found to form strong hydrogen bonds with the phosphate group. In addition, another water molecule can be identified to form bridging hydrogen bonds between the phosphate group and Asp‐129, which may act as the nucleophile in the subsequent phosphate hydrolysis reaction, with Asp‐129 serving as a general base. The structural difference at the active site between the wild‐type and C12S mutant has been examined. It was found that the alkoxide anion is significantly shifted toward one side of the phosphate binding loop, away from the optimal position enjoyed by the thiolate anion of the wild‐type enzyme in an S_N2 process. This, coupled with the high pK_a value of an alcoholic residue, makes the C12S mutant catalytically inactive. These molecular dynamics simulations provided details of hydrogen bonding interactions in the active site of BPTP, and a structural basis for further studies using combined quantum mechanical and molecular mechanical potential to model the entire dephosphorylation reaction by BPTP. © 2000 John Wiley & Sons, Inc. J Comput Chem 21: 1192–1203, 2000     

Theoretical studies on the mechanism of inhibition of Ribonucleotide Reductase by (E)-2'-Fluoromethylene-2'-deoxycitidine-5'-diphosphate

(E)-2'-Fluoromethylene-2'-deoxycitidine-5'-diphosphate (FMCDP) is a potent time-dependent inactivator of the enzyme Ribonucleotide Reductase, which operates by destructing an essential tyrosil radical and performing a covalent addition to an active site residue. Considerable effort to elucidate the inhibition mechanism has been undertaken in recent years, and some insights have been obtained. Although a mechanistic proposal has been put forward, based on a general paradigm of inhibition of RNR by 2' substituted substrate analogues, details about the mechanism have remained elusive. Namely, the exact residue that adds to the inhibitor is still not identified, although mutagenesis experiments suggest that it should correspond to the E441 residue. In this work, we performed an extensive theoretical exploration of the potential energy surface of a model system representing the active site of RNR with FMCDP, using Density Functional Theory. This study establishes the detailed mechanism of inhibition, which is considerably different from the one proposed earlier. The proposed mechanism is fully consistent with available experimental data. Energetic results reveal unambiguously that the residue adding to the inhibitor is a cysteine, most probably C439, and exclude the possibility of the addition of E441. However, the E441 residue is shown to be essential for inhibition, catalyzing both the major and minor inhibition pathways, in agreement with mutagenesis results. It is shown also that the major mode of inactivation mimics the early stages of the natural substrate pathway.     

Copper chemistry of beta-diketiminate ligands: monomer/dimer equilibria and a new class of bis(mu-oxo)dicopper compounds

A series of Cu(I) and Cu(II) complexes of a variety of beta-diketiminate ligands (L(-)) with a range of substitution patterns were prepared and characterized by spectroscopic, electrochemical, and, in several cases, X-ray crystallographic methods. Specifically, complexes of the general formula [LCuCl](2) were structurally characterized and their magnetic properties assessed through EPR spectroscopy of solutions and, in one instance, by variable-temperature SQUID magnetization measurements on a powder sample. UV-vis spectra indicated reversible dissociation to 3-coordinate monomers LCuCl in solution at temperatures above -55 degrees C. The Cu(I) complexes LCu(MeCN) exhibited reversible Cu(I)/Cu(II) redox couples with E(1/2) values between +300 and +520 mV versus NHE (cyclic voltammetry, MeCN solutions). These complexes were highly reactive with O(2), yielding intermediates that were identified as rare examples of neutral bis(mu-oxo)dicopper complexes on the basis of their EPR silence, diagnostic UV-vis absorption data, and O-isotope-sensitive resonance Raman spectroscopic features. The structural features of the compounds [LCuCl](2) and LCu(MeCN) as well as the proclivity to form bis(mu-oxo)dicopper products upon oxygenation of the Cu(I) complexes are compared to data previously reported for complexes of more sterically hindered beta-diketiminate ligands (Aboelella, N. W.; Lewis, E. A.; Reynolds, A. M.; Brennessel, W. W.; Cramer, C. J.; Tolman, W. B. J. Am. Chem. Soc. 2002, 124, 10600. Spencer, D. J. E.; Aboelella, N. W.; Reynolds, A. M.; Holland, P. L.; Tolman, W. B. J. Am. Chem. Soc. 2002, 124, 2108. Holland, P. L.; Tolman, W. B. J. Am. Chem. Soc. 1999, 121, 7270). The observed structural and reactivity differences are rationalized by considering the steric influences of both the substituents on the flanking aromatic rings and those present on the beta-diketiminate backbone.     

Do spin state changes matter in organometallic chemistry? A computational study

Spin changes occur often in organometallic chemistry, and their effect on kinetics is not well understood. We report computations on the singlet and triplet potential energy surfaces of several processes of this type and show that the topology of the individual surfaces, as well as of the crossing regions between them, can be used to rationalize the observed reactivity in all cases. In particular, the slow addition of dihydrogen to W[N(CH(2)CH(2)NSiMe(3))(3)]H (Schrock, R. R.; Shih, K. Y.; Dobbs, D. A.; Davis, W. M. J. Am. Chem. Soc. 1995, 117, 6609) is shown to be a "spin-blocked" reaction with a high barrier due to the crossing between reactant triplet and product singlet surfaces. In contrast, addition of CO to TpCo(CO) (Detrich, J. L.; Reinaud, O. M.; Rheingold, A. L.; Theopold, K. H. J. Am. Chem. Soc. 1995, 117, 11745) is fast because the triplet and singlet surfaces cross at low energy. Particular care is taken to use DFT methods which are in adequate agreement with experimental and high-level computational energetics for these systems.     

Mechanism of inactivation of inducible nitric oxide synthase by amidines. Irreversible enzyme inactivation without inactivator modification

Nitric oxide synthases (NOS) are hemoproteins that catalyze the reaction of L-arginine to L-citrulline and nitric oxide. N-(3-(Aminomethyl)benzyl)acetamidine (1400W) was reported to be a slow, tight-binding, and highly selective inhibitor of iNOS in vitro and in vivo. Previous mechanistic studies reported that 1400W was recovered quantitatively after iNOS fully lost its activity and modification to iNOS was not detected. Here, it is shown that 1400W is a time-, concentration-, and NADPH-dependent irreversible inactivator of iNOS. HPLC-electrospray mass spectrometric analysis of the incubation mixture of iNOS with 1400W shows both loss of heme cofactor and formation of biliverdin, as was previously observed for iNOS inactivation by another amidine-containing compound, N5-(1-iminoethyl)-L-ornithine (L-NIO). The amount of biliverdin produced corresponds to the amount of heme lost by 1400W inactivation of iNOS. A convenient MS/MS-HPLC methodology was developed to identify the trace amount of biliverdin produced by inactivation of iNOS with either 1400W or L-NIO to be biliverdin IXalpha out of the four possible regioisomers. Two mechanisms were previously proposed for iNOS inactivation by L-NIO: (1) uncoupling of the heme peroxide intermediate, leading to destruction of the heme to biliverdin; (2) abstraction of a hydrogen atom from the amidine methyl group followed by attachment to the heme cofactor, which causes the enzyme to catalyze the heme oxygenase reaction. The second mechanistic proposal was ruled out by inactivation of iNOS with d3-1400W, which produced no d2-1400W. Detection of carbon monoxide as one of the heme-degradation products further excludes the covalent heme adduct mechanism. On the basis of these results, a third mechanism is proposed in which the amidine inactivators of iNOS bind as does substrate L-arginine, but because of the amidine methyl group, the heme peroxy intermediate cannot be protonated, thereby preventing its conversion to the heme oxo intermediate. This leads to a change in the enzyme mechanism to one that resembles that of heme oxygenase, an enzyme known to convert heme to biliverdin IXalpha. This appears to be the first example of a compound that causes irreversible inactivation of an enzyme without itself becoming modified in any way.     

Covalent modification of actin by 4-hydroxy-trans-2-nonenal (HNE): LC-ESI-MS/MS evidence for Cys374 Michael adduction

We demonstrate for the first time, by a combined mass spectrometric and computational approach, that G- and F-actin can be covalently modified by the lipid-derived aldehyde, 4-hydroxy-trans-2-nonenal, providing information on the molecular mass of modified protein and the mechanism and site of adduction.ESI-MS analysis of actin treated with different molar ratios of HNE (1 : 1 to 1 : 20) showed the formation of a protein derivative in which there was an increase of 156 Da (42028 Da) over native actin (41872 Da), consistent with the adduction of one HNE residue through Michael addition. To identify the site of HNE adduction, G- and F-actin were stabilized by NaBH(4) reduction and digested with trypsin. LC-ESI-MS/MS analysis in data-dependent scan mode of the resulting peptides unequivocally indicated that Cys374 is the site of HNE adduction. Computational studies showed that the reactivity of Cys374 residue is due to a significant accessible surface and substantial thiol acidity due to the particular microenvironment surrounding Cys374.     

Mechanism of inhibition of the class C beta-lactamase of Enterobacter cloacae P99 by cyclic acyl phosph(on)ates: rescue by return.

As previously described (Pratt, R. F.; Hammar, N. J. J. Am. Chem. Soc. 1998, 120, 3004.), 1-hydroxy-4,5-benzo-2,6-dioxaphosphorinone(3)-1-oxide (salicyloyl cyclic phosphate) inactivates the class C beta-lactamase of Enterobacter cloacae P99 in a covalent fashion. The inactivated enzyme slowly reverts to the active form. This paper shows that reactivation involves a recyclization reaction that regenerates salicyloyl cyclic phosphate rather than hydrolysis of the covalent intermediate. The inactivation, therefore, is a slowly reversible covalent modification of the active site. The thermodynamic dissociation constant of the inhibitor from the inactivated enzyme is 0.16 microM. Treatment of the inactivated enzyme with alkali does not produce salicylic acid but does, after subsequent acid hydrolysis, yield one molar equivalent of lysinoalanine. This result proves that salicyloyl cyclic phosphate inactivates the enzyme by (slowly reversible) phosphorylation of the active site serine residue. This result contrasts sharply with the behavior of acyclic acyl phosphates which transiently inactivate the P99 beta-lactamase by acylation (Li, N.; Pratt, R. F. J. Am. Chem. Soc. 1998, 120, 4264.). This chemoselectivity difference is explored by means of molecular modeling. Rather counterintuitively, in view of the relative susceptibility of phosphates and phosphonates to nucleophilic attack at phosphorus, 1-hydroxy-4,5-benzo-2-oxaphosphorinanone(3)-1-oxide, the phosphonate analogue of salicyloyl cyclic phosphate, did not appear to inactivate the P99 beta-lactamase in a time-dependent fashion. It was found, however, to act as a fast reversible inhibitor (K(i) = 10 microM). A closer examination of the kinetics of inhibition revealed that both on and off rates (9.8 x 10(3) s(-1) x M(-1) and 0.098 s(-1), respectively) were much slower than expected for noncovalent binding. This result strongly indicates that the inhibition reaction of the phosphonate also involves phosphylation of the active site. Hence, unlike the situation with bacterial DD-peptidases covalently inactivated by beta-lactams, the P99 beta-lactamase inactivated by the above cyclic acyl phosph(on)ates can be rescued by return. Elimination of the recyclization reaction would lead to more effective inhibitors.