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In our efforts to develop inhibitors selective for neuronal nitric oxide synthase (nNOS) over endothelial nitric oxide synthase (eNOS), we found that nNOS can undergo conformational changes in response to inhibitor binding that does not readily occur in eNOS. One change involves movement of a conserved tyrosine, which hydrogen bonds to one of the heme propionates, but in the presence of an inhibitor, changes conformation, enabling part of the inhibitor to hydrogen bond with the heme propionate. This movement does not occur as readily in eNOS and may account for the reason why these inhibitors bind more tightly to nNOS. A second structural change occurs upon the binding of a second inhibitor molecule to nNOS, displacing the pterin cofactor. Binding of this second site inhibitor requires structural changes at the dimer interface, which also occurs more readily in nNOS than in eNOS. Here, we used a combination of crystallography, mutagenesis, and computational methods to better understand the structural basis for these differences in NOS inhibitor binding. Computational results show that a conserved tyrosine near the primary inhibitor binding site is anchored more tightly in eNOS than in nNOS, allowing for less flexibility of this residue. We also find that the inefficiency of eNOS to bind a second inhibitor molecule is likely due to the tighter dimer interface in eNOS compared with nNOS. This study provides a better understanding of how subtle structural differences in NOS isoforms can result in substantial dynamic differences that can be exploited in the development of isoform-selective inhibitors.

The overproduction of nitric oxide in the brain by neuronal nitric oxide synthase (nNOS) is associated with a number of neurodegenerative diseases. Although inhibiting nNOS is an important therapeutic goal, it is important not to inhibit endothelial NOS (eNOS) because of the critical role played by eNOS in maintaining vascular tone. While it has been possible to develop nNOS selective aminopyridine inhibitors, many of the most potent and selective inhibitors exhibit poor bioavailability properties. Our group and others have turned to more biocompatible thiophene-2-carboximidamide (T2C) inhibitors as potential nNOS selective inhibitors. We have used crystallography and computational methods to better understand how and why two commercially developed T2C inhibitors exhibit selectivity for human nNOS over human eNOS. As with many of the aminopyridine inhibitors, a critical active site Asp residue in nNOS versus Asn in eNOS is largely responsible for controlling selectivity. We also present thermodynamic integration results to better understand the change in p K a and thus the charge of inhibitors once bound to the active site. In addition, relative free energy calculations underscore the importance of enhanced electrostatic stabilization of inhibitors bound to the nNOS active site compared to eNOS.

Potent mechanism-based inactivators can be rationally designed against pyridoxal 5'-phosphate (PLP)-dependent drug targets, such as ornithine aminotransferase (OAT) or γ-aminobutyric acid aminotransferase (GABA-AT). An important challenge, however, is the lack of selectivity toward other PLP-dependent, off-target enzymes, because of similarities in mechanisms of all PLP-dependent aminotransferase reactions. On the basis of complex crystal structures, we investigate the inactivation mechanism of OAT, a hepatocellular carcinoma target, by (1R,3S,4S)-3-amino-4-fluorocyclopentane-1-carboxylic acid (FCP), a known inactivator of GABA-AT. A crystal structure of OAT and FCP showed the formation of a ternary adduct. This adduct can be rationalized as occurring via an enamine mechanism of inactivation, similar to that reported for GABA-AT. However, the crystal structure of an off-target, PLP-dependent enzyme, aspartate aminotransferase (Asp-AT), in complex with FCP, along with the results of attempted inhibition assays, suggests that FCP is not an inactivator of Asp-AT, but rather an alternate substrate. Turnover of FCP by Asp-AT is also supported by high-resolution mass spectrometry. Amid existing difficulties in achieving selectivity of inactivation among a large number of PLP-dependent enzymes, the obtained results provide evidence that a desirable selectivity could be achieved, taking advantage of subtle structural and mechanistic differences between a drug-target enzyme and an off-target enzyme, despite their largely similar substrate binding sites and catalytic mechanisms.

Nitric oxide is produced in Gram-positive pathogens Bacillus anthracis and Staphylococcus aureus by the bacterial isoform of nitric oxide synthase (NOS). Inhibition of bacterial nitric oxide synthase (bNOS) has been identified as a promising antibacterial strategy for targeting methicillin-resistant S. aureus [Holden, J. K., et al. (2015) Chem. Biol. 22, 785-779]. One class of NOS inhibitors that demonstrates antimicrobial efficacy utilizes an aminoquinoline scaffold. Here we report on a variety of aminoquinolines that target the bacterial NOS active site, in part, by binding to a hydrophobic patch that is unique to bNOS. Through mutagenesis and crystallographic studies, our findings demonstrate that aminoquinolines are an excellent scaffold for further aiding in the development of bNOS specific inhibitors.

Development of potent and isoform selective nitric oxide synthase (NOS) inhibitors is challenging because of the structural similarity in the heme active sites. One amino acid difference between NOS isoforms, Asp597 in rat neuronal NOS (nNOS) versus Asn368 in bovine endothelial NOS (eNOS), has been identified as the structural basis for why some dipeptide amide inhibitors bind more tightly to nNOS than to eNOS. We now have found that the same amino acid variation is responsible for substantially different binding modes and affinity for a new class of aminopyridine-based inhibitors.

Nitric oxide generated by bacterial nitric oxide synthase (NOS) increases the susceptibility of Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis to oxidative stress, including antibiotic-induced oxidative stress. Not surprisingly, NOS inhibitors also improve the effectiveness of antimicrobials. Development of potent and selective bacterial NOS inhibitors is complicated by the high active site sequence and structural conservation shared with the mammalian NOS isoforms. To exploit bacterial NOS for the development of new therapeutics, recognition of alternative NOS surfaces and pharmacophores suitable for drug binding is required. Here, we report on a wide number of inhibitor-bound bacterial NOS crystal structures to identify several compounds that interact with surfaces unique to the bacterial NOS. Although binding studies indicate that these inhibitors weakly interact with the NOS active site, many of the inhibitors reported here provide a revised structural framework for the development of new antimicrobials that target bacterial NOS. In addition, mutagenesis studies reveal several key residues that unlock access to bacterial NOS surfaces that could provide the selectivity required to develop potent bacterial NOS inhibitors.

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L-citrulline and nitric oxide. N(5)-(1-Iminoethyl)-L-ornithine (L-NIO), an amidine-containing molecule, is a natural product known to be an inactivator of inducible NOS (iNOS). Because of the presence of the amidine methyl group in place of the guanidine amino group of substrate L-arginine, the active site heme peroxy intermediate sometimes cannot be protonated, thereby preventing its conversion to the heme oxo intermediate; instead, a heme oxygenase-type mechanism occurs, leading to conversion of the heme to biliverdin. This might be a new and general inactivation mechanism for heme-containing enzymes. In the studies described here, we attempted to provide support for amidines as substrates and inactivators of iNOS by the design and synthesis of amidine analogues of L-NIO having groups other than the amidine methyl group. No nitric oxide- or enzyme-catalyzed products could be detected by incubation of these amidines with iNOS. Although none of the L-NIO analogues acted as substrates, they all inhibited iNOS; increased inhibitory potency correlated with decreased substituent size. Computer modeling and molecular dynamics simulations were run on 10 and 11 to rationalize why these compounds do not act as substrates. Unlike the methyl amidine (L-NIO), the other alkyl groups block binding of O2 at the heme iron. Compounds 8, 9, and 11 were inactivators; however, no heme was lost, and no biliverdin was formed. No kinetic isotope effect on inactivation was observed with perdeuterated ethyl 8. A small amount of dimer disruption occurred with these inactivators, although the amount would not account for complete enzyme inactivation. The L-NIO analogues inactivate iNOS by a yet unknown mechanism; however, it is different from that of L-NIO, and the inactivation mechanism previously reported for L-NIO appears to be unique to methyl amidines.

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L-citrulline and NO in a two-step process involving the intermediate N(ω)-hydroxy-L-arginine (NHA). It was shown that Cpd I is the oxygenating species for L-arginine; the hydroperoxo ferric intermediate is the reactive intermediate with NHA. Methylation of the N(ω)-OH and N(ω)-H of NHA significantly inhibits the conversion of NHA into NO and L-citrulline by mammalian NOS. Kinetic studies now show that N(ω)-methylation of NHA has a qualitatively similar effect on H₂O₂-dependent catalysis by bacterial gsNOS. To elucidate the effect of methylating N(ω)-hydroxy L-arginine on the properties and reactivity of the one-electron-reduced oxy-heme center of NOS, we have applied cryoreduction/annealing/EPR/ENDOR techniques. Measurements of solvent kinetic isotope effects during 160 K cryoannealing cryoreduced oxy-gsNOS/NHA confirm the hydroperoxo ferric intermediate as the catalytically active species of step two. Product analysis for cryoreduced samples with methylated NHA's, NHMA, NMOA, and NMMA, annealed to 273 K, show a correlation of yields of L-citrulline with the intensity of the g 2.26 EPR signal of the peroxo ferric species trapped at 77 K, which converts to the reactive hydroperoxo ferric state. There is also a correlation between the yield of L-citrulline in these experiments and k(obs) for the H₂O₂-dependent conversion of the substrates by gsNOS. Correspondingly, no detectable amount of cyanoornithine, formed when Cpd I is the reactive species, was found in the samples. Methylation of the NHA guanidinium N(ω)-OH and N(ω)-H inhibits the second NO-producing reaction by favoring protonation of the ferric-peroxo to form unreactive conformers of the ferric-hydroperoxo state. It is suggested that this is caused by modification of the distal-pocket hydrogen-bonding network of oxy gsNOS and introduction of an ordered water molecule that facilitates delivery of the proton(s) to the one-electron-reduced oxy-heme moiety. These results illustrate how variations in the properties of the substrate can modulate the reactivity of a monooxygenase.

Many pyrrolidine-based inhibitors highly selective for neuronal nitric oxide synthase (nNOS) over endothelial NOS (eNOS) exhibit dramatically different binding modes. In some cases, the inhibitor binds in a 180° flipped orientation in nNOS relative to eNOS. From the several crystal structures we have determined, we know that isoform selectivity correlates with the rotamer position of a conserved tyrosine residue that H-bonds with a heme propionate. In nNOS, this Tyr more readily adopts the out-rotamer conformation, while in eNOS, the Tyr tends to remain fixed in the original in-rotamer conformation. In the out-rotamer conformation, inhibitors are able to form better H-bonds with the protein and heme, thus increasing inhibitor potency. A segment of polypeptide that runs along the surface near the conserved Tyr has long been thought to be the reason for the difference in Tyr mobility. Although this segment is usually disordered in both eNOS and nNOS, sequence comparisons and modeling from a few structures show that this segment is structured quite differently in eNOS and nNOS. In this study, we have probed the importance of this surface segment near the Tyr by making a few mutants in the region followed by crystal structure determinations. In addition, because the segment near the conserved Tyr is highly ordered in iNOS, we also determined the structure of an iNOS-inhibitor complex. This new structure provides further insight into the critical role that mobility plays in isoform selectivity.

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L-citrulline through the intermediate N(ω)-hydroxy-L-arginine (NHA), producing nitric oxide, an important mammalian signaling molecule. Several disease states are associated with improper regulation of nitric oxide production, making NOS a therapeutic target. The first step of the NOS reaction has been well-characterized and is presumed to proceed through a compound I heme species, analogous to the cytochrome P450 mechanism. The second step, however, is enzymatically unprecedented and is thought to occur via a ferric peroxo heme species. To gain insight into the details of this unique second step, we report here the synthesis of NHA analogues bearing guanidinium methyl or ethyl substitutions and their investigation as either inhibitors of or alternate substrates for NOS. Radiolabeling studies reveal that N(ω)-methoxy-L-arginine, an alternative NOS substrate, produces citrulline, nitric oxide, and methanol. On the basis of these results, we propose a mechanism for the second step of NOS catalysis in which a methylated nitric oxide species is released and is further metabolized by NOS. Crystal structures of our NHA analogues bound to nNOS have been determined, revealing the presence of an active site water molecule only in the presence of singly methylated analogues. Bulkier analogues displace this active site water molecule; a different mechanism is proposed in the absence of the water molecule. Our results provide new insights into the steric and stereochemical tolerance of the NOS active site and substrate capabilities of NOS.

In previous studies [Delker, S. L., et al. (2010), J. Am. Chem. Soc. 132, 5437-5442], we determined the crystal structures of neuronal nitric oxide synthase (nNOS) in complex with nNOS-selective chiral pyrrolidine inhibitors, designed to have an aminopyridine group bound over the heme where it can electrostatically interact with the conserved active site Glu residue. However, in addition to the expected binding mode with the (S,S)-cis inhibitors, an unexpected "flipped" orientation was observed for the (R,R)-cis enantiomers. In the flipped mode, the aminopyridine extends out of the active site where it interacts with one heme propionate. This prompted us to design and synthesize symmetric "double-headed" inhibitors with an aminopyridine at each end of a bridging ring structure [Xue, F., Delker, S. L., Li, H., Fang, J., Jamal, J., Martásek, P., Roman, L. J., Poulos, T. L., and Silverman, R. B. Symmetric double-headed aminopyridines, a novel strategy for potent and membrane-permeable inhibitors of neuronal nitric oxide synthase. J. Med. Chem. (submitted for publication)]. One aminopyridine should interact with the active site Glu and the other with the heme propionate. Crystal structures of these double-headed aminopyridine inhibitors in complexes with nNOS show unexpected and significant protein and heme conformational changes induced by inhibitor binding that result in removal of the tetrahydrobiopterin (H(4)B) cofactor and creation of a new Zn(2+) site. These changes are due to binding of a second inhibitor molecule that results in the displacement of H(4)B and the placement of the inhibitor pyridine group in position to serve as a Zn(2+) ligand together with Asp, His, and a chloride ion. Binding of the second inhibitor molecule and generation of the Zn(2+) site do not occur in eNOS. Structural requirements for creation of the new Zn(2+) site in nNOS were analyzed in detail. These observations open the way for the potential design of novel inhibitors selective for nNOS.

As a potential drug to treat neurological diseases, the mechanism-based inhibitor (S)-4-amino-4,5-dihydro-2-furancarboxylic acid (S-ADFA) has been found to inhibit the γ-aminobutyric acid aminotransferase (GABA-AT) reaction. To circumvent the difficulties in structural studies of a S-ADFA-enzyme complex using GABA-AT, l-aspartate aminotransferase (l-AspAT) from Escherichia coli was used as a model PLP-dependent enzyme. Crystal structures of the E. coli aspartate aminotransferase with S-ADFA bound to the active site were obtained via cocrystallization at pH 7.5 and 8. The complex structures suggest that S-ADFA inhibits the transamination reaction by forming adducts with the catalytic lysine 246 via a covalent bond while producing 1 equiv of pyridoxamine 5'-phosphate (PMP). Based on the structures, formation of the K246-S-ADFA adducts requires a specific initial binding configuration of S-ADFA in the l-AspAT active site, as well as deprotonation of the ε-amino group of lysine 246 after the formation of the quinonoid and/or ketimine intermediate in the overall inactivation reaction.

Mechanism-based inhibitors such as cycloserine and gabaculine can inactivate aminotransferases via reactions of the compounds with the pyridoxal phosphate cofactor forming an irreversible adduct. The reaction is chirally specific in that any one enzyme usually only recognizes one enantiomer of the inactivator. For instance, l-aspartate aminotransferase (l-AspAT) is inactivated by 4-amino-4,5-dihydro-2-thiophenecarboxylic acid (ADTA), however, only by the S-isomer. We have now shown that d-amino acid aminotransferase (d-a-AT) is irreversibly inactivated by the R-isomer of the same compound. The X-ray crystal structure (PDB code: 3LQS ) of the inactivated enzyme shows that in the product the enzyme no longer makes a Schiff base linkage to the pyridoxal 5'-phosphate (PLP) cofactor, and instead the compound has formed a derivative of the cofactor. The adduct is similar to that formed between d-cycloserine and d-a-AT or alanine racemase (Ala-Rac) in that the thiophene ring of R-ADTA is intact and seems to be aromatic. The plane of the ring is rotated by nearly 90 degrees with respect to the plane of the pyridine ring of the cofactor, in comparison with the enzyme inactivated by cycloserine. Based on the structure of the product, the mechanism of inactivation most probably involves a transamination followed by aromatization to form an aromatic thiophene ring.

Despite the essential biological importance of reactions that involve heme, mechanisms of heme reactions in enzymes like nitric oxide synthase (NOS), heme oxygenase (HO), and cytochrome P450s (CYP450s) are still not well-understood. This Perspective on NOS, HO, and CYP450 mechanisms is written from the point of view of the heme chemistry. Steps in the classical heme catalytic cycle are discussed based on the specific environment within each of these enzymes. Elucidation of the mechanisms of NOS inactivation by some substrate analogues provides important mechanistic clues to the NOS catalytic mechanism. On the basis of mechanistic studies of NOS inactivation by amidine analogues of l-arginine and other previous mechanistic results, a new mechanism for NOS-catalyzed l-arginine NG-hydroxylation (the first half of the catalytic reaction) is proposed in this Perspective. The key step in the second half of the NOS catalytic reaction, the internal electron transfer between the substrate and heme, is discussed on the basis of mechanistic results of NOS inactivation by NG-allyl-l-arginine and the structures of the substrate intermediates. Elucidation of the mechanism of NOS inactivation by amidines, which leads to heme degradation, also provides important mechanistic implications for heme oxygenase-catalyzed heme catabolism. Focusing on the meso-hydroxylation step during inactivation of NOS by amidines as well as the HO-catalyzed reaction, the essential nature of the heme-oxygen species responsible for porphyrin meso-hydroxylation is discussed. Finally, on the basis of the proposed heme degradation mechanism during NOS inactivation and the HO-catalyzed reaction, the mechanism for the formation of the monooxygenated heme species in P450-catalyzed reactions is discussed.

Gamma-aminobutyric acid aminotransferase (GABA-AT), a pyridoxal 5'-phosphate dependent enzyme, catalyzes the degradation of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) to succinic semialdehyde with concomitant conversion of pyridoxal 5'-phosphate (PLP) to pyridoxamine 5'-phosphate (PMP). The enzyme then catalyzes the conversion of alpha-ketoglutarate to the excitatory neurotransmitter L-glutamate. Racemic 4-amino-3-fluorobutanoic acid (3-F-GABA) was shown previously to act as a substrate for GABA-AT, not for transamination, but for HF elimination. Here we report studies of the reaction catalyzed by GABA-AT on (R)- and (S)-3-F-GABA. Neither enantiomer is a substrate for transamination. Very little elimination from the (S)-enantiomer was detected using a coupled enzyme assay; The rate of elimination of HF from the (R)-enantiomer is at least 10 times greater than that for the (S)-enantiomer. The (R)-enantiomer is about 20 times more efficient as a substrate for GABA-AT catalyzed HF elimination than GABA is a substrate for transamination. The (R)-enantiomer also inhibits the transamination of GABA 10 times more effectively than the (S)-enantiomer. Using a combination of computer modeling and the knowledge that vicinal C-F and C-NH3+ bonds have a strong preference to align gauche rather than anti to each other, it is concluded that on binding of free 3-F-GABA to GABA-AT the optimal conformation places the C-NH3+ and C-F bonds gauche in the (R)-enantiomer but anti in the (S)-enantiomer. Furthermore, the dynamic binding process and the bioactive conformation of GABA bound to GABA-AT have been inferred on the basis of the different biological behavior of the two enantiomers of 3-F-GABA when they bind to the enzyme. The present study suggests that the C-F bond can be utilized as a conformational probe to explore the dynamic binding process and provide insight into the bioactive conformation of substrates, which cannot be easily determined by other biophysical approaches.

As a mechanism-based inactivator of PLP-enzymes, (S)-4-amino-4,5-dihydro-2-thiophenecarboxylic acid (SADTA) was cocrystallized with Escherichia coli aspartate aminotransferase (l-AspAT) at a series of pH values ranging from 6 to 8. Five structural models with high resolution (1.4-1.85 A) were obtained for l-AspAT-SADTA complexes at pH 6.0, 6.5, 7.0, 7.5, and 8.0. Electron densities of the models showed that two different adducts had formed in the active sites. One adduct was formed from SADTA covalently linked to pyridoxal 5'-phosphate (PLP) while the other adduct was formed with the inhibitor covalently linked to Lysine246,1 the active site lysine. Moreover, there is a strong indication based on the electron densities that the occurrence of the two adducts is pH dependent. We conclude that SADTA inactivates l-AspAT via two different mechanisms based on the binding direction of the inactivator. Additionally, the structural models also show pH dependence of the protein structure itself, which provided detailed mechanistic implications for l-AspAT.

Inhibition of gamma-aminobutyric acid aminotransferase (GABA-AT) increases the concentration of GABA, an inhibitory neurotransmitter in human brain, which could have therapeutic applications for a variety of neurological diseases, including epilepsy. On the basis of studies of several previously synthesized conformationally restricted GABA-AT inhibitors, (+/-)-(1S,2R,5S)-5-amino-2-fluorocyclohex-3-enecarboxylic acid (12) was designed as a mechanism-based inactivator. This compound was shown to irreversibly inhibit GABA-AT; substrate protects the enzyme from inactivation. Mechanistic experiments demonstrated the loss of one fluoride ion per active site during inactivation and the formation of N-m-carboxyphenylpyridoxamine 5'-phosphate (26), the same product generated by inactivation of GABA-AT by gabaculine (8). An elimination-aromatization mechanism is proposed to account for these results.

A series of L-nitroarginine-based dipeptide inhibitors are highly selective for neuronal nitric oxide synthase (nNOS) over the endothelial isoform (eNOS). Crystal structures of these dipeptides bound to both isoforms revealed two different conformations, curled in nNOS and extended in eNOS, corresponding to higher and lower binding affinity to the two isoforms, respectively. In previous studies we found that the primary reason for selectivity is that Asp597 in nNOS, which is Asn368 in eNOS, provides greater electrostatic stabilization in the inhibitor complex. While this is the case for smaller dipeptide inhibitors, electrostatic stabilization may no longer be the sole determinant for isoform selectivity with bulkier dipeptide inhibitors. Another residue farther away from the active site, Met336 in nNOS (Val106 in eNOS), is in contact with bulkier dipeptide inhibitors. Double mutants were made to exchange the D597/M336 pair in nNOS with N368/V106 in eNOS. Here we report crystal structures and inhibition constants for bulkier dipeptide inhibitors bound to nNOS and eNOS that illustrate the important role played by residues near the entry to the active site in isoform selective inhibition.

(1R,3S,4S)-3-Amino-4-fluorocyclopentane-1-carboxylic acid (7) was previously shown to be a mechanism-based inactivator of gamma-aminobutyric acid aminotransferase (GABA-AT) [Qiu, J. and Silverman, R. B. (2000) J. Med. Chem. 43, 706-720]. Two mechanisms were considered as reasonable possibilities, a Michael addition mechanism and an enamine mechanism. On the basis of a variety of chemical studies, including tedious radiolabeling experiments, it was concluded that inactivation by 7 proceeds by a Michael addition mechanism. Here, a crystal structure of 7 bound to pig liver GABA-AT is reported, which clearly demonstrates that the adduct formed is derived from an enamine mechanism. This represents another example of how crystallography is an important tool for elucidation of inactivation mechanisms.

In a continuing effort to unravel the structural basis for isoform-selective inhibition of nitric oxide synthase (NOS) by various inhibitors, we have determined the crystal structures of the nNOS and eNOS heme domain bound with two D-nitroarginine-containing dipeptide inhibitors, D-Lys-D-Arg(NO)2-NH(2) and D-Phe-D-Arg(NO)2-NH(2). These two dipeptide inhibitors exhibit similar binding modes in the two constitutive NOS isozymes, which is consistent with the similar binding affinities for the two isoforms as determined by K(i) measurements. The D-nitroarginine-containing dipeptide inhibitors are not distinguished by the amino acid difference between nNOS and eNOS (Asp 597 and Asn 368, respectively) which is key in controlling isoform selection for nNOS over eNOS observed for the L-nitroarginine-containing dipeptide inhibitors reported previously [Flinspach, M., et al. (2004) Nat. Struct. Mol. Biol. 11, 54-59]. The lack of a free alpha-amino group on the D-nitroarginine moiety makes the dipeptide inhibitor steer away from the amino acid binding pocket near the active site. This allows the inhibitor to extend into the solvent-accessible channel farther away from the active site, which enables the inhibitors to explore new isoform-specific enzyme-inhibitor interactions. This might be the structural basis for why these D-nitroarginine-containing inhibitors are selective for nNOS (or eNOS) over iNOS.

Trimethylamine dehydrogenase (TMADH) from the bacterium Methylophilus methylotrophus (sp. W(3)A(1)) and its C30A mutant were inactivated with three known inactivators of monoamine oxidase, namely, phenylhydrazine, N-cyclopropyl-alpha-methylbenzylamine, and 1-phenylcyclopropylamine. All three compounds irreversibly inactivated both the wild-type and C30A mutant enzymes, although phenylhydrazine was 10 times more potent than N-cyclopropyl-alpha-methylbenzylamine, which was much more potent than 1-phenylcyclopropylamine. The change in the UV--visible absorption spectra upon modification indicated that the flavin was modified. In the case of the C30A mutant, the absence of a covalent attachment of the flavin to the polypeptide has permitted LC-electrospray mass spectrometry of the reaction product to be undertaken, demonstrating new mass peaks corresponding to various chemically modified forms of the flavin cofactor. In the case of N-cyclopropyl-alpha-methylbenzylamine, masses corresponding to hydroxy-FMN and hydroxyriboflavin were detected. 1-Phenylcyclopropylamine inactivation of the C30A mutant produced three modified flavins, as evidenced by the electrospray mass spectrum: hydroxy-FMN, FMN plus C(6)H(5)COCH(2)CH(2), and hydroxy-FMN plus C(6)H(5)COCH(2)CH(2). Phenylhydrazine inactivation of the C30A mutant gave at least seven different modified flavins: hydroxyriboflavin, hydroxy-FMN, two apparently isomeric compounds corresponding to hydroxy-FMN plus one phenyl group, two apparently isomeric compounds corresponding to FMN plus one phenyl group, and FMN plus two phenyl groups. Covalent flavin adduct formation appears to be the only modification because dialysis of the inactive enzyme followed by reconstitution with FMN restores the enzyme activity to that of a noninactivated control.

1-Phenylcyclopropylamine (1-PCPA) is shown to be an inactivator of the fungal flavoenzyme monoamine oxidase (MAO) N. Inactivation results in an increase in absorbance at 410 nm and is accompanied by the concomitant loss of the flavin absorption band at 458 nm. The spectral properties of the covalent adduct formed between the flavin cofactor of MAO N and 1-PCPA are similar to those reported for the irreversible inactivation product formed with 1-PCPA and mammalian mitochondrial monoamine oxidase B [Silverman, R. B., and Zieske, P. A. (1985) Biochemistry 24, 2128-2138]. There is a hypsochromic shift of the 410 nm band upon lowering the pH to 2, indicating that an N(5)-flavin adduct formed upon inactivation. Use of the fungal enzyme, MAO N, which lacks the covalent attachment to the flavin adenine dinucleotide (FAD) cofactor present in the mammalian forms MAO A and MAO B, has allowed for the isolation and further structural identification of the flavin-inactivator adduct. The incorporation of two (13)C labels into the inactivator, [2,3-(13)C(2)]-1-PCPA, followed by analysis using on-line liquid chromatography/electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy, provided a means to explore the structure of the flavin-inactivator adduct of MAO N. The spectral evidence supports covalent attachment of the 1-PCPA inactivator to the cofactor as N(5)-3-oxo-3-phenylpropyl-FAD.

Recently, we used 35 GHz pulsed 15N ENDOR spectroscopy to determine the position of the reactive guanidino nitrogen of substrate L-arginine relative to the high-spin ferriheme iron of holo-neuronal nitric oxide synthase (nNOS) [Tierney, D. L., et al. (1998) J. Am. Chem. Soc. 120, 2983-2984]. Analogous studies of the enzyme-bound reaction intermediate, NG-hydroxy-L-arginine (NOHA), singly labeled with 15N at the hydroxylated nitrogen (denoted NR), show that NR is held 3.8 A from the Fe, closer than the corresponding guanidino N of L-Arg (4.05 A). 1,2H ENDOR of NOHA bound to holo-nNOS in H2O and D2O discloses the presence of a single resolved exchangeable proton (H1) 4.8 A from Fe and very near the heme normal. The ENDOR data indicate that NOHA does not bind as the resonance-stabilized cation in which the terminal nitrogens share a positive charge. ENDOR-determined structural constraints permit two alternate structural models for the interaction of NOHA with the high-spin heme iron. In one model, H1 is assigned to the O-H proton; in the other, it is the NR-H proton. However, the alternatives differ in the placement of the N-O bond relative to the heme iron. Thus, a combination of the ENDOR data with appropriate diffraction studies can achieve a definitive determination of the protonation state of NR and thus of the tautomeric form that is present in the enzyme-NOHA complex. The mechanistic implications of this result are further discussed.

Monoamine oxidase (MAO) plays an essential role in the regulation of various neurotransmitter and xenobiotic amines. Inhibitors of MAO have been employed in the treatment of depression and as adjuncts in Parkinson's disease therapy. X-Band and Q-band electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopic techniques are employed to characterize a signal assigned as a stable red anionic semiquinone radical in the resting state of MAO B. It is shown that the radical signal is not affected during substrate (either benzylamine or phenylethylamine) turnover, by anaerobic incubation with substrate, or by covalent modification of the active site flavin cofactor in the catalytically active dimer. Upon denaturation, however, the semiquinone absorbances and EPR signals are lost. Photoreduction of the native enzyme in the presence of ethylenediaminetetraacetate generates an EPR signal that is not the same as that obtained in the resting state and shows different proton ENDOR signals. These results suggest that the two flavin prosthetic groups that exist in catalytically active monoamine oxidase B are physically distinct.

Three known mechanism-based inactivators of beef liver mitochondrial monoamine oxidase (MAO) B are tested as inactivators of human placental mitochondrial MAO A. 1-Phenylcyclopropylamine (1-PCPA), 1-benzylcyclopropylamine (1-BCPA), and N-cyclopropyl-alpha-methylbenzylamine (N-C alpha MBA) are time-dependent irreversible inactivators of MAO A. The KI values for 1-PCPA and N-C alpha MBA, analogues of the MAO B substrate benzylamine, are much higher with MAO A than with MAO B. Evidence is presented to show that 1-PCPA inactivates MAO A by attachment to the flavin cofactor, unlike the reaction with MAO B in which 1-PCPA can attach to both a cysteine residue and the flavin [Silverman, R.B., & Zieske, P.A. (1985) Biochemistry 24, 2128-2138]. The reaction of 1-BCPA with MAO A was too slow to study in detail. N-C alpha MBA exhibits the same properties toward inactivation of MAO A that it does for inactivation of MAO B. Attachment in both cases is shown to be to one cysteine residue per enzyme molecule. The results with 1-PCPA indicate that the active site topographies of MAO A and MAO B are different. The ability of N-C alpha MBA to undergo attachment to a cysteine residue in both MAO A and MAO B may lead the way toward peptide mapping of the two isozymes in order to determine differences in their primary structures.

The effect of 18 different amines, two mercaptans, and two alcohols on the reactivation of N-cyclopropylbenzylamine- (N-CBA-) inactivated bovine liver monoamine oxidase (MAO) is described. All of the compounds that reactivate the enzyme produce a time-dependent pseudo-first-order return of enzyme activity and exhibit saturation kinetics. There is no direct correlation between the ability of a compound to serve as a substrate for native MAO and its ability to reactivate N-CBA-inactivated MAO. Amines containing an aromatic moiety, in general, are better reactivators than the aliphatic amines. The amine must be primary or secondary in order for reactivation to occur. The distance between the aromatic portion and the amino group is critical to the reactivation properties of the compound. The mercaptans and alcohols do not reactivate N-CBA-inactivated MAO, nor do they interfere with the reactivation reaction by benzylamine. Three mechanisms for the reactivation reaction are considered. One involves initial Schiff base formation with the active site adduct produced by N-CBA inactivation of MAO followed by base-catalyzed beta-elimination to the imine of acrolein. The second mechanism is the same as the first except no prior Schiff base formation is invoked. The third mechanism is an SN2 displacement by the amine of the active site amino acid residue attached to the adduct. Experiments are carried out to exclude the SN2 mechanism. The results of the reactivation experiments favor the Shiff base mechanism.

(S)-4-Amino-5-halopentanoic acids were previously shown to irreversibly inhibit pig brain gamma-aminobutyric acid--alpha-ketoglutaric acid aminotransferase, and a mechanism for the inactivation was proposed (Silverman, R. B., & Levy M. A. (1980) Biochem. Biophys. Res. Commun. 95, 250). Evidence is presented to show that these compounds are mechanism-based inactivators, and experiments are described to elucidate their mechanism of action. The enzyme must be in the pyridoxal phosphate form for inactivation to occur, the gamma proton of the inactivators is removed in a rate-determining step, and one fluoride ion is released from 4-amino-5-fluoropentanoic acid per active site labeled. The change in the optical spectrum of the enzyme during inactivation suggests that the coenzyme is converted into the pyridoxamine phosphate form. Every turnover of inactivator leads to an inactivation event, i.e., the partition ratio is zero.

beta,beta-Dichloro- and beta,beta,beta-trifluoroalanine irreversibly inactivate a number of pyridoxal phosphate dependent enzymes which catalyze beta- or gamma-elimination reactions. The inactivation is time dependent and the rate of inactivation is first order in enzyme concentration. This suggests that inactivation is due to covalent modification of the enzyme by a species generated at the active site from the polyhaloalanine (i.e., suicide inactivation). Monohaloalanines are substrates and do not inactivate. For gamma-cystathionase, covalent and stoichiometric attachment of [1-14C]beta,beta,beta-trifluoroalanine was shown. It is proposed that the mechanism of inactivation involves Schiff base formation between inactivator and enzyme-bound pyridoxal and subsequent elimination of HC1 from dichloroalanine or HF from trifluoroalanine. This results in the formation of a beta-halo-alpha,beta unsaturated imine, an activated Michael acceptor. Michael addition of a nucleophile at the active site leads to covalent labeling of the enzyme and inactivation. Alanine racemase is also inactivated by the two polyhaloalanines. Glutamate-pyruvate and gultamate-oxaloacetate transaminase are inactivated by monohaloalanines but not by polyhaloalanines.

Three different radioactively labeled N-(1-methylcyclopropyl)benzylamines [N-(1-Me)CBA] were synthesized and used to show which atoms of the inactivator remain bound to monoamine oxidase (MAO) after inactivation. Organic chemical reactions were employed to elucidate the structure of the enzyme adduct and clarify the mechanism of inactivation. Following inactivation and dialysis, the benzyl substituent is lost, but the methyl group and cyclopropyl carbons remain attached to the enzyme even after further dialysis against solutions containing 1 mM benzylamine or 8 M urea. Treatment of inactivated enzyme with sodium cyanoborohydride prior to dialysis results in the retention of the benzyl group, suggesting an imine linkage. One hydride from sodium boro[3H]hydride is incorporated into the dialyzed inactivated enzyme consistent with a ketone functional group. When Pronase-digested N-(1-Me)CBA-inactivated MAO is treated with basic potassium triiodide, iodoform is isolated, indicating the presence of a methyl ketone. During inactivation, the optical spectrum of the covalently bound active site flavin changes from that of oxidized to reduced flavin. After urea denaturation, the flavin remains reduced, suggesting covalent linkage of the inactivator to the cofactor. On the basis of previous results [Silverman, R. B., Hoffman, S. J., & Catus, W. B., III (1980) J. Am. Chem. Soc. 102, 7126-7128], it is proposed that the mechanism of inactivation involves transfer of one electron from N-(1-Me)CBA to the flavin, resulting in an amine radical cation and a flavin radical. Then, either the cyclopropyl ring is attacked by the flavin radical or the cyclopropyl ring opens, and the radical generated is captured by the flavin radical. The product of this mechanism is the imine of benzylamine and 4-flavinyl-2-butanone, the proposed enzyme-inactivator adduct.

Monoamine oxidase (MAO) was shown previously [Silverman, R. B., & Hoffman, S. J. (1980) J. Am. Chem. Soc. 102, 7126-7128] to catalyze the oxidation of N-cyclopropylbenzylamine (N-CBA) at two sites on the molecule. Oxidation at the benzyl methylene gave benzaldehyde and cyclopropylamine; oxidation of the cyclopropyl group, which involved cyclopropyl ring cleavage, led to inactivation of the enzyme. In this paper it is shown that methylation of the benzyl methylene dramatically alters this partition ratio in favor of enzyme inactivation. Contrary to a previous report [Alles, G., & Heegaard, E. V. (1943) J. Biol. Chem. 147, 487-503], it is shown here that alpha-methylbenzylamine is a substrate for MAO; consequently, N-cyclopropyl-alpha-methylbenzylamine (N-C alpha MBA) is a good candidate for mechanism-based inactivation. N-Cyclopropyl[7-14C]benzylamine, N-cyclopropyl-alpha-methyl[phenyl-14C]benzylamine, N-[1-3H]-cyclopropylbenzylamine, and N-[1-3H]cyclopropyl-alpha-methylbenzylamine are synthesized, and product formation following MAO inactivation is quantified. The results obtained with these compounds indicate that with N-C alpha MBA, alpha-methylbenzyl oxidation (which produces acetophenone and cyclopropylamine) is only 1% that of cyclopropyl oxidation (which gives enzyme inactivation), whereas with N-CBA the amount of oxidation at the corresponding sites is equal. It also is shown that the Ki values for (R)-(+)- and (S)-(-)-alpha-methylbenzylamine are similar, suggesting that dimethylation of N-CBA should not interfere with binding to MAO.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Phenylcyclopropylamine (1-PCPA) is shown to be a mechanism-based inactivator of mitochondrial monoamine oxidase (MAO). The strained cyclopropyl ring is important to inactivation since alpha,alpha-dimethylbenzylamine, the acyclic analogue of 1-PCPA, is neither an inactivator nor a substrate of MAO. Two different pathways occur during inactivation by 1-PCPA, both believed to be derived from a common intermediate. One pathway leads to irreversible inactivation of the enzyme and a 1:1 stoichiometry of radioactivity to the active site when 1-[phenyl-14C]PCPA is used as the inactivator; the other pathway results in a covalent reversible adduct. Three organic reactions are carried out on the irreversibly labeled enzyme in order to determine the structure of the active site adduct. Sodium boro[3H]hydride reduction results in the incorporation of 0.73 equiv of tritium, suggesting a carbonyl functionality. Baeyer-Villiger oxidation followed by saponification gives 0.8 equiv of phenol, indicating the presence of a phenyl ketone. Treatment of the labeled enzyme with hydroxide produces acrylophenone, as would be expected from the retro-Michael reaction of beta-X-propiophenone. The identity of X is determined in two ways. The optical spectrum of the flavin cofactor is reduced during inactivation; no reoxidation occurs upon denaturation. Pronase treatment of the radioactively labeled enzyme produces fragments that contain both the radioactivity and the flavin. The X group, therefore, is the flavin. The results of two tests designed to differentiate N5 from C4a attachment to the flavin suggest an N5 adduct. In addition to formation of this stable covalent adduct, another pathway occurs 7 times as often. This alternate reaction of 1-[phenyl-14C]PCPA with MAO produces 7 equiv of [14C]acrylophenone during the course of irreversible inactivation and is believed to arise from formation of the same type of adduct as described above except that X is something other than the N5-flavin (Y). Upon denaturation of this labeled enzyme, the flavin is completely oxidized when most of the radioactivity is still bound to the enzyme. This indicates that Y is not a C4a-flavin adduct and suggests attachment to an active site amino acid residue. More facile elimination of Y from this beta-substituted propiophenone adduct would give acrylophenone on the time scale of the inactivation. Treatment of the reversible adduct with sodium borohydride prior to denaturation prevents release of radioactivity.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Phenylcyclobutylamine (PCBA) is shown to be both a substrate and a time-dependent irreversible inactivator of monoamine oxidase (MAO). Inactivation results in attachment to the flavin cofactor. For every molecule of PCBA leading to inactivation, 325 molecules are converted to product. The first metabolite formed is identified as 2-phenyl-1-pyrroline; then after a lag time, 3-benzoylpropanal and 3-benzoylpropionic acid are generated. The 3-benzoylpropanal is a product of MAO-catalyzed oxidation of 2-phenyl-1-pyrroline (presumably, of its hydrolysis product, gamma-aminobutyrophenone). The aldehyde is nonenzymatically oxidized by nascent hydrogen peroxide to the carboxylic acid. These results are consistent with a one-electron oxidation of PCBA to the amine radical cation followed by homolytic cyclobutane ring cleavage. The resulting radical can partition between cyclization (an intramolecular radical trap) to the 2-phenylpyrrolinyl radical and attachment to the flavin. The cyclic radical can be further oxidized by one electron to 2-phenyl-1-pyrroline. PCBA represents the first in the cyclobutylamine class of MAO inactivators and strongly supports involvement of a radical mechanism for MAO-catalyzed amine oxidations.

The mechanism of inactivation of pig brain gamma-aminobutyric acid aminotransferase (GABA-T) by (S)-4-amino-5-fluoropentanoic acid (1, R = CH2CH2COOH, X = F) previously proposed [Silverman, R. B., & Levy, M. A. (1981) Biochemistry 20, 1197-1203] is revised. apo-GABA-T is reconstituted with [4-3H]pyridoxal 5'-phosphate and inactivated with 1 (R = CH2CH2COOH, X = F). Treatment of inactivated enzyme with base followed by acid denaturation leads to the complete release of radioactivity as 6-[2-hydroxy-3-methyl-6-(phosphonoxymethyl)-4-pyridinyl]-4-oxo-5-+ ++hexenoic acid (4, R = CH2CH2COOH). Alkaline phosphatase treatment of this compound produces dephosphorylated 4 (R = CH2CH2COOH). These results support a mechanism that was suggested by Metzler and co-workers [Likos, J. J., Ueno, H., Feldhaus, R. W., & Metzler, D. E. (1982) Biochemistry 21, 4377-4386] for the inactivation of glutamate decarboxylase by serine O-sulfate (Scheme I, pathway b, R = COOH, X = OSO3-).

A mechanism previously proposed for inactivation of monoamine oxidase (MAO) by N-cyclopropylbenzylamine (N-CBA) [Silverman, R. B., & Hoffman, S. J. (1980) J. Am. Chem. Soc. 102, 884-886] is revised. Inactivation of MAO by N-[1-3H]CBA results in incorporation of about 3 equiv of tritium into the enzyme and release of [3H]acrolein. Treatment of inactivated enzyme with benzylamine, a reactivator for N-CBA-inactivated MAO, releases only 1 equiv of tritium as [3H]acrolein concomitant with reactivation of the enzyme. Even after MAO is inactivated by N-[1-3H]CBA, the reaction continues. At pH 7.2, a linear release of [3H]acrolein is observed for 70 h, which produces 55 equiv of [3H]acrolein while 2.3 equiv of tritium is incorporated into the enzyme. At pH 9, only 3.5 equiv of [3H]acrolein is detected in solution after 96 h, but 40 equiv of tritium is incorporated into the enzyme, presumably as a result of greater ionization of protein nucleophiles at the higher pH. N-[1-3H]Cyclopropyl-alpha-methylbenzylamine (N-C alpha MBA) produces the same adduct as N-CBA but gives only 1-1.35 equiv of tritium bound after inactivation of the enzyme. Denaturation of labeled enzyme results in reoxidation of the flavin without release of tritium, indicating attachment is not to the flavin but rather to an amino acid residue. Enzyme inactivated with N-[1-3H]C alpha MBA is reactivated by benzylamine with the release of 1 equiv of [3H]acrolein, which must have come from an adduct attached to an active site amino acid residue.(ABSTRACT TRUNCATED AT 250 WORDS)

(Z)-4-Amino-2-fluorobut-2-enoic acid (1) is shown to be a mechanism-based inactivator of pig brain gamma-aminobutyric acid aminotransferase. Approximately 750 inactivator molecules are consumed prior to complete enzyme inactivation. Concurrent with enzyme inactivation is the release of 708 +/- 79 fluoride ions; transamination occurs 737 +/- 15 times per inactivation event. Inactivation of [3H]pyridoxal 5'-phosphate ([3H]PLP) reconstituted GABA aminotransferase by 1 followed by denaturation releases [3H]PMP with no radioactivity remaining attached to the protein. A similar experiment carried out with 4-amino-5-fluoropent-2-enoic acid [Silverman, R. B., Invergo, B. J., & Mathew, J. (1986) J. Med. Chem. 29, 1840-1846] as the inactivator produces no [3H]PMP; rather, another radioactive species is released. These results support an inactivation mechanism for 1 that involves normal catalytic isomerization followed by active site nucleophilic attack on the activated Michael acceptor. A general hypothesis for predicting the inactivation mechanism (Michael addition vs enamine addition) of GABA aminotransferase inactivators is proposed.