Unexpected formation of an epoxide-derived multisubstrate adduct inhibitor on the active site of GAR transformylase.

Multisubstrate adduct inhibitors (MAI) of glycinamide ribonucleotide transformylase (GAR Tfase), which incorporate key features of the folate cofactor and the beta-GAR substrate, typically exhibit K(i)'s in the picomolar range. However, these compounds have reduced bioavailability due to the incorporation of a negatively charged phosphate moiety that prevents effective cellular uptake. Thus, a folate analogue that is capable of adduct formation with the substrate on the enzyme active site could lead to a potent GAR Tfase inhibitor that takes advantage of the cellular folate transport systems. We synthesized a dibromide folate analogue, 10-bromo-10-bromomethyl-5,8,10-trideazafolic acid, that was an intermediate designed to assemble with the substrate beta-GAR on the enzyme active site. We have now determined the crystal structure of the Escherichia coli GAR Tfase/MAI complex at 1.6 A resolution to ascertain the nature and mechanism of its time-dependent inhibition. The high-resolution crystal structure clearly revealed the existence of a covalent adduct between the substrate beta-GAR and the folate analogue (K(i) = 20 microM). However, the electron density map surprisingly indicated a C10 hydroxyl in the adduct rather than a bromide and suggested that the multisubstrate adduct is not formed directly from the dibromide but proceeds via an epoxide. Subsequently, we demonstrated the in situ conversion of the dibromide to the epoxide. Moreover, synthesis of the authentic epoxide confirmed that its inhibitory, time-dependent, and cytotoxic properties are comparable to those of the dibromide. Further, inhibition was strongest when the dibromide or epoxide is preincubated with both enzyme and substrate, indicating that inhibition occurs via the enzyme-dependent formation of the multisubstrate adduct. Thus, the crystal structure revealed the successful formation of an enzyme-assembled multisubstrate adduct and highlighted a potential application for epoxides, and perhaps aziridines, in the design of efficacious GAR Tfase inhibitors.     

Rational design,synthesis, evaluation,and crystal structure of a potent inhibitor of human GAR Tfase: 10-(trifluoroacetyl)-5,10-dideazaacyclic-5,6,7,8-tetrahydrofolic acid

Glycinamide ribonucleotide transformylase (GAR Tfase) has been the target of anti-neoplastic intervention for almost two decades. Here, we use a structure-based approach to design a novel folate analogue, 10-(trifluoroacetyl)-5,10-dideazaacyclic-5,6,7,8-tetrahydrofolic acid (10-CF(3)CO-DDACTHF, 1), which specifically inhibits recombinant human GAR Tfase (K(i) = 15 nM), but is inactive (K(i) > 100 microM) against other folate-dependent enzymes that have been examined. Moreover, compound 1 is a potent inhibitor of tumor cell proliferation (IC(50) = 16 nM, CCRF-CEM), which represents a 10-fold improvement over Lometrexol, a GAR Tfase inhibitor that has been in clinical trials. Thus, this folate analogue 1 is among the most potent and selective inhibitors known toward GAR Tfase. Contributing to its efficacious activity, compound 1 is effectively transported into the cell by the reduced folate carrier and intracellularly sequestered by polyglutamation. The crystal structure of human GAR Tfase with folate analogue 1 at 1.98 A resolution represents the first structure of any GAR Tfase to be determined with a cofactor or cofactor analogue without the presence of substrate. The folate-binding loop of residues 141-146, which is highly flexible in both Escherichia coli and unliganded human GAR Tfase structures, becomes highly ordered upon binding 1 in the folate-binding site. Computational docking of the natural cofactor into this and other apo or complexed structures provides a rational basis for modeling how the natural cofactor 10-formyltetrahydrofolic acid interacts with GAR Tfase, and suggests that this folate analogue-bound conformation represents the best template to date for inhibitor design.     

10-Formyl-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic acid (10-formyl-DDACTHF): a potent cytotoxic agent acting by selective inhibition of human GAR Tfase and the de novo purine biosynthetic pathway

The synthesis of 10-formyl-DDACTHF (3) as a potential inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) is reported. Aldehyde 3, the corresponding gamma- and alpha-pentaglutamates 21 and 25 and related agents were evaluated for inhibition of folate-dependent enzymes including GAR Tfase and AICAR Tfase. The inhibitors were found to exhibit potent cytotoxic activity (CCRF-CEM IC(50) for 3=60nM) that exceeded their enzyme inhibition potency [K(i) (3)=6 and 1 microM for Escherichia coli GAR and human AICAR Tfase, respectively]. Cytotoxicity rescue by medium purines, but not pyrimidines, indicated that the potent cytotoxic activity is derived from selective purine biosynthesis inhibition and rescue by AICAR monophosphate established that the activity is derived preferentially from GAR versus AICAR Tfase inhibition. The potent cytotoxic compounds including aldehyde 3 lost activity against CCRF-CEM cell lines deficient in the reduced folate carrier (CCRF-CEM/MTX) or folylpolyglutamate synthase (CCRF-CEM/FPGS(-)) establishing that their potent activity requires both reduced folate carrier transport and polyglutamation. Unexpectedly, the pentaglutamates displayed surprisingly similar K(i)'s versus E. coli GAR Tfase and only modestly enhanced K(i)'s versus human AICAR Tfase. On the surface this initially suggested that the potent cytotoxic activity of 3 and related compounds might be due simply to preferential intracellular accumulation of the inhibitors derived from effective transport and polyglutamation (i.e., ca. 100-fold higher intracellular concentrations). However, a subsequent examination of the inhibitors against recombinant human GAR Tfase revealed they and the corresponding gamma-pentaglutamates were unexpectedly much more potent against the human versus E. coli enzyme (K(i) for 3, 14nM against rhGAR Tfase versus 6 microM against E. coli GAR Tfase) which also accounts for their exceptional cytotoxic potency.     

10-(2-benzoxazolcarbonyl)-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic acid: a potential inhibitor of GAR transformylase and AICAR transformylase

The design and synthesis of 10-(2-benzoxazolcarbonyl)-DDACTHF (1) as an inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide transformylase (AICAR Tfase) are reported. Ketone 1 and the corresponding alcohol 13 were evaluated for inhibition of GAR Tfase and AICAR Tfase and the former was found to be a potent inhibitor of recombinant human (rh) GAR Tfase (Ki=600 nM).     

Synthesis and biological evaluation of alpha- and gamma-carboxamide derivatives of 10-CF3CO-DDACTHF

Structurally-related, but non-polyglutamylatable, derivatives of 10-CF3CO-DDACTHF (1), which incorporate L-glutamine (2) and L-isoglutamine (3) in place of L-glutamate, were prepared and evaluated as inhibitors of recombinant human (rh) GAR Tfase. While the L-glutamate alpha-carboxamide derivative 3 was much less effective as a rhGAR Tfase inhibitor (K(i) = 4.8 microM) and inactive in cellular functional assays, the gamma-carboxamide derivative 2 was found to be a potent and selective rhGAR Tfase inhibitor (K(i) = 0.056 microM) being only 4-fold less potent than 1 (K(i) = 0.015 microM). Moreover, 2 was effective in cellular functional assays exhibiting purine sensitive cytotoxic activity (IC50 = 300 nM, CCRF-CEM) only 20-fold less potent than 1 (IC50 = 16 nM), consistent with inhibition of de novo purine biosynthesis via selective inhibition of GAR Tfase. Like 1, 2 is transported into the cell by the reduced folate carrier. Unlike 1, the functional activity of 2 is not dependent upon FPGS polyglutamylation.     

Structural insights into the avian AICAR transformylase mechanism

ATIC encompasses both AICAR transformylase and IMP cyclohydrolase activities that are responsible for the catalysis of the penultimate and final steps of the purine de novo synthesis pathway. The formyl transfer reaction catalyzed by the AICAR Tfase domain is substantially more demanding than that catalyzed by the other folate-dependent enzyme of the purine biosynthesis pathway, GAR transformylase. Identification of the AICAR Tfase active site and key catalytic residues is essential to elucidate how the non-nucleophilic AICAR amino group is activated for formyl transfer. Hence, the crystal structure of dimeric avian ATIC was determined as a complex with the AICAR Tfase substrate AICAR, as well as with an IMP cyclohydrolase inhibitor, XMP, to 1.93 A resolution. AICAR is bound at the dimer interface of the transformylase domains and forms an extensive hydrogen bonding network with a multitude of active site residues. The crystal structure suggests that the conformation of the 4-carboxamide of AICAR is poised to increase the nucleophilicity of the C5 amine, while proton abstraction occurs via His(268) concomitant with formyl transfer. Lys(267) is likely to be involved in the stabilization of the anionic formyl transfer transition state and in subsequent protonation of the THF leaving group.     

Design, synthesis and biological evaluation of 10-CF3CO-DDACTHF analogues and derivatives as inhibitors of GAR Tfase and the de novo purine biosynthetic pathway

The synthesis and evaluation of analogues and key derivatives of 10-CF3CO-DDACTHF as inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide transformylase (AICAR Tfase) are reported. Polyglutamate analogues of 1 were evaluated as inhibitors of Escherichia coli and recombinant human (rh) GAR Tfase, and AICAR Tfase. Although the pentaglutamate 6 was found to be the most active inhibitor of the series tested against rhGAR Tfase (Ki=0.004 microM), little distinction between the mono-pentaglutamate derivatives was observed (Ki=0.02-0.004 microM), suggesting that the principal role of the required polyglutamation of 1 is intracellular retention. In contrast, 1 and its defined polyglutamates 3-6 were much less inactive when tested against rhAICAR Tfase (Ki=65-0.120 microM) and very selective (> or =100-fold) for rh versus E. coli GAR Tfase. Additional key analogues of 1 were examined (7 and 8) and found to be much less active (1000-fold) highlighting the exceptional characteristics of 1.     

Structural evidence for a ligand coordination switch in liver alcohol dehydrogenase

The use of substrate analogues as inhibitors provides a way to understand and manipulate enzyme function. Here we report two 1 A resolution crystal structures of liver alcohol dehydrogenase in complex with NADH and two inhibitors: dimethyl sulfoxide and isobutyramide. Both structures present a dynamic state of inhibition. In the dimethyl sulfoxide complex structure, the inhibitor is caught in transition on its way to the active site using a flash-freezing protocol and a cadmium-substituted enzyme. One inhibitor molecule is partly located in the first and partly in the second coordination sphere of the active site metal. A hydroxide ion bound to the active site metal lies close to the pyridine ring of NADH, which is puckered in a twisted boat conformation. The cadmium ion is coordinated by both the hydroxide ion and the inhibitor molecule, providing structural evidence of a coordination switch at the active site metal ion. The structure of the isobutyramide complex reveals the partial formation of an adduct between the isobutyramide inhibitor and NADH. It provides evidence of the contribution of a shift from the keto to the enol tautomer during aldehyde reduction. The different positions of the inhibitors further refine the knowledge of the dynamics of the enzyme mechanism and explain how the crowded active site can facilitate the presence of a substrate and a metal-bound hydroxide ion.     

Design, synthesis, and biological evaluation of 10-methanesulfonyl-DDACTHF, 10-methanesulfonyl-5-DACTHF, and 10-methylthio-DDACTHF as potent inhibitors of GAR Tfase and the de novo purine biosynthetic pathway

The synthesis and evaluation of 10-methanesulfonyl-DDACTHF (1), 10-methanesulfonyl-5-DACTHF (2), and 10-methylthio-DDACTHF (3) as potential inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) are reported. The compounds 10-methanesulfonyl-DDACTHF (1, K(i) = 0.23 microM), 10-methanesulfonyl-5-DACTHF (2, K(i) = 0.58 microM), and 10-methylthio-DDACTHF (3, K(i) = 0.25 microM) were found to be selective and potent inhibitors of recombinant human GAR Tfase. Of these, 3 exhibited exceptionally potent, purine sensitive growth inhibition activity (3, IC50 = 100 nM) against the CCRF-CEM cell line being 3-fold more potent than Lometrexol and 30-fold more potent than the parent, unsubstituted DDACTHF, whereas 1 and 2 exhibited more modest growth inhibition activity (1, IC50 = 1.0 microM and 2, IC50 = 2.0 microM).     

Evidence for a tetrahedral intermediate complex during serpin-proteinase interactions

Proteinase inhibitors in the serpin family form complexes with serine proteinases by interactions between the gamma-OH group at serine 195 of the enzyme and a specific peptide bond within the reactive site loop of the inhibitor. However, the type of complex formed (i.e. Michaelis, acyl, or tetrahedral) is unknown. Until now, 13C NMR spectroscopy studies have only been useful in examining complexes formed with either peptide-related or small protein inhibitors, where 13C-labeled amino acids can be inserted semi-synthetically. Recombinant DNA technology has, however, made it possible to specifically enrich larger proteins with 13C. In the case of serpins we have examined the structure of the complex formed between human alpha 1-proteinase inhibitor uniformally labeled with [13C]methionine and porcine pancreatic elastase. 13C NMR spectroscopic studies revealed a large upfield chemical shift of the carbonyl signal of Met-358 upon complex formation suggesting for the first time that a tetrahedral adduct is formed between a serpin inhibitor and a serine proteinase.     

Design, synthesis, and biological evaluation of simplified alpha-keto heterocycle, trifluoromethyl ketone, and formyl substituted folate analogues as potential inhibitors of GAR transformylase and AICAR transformylase

A series of simplified alpha-keto heterocycle, trifluoromethyl ketone, and formyl substituted folate analogues lacking the benzoylglutamate subunit were prepared and examined as potential inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide transformylase (AICAR Tfase).     

Proton transfer dynamics of GART: the pH-dependent catalytic mechanism examined by electrostatic calculations

The enzyme glycinamide ribonucleotide transformylase (GART) catalyzes the transfer of a formyl group from formyl tetrahydrofolate (fTHF) to glycinamide ribonucleotide (GAR), a process that is pH-dependent with pK(a) of approximately 8. Experimental studies of pH-rate profiles of wild-type and site-directed mutants of GART have led to the proposal that His108, Asp144, and GAR are involved in catalysis, with His108 being an acid catalyst, while forming a salt bridge with Asp144, and GAR being a nucleophile to attack the formyl group of fTHF. This model implied a protonated histidine with pK(a) of 9.7 and a neutral GAR with pK(a) of 6.8. These proposed unusual pK(a)s have led us to investigate the electrostatic environment of the active site of GART. We have used Poisson-Boltzmann-based electrostatic methods to calculate the pK(a)s of all ionizable groups, using the crystallographic structure of a ternary complex of GART involving the pseudosubstrate 5-deaza-5,6,7,8-THF (5dTHF) and substrate GAR. Theoretical mutation and deletion analogs have been constructed to elucidate pairwise electrostatic interactions between key ionizable sites within the catalytic site. Also, a construct of a more realistic catalytic site including a reconstructed pseudocofactor with an attached formyl group, in an environment with optimal local van der Waals interactions (locally minimized) that imitates closely the catalytic reactants, has been used for pK(a) calculations. Strong electrostatic coupling among catalytic residues His108, Asp144, and substrate GAR was observed, which is extremely sensitive to the initial protonation and imidazole ring flip state of His108 and small structural changes. We show that a proton can be exchanged between GAR and His108, depending on their relative geometry and their distance to Asp144, and when the proton is attached on His108, catalysis could be possible. Using the formylated locally minimized construct of GART, a high pK(a) for His108 was calculated, indicating a protonated histidine, and a low pK(a) for GAR(NH(2)) was calculated, indicating that GAR is in neutral form. Our results are in qualitative agreement with the current mechanistic picture of the catalytic process of GART deduced from the experimental data, but they do not reproduce the absolute magnitude of the pK(a)s extracted from fits of k(cat)-pH profiles, possibly because the static time-averaged crystallographic structure does not describe adequately the dynamic nature of the catalytic site during binding and catalysis. In addition, a strong effect on the pK(a) of GAR(NH(2)) is produced by the theoretical mutations of His108Ala and Asp144Ala, which is not in agreement with the observed insensitivity of the pK(a) of GAR(NH(2)) modeled from the experimental data using similar mutations. Finally, we show that important three-way electrostatic interactions between highly conserved His137, with His108 and Asp144, are responsible for stabilizing the electrostatic microenvironment of the catalytic site. In conclusion, our data suggest that further detailed computational and experimental work is necessary.     

Evidence for the accumulation of a stable intermediate in the nonenzymatic hydrolysis of 5,10-methenyltetrahydropteroylglutamate to 5-formyltetrahydropteroylglutamate.

Solutions of 5,10-methenyltetrahydropteroylglutamate can be converted to a stable hydrated adduct by heating solutions at 50 degrees C at pH values of 3-5 for several hours. The adduct is stable at pH values from 4 to 9 for hours, but at pH values below 2 it is converted to 5,10-methenyltetrahydropteroylglutamate and at pH values above 8 it is converted to 5-formyltetrahydropteroylglutamate. Arguments are presented that the adduct is (11R)-5,10-hydroxymethylenetetrahydropteroylglutamate formed from (11S)-5,10-hydroxymethylenetetrahydropteroylglutamate by formation of an ylide at C-11 which undergoes inversion of the electron pair to form the (11R) isomer. The (11R) hydrated adducted is believed to be the isomer of 5,10-methenyltetrahydropteroylglutamate referred to as anhydroleucovorin B by Cosulich et al. [Cosulich, D. C., Roth, B., Smith, J. M., Hultquist, M. E., & Parker, R. P. (1952) J. Am. Chem. Soc. 74, 3252-3263]. In addition, a new mechanism for the formation of 5-formyltetrahydropteroylglutamate from either 5,10-methenyltetrahydropteroylglutamate or 10-formyltetrahydropteroylglutamate via (11R)-5,10-hydroxymethylenetetrahydropteroylglutamate is proposed. A requirement for this pathway is that the formyl proton of 10-formyltetrahydropteroylglutamate exchange with solvent protons. The exchange of this formyl proton was observed at all pH values from 5.5 to 11.5 at a rate which exceeded by more than an order of magnitude the rate of formation of 5-formyltetrahydropteroylglutamate.     

Determination of the structure of tetrahedral transition state analogues bound at the active site of chymotrypsin using 18O and 2H isotope shifts in the 13C NMR spectra of glyoxal inhibitors

The peptide-derived glyoxal inhibitor Z-Ala-Pro-Phe-glyoxal, where Z is benzyloxycarbonyl, is an extremely potent inhibitor of chymotrypsin. When it is bound to chymotrypsin both the glyoxal (RCOCHO) keto and aldehyde carbons are sp3 hybridized with chemical shifts of 100.7 and 91.4 ppm, respectively. However it is has not been shown whether these carbons are bound as hydrates or whether the active-site serine has reacted with them to form the corresponding hemiketal or hemiacetal. In this study we use 18O isotope shifts to determine whether one or two exchangeable oxygen atoms are attached to the glyoxal keto or aldehyde carbons when it is free in water or bound to alpha-chymotrypsin. Both the 18O isotope shifts at the free and enzyme-bound aldehyde carbons were approximately 0.04 ppm showing that it is hydrated in both the free and bound forms. The 18O isotope shift for the free hydrated keto carbon at 96.6 ppm was 0.046-0.049 ppm, but this was reduced to 0.026 ppm when the glyoxal inhibitor was bound to alpha-chymotrypsin showing that the nonexchangeable serine hydroxyl group has formed a hemiketal with glyoxal keto carbon. Deuterium isotope shifts on the 13C NMR signals from the glyoxal inhibitor when it free and hydrated, when it is bound to chymotrypsin, as well as when it forms a model hemiketal confirm that the serine hydroxyl group has formed a hemiketal with the glyoxal keto carbon. The reasons for the different reaction specificities of glyoxal inhibitors for the active-site nucleophiles of serine and cysteine proteases are discussed.     

alpha-Bromo ketone substrate analogues are powerful reversible inhibitors of carboxypeptidase A

The aldehyde (RS)-2-benzyl-4-oxobutanoic acid, which is 25% hydrated at pH 7.5, has recently been shown to be a strong reversible competitive inhibitor of carboxypeptidase A [Ki = 0.48 nM; Galardy, R. E., & Kortylewicz, Z. P. (1984) Biochemistry 23, 2083-2087]. The ketone analogue of this aldehyde (RS)-2-benzyl-4-oxopentanoic acid (IV) is not detectably hydrated under the same conditions and is 1500-fold less potent (Ki = 730 microM). The ketone homologue (RS)-2-benzyl-5-oxohexanoic acid (XIII) is also a weak inhibitor (Ki = 1.3 mM). The alpha-monobrominated derivatives of these two ketones are, however, strong competitive inhibitors with Ki's of 0.57 microM and 1.3 microM, respectively. Oximes derived from the aldehyde, the ketones IV and XIII, and a homologue of the aldehyde are weak inhibitors with Ki's ranging from 480 to 7900 microM. The inhibition of carboxypeptidase A by the alpha-monobrominated ketones is reversible and independent of the time (up to 6 h) of incubation of enzyme and inhibitor together. Bromoacetone at a concentration of 30 mM does not inhibit carboxypeptidase A. Incubation of an equimolar mixture of 2-benzyl-4-bromo-5-oxohexanoic acid (XV) and enzyme for 1 h led to the recovery of 82% of XV, demonstrating that it is the major species reversibly bound during assay of inhibition. Taken together, these results indicate that tight binding of carbonyl inhibitors to carboxypeptidase A requires specific binding of inhibitor functional groups such as benzyl and an electrophilic carbonyl carbon such as that of an alpha-bromo ketone or aliphatic aldehyde.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new lysine derived glyoxal inhibitor of trypsin,its properties and utilization for studying the stabilization of tetrahedral adducts by trypsin

New trypsin inhibitors Z-Lys-COCHO and Z-Lys-H have been synthesised. K_i values for Z-Lys-COCHO, Z-Lys-COOH, Z-Lys-H and Z-Arg-COOH have been determined. The glyoxal group (–COCHO) of Z-Lys-COCHO increases binding ~300 fold compared to Z-Lys-H. The α-carboxylate of Z-Lys-COOH has no significant effect on inhibitor binding. Z-Arg-COOH is shown to bind ~2 times more tightly than Z-Lys-COOH. Both Z-Lys-¹³COCHO and Z-Lys-CO¹³CHO have been synthesized. Using Z-Lys-¹³COCHO we have observed a signal at 107.4 ppm by ¹³C NMR which is assigned to a terahedral adduct formed between the hydroxyl group of the catalytic serine residue and the ¹³C-enriched keto-carbon of the inhibitor glyoxal group. Z-Lys-CO¹³CHO has been used to show that in this tetrahedral adduct the glyoxal aldehyde carbon is not hydrated and has a chemical shift of 205.3 ppm. Hemiketal stabilization is similar for trypsin, chymotrypsin and subtilisin Carlsberg. For trypsin hemiketal formation is optimal at pH 7.2 but decreases at pHs 5.0 and 10.3. The effective molarity of the active site serine hydroxyl group of trypsin is shown to be 25300 M. At pH 10.3 the free glyoxal inhibitor rapidly (t_1/2=0.15 h) forms a Schiff base while at pH 7 Schiff base formation is much slower (t_1/2=23 h). Subsequently a free enol species is formed which breaks down to form an alcohol product. These reactions are prevented in the presence of trypsin and when the inhibitor is bound to trypsin it undergoes an internal Cannizzaro reaction via a C2 to C1 alkyl shift producing an α-hydroxycarboxylic acid.     

Crystal structures of human GAR Tfase at low and high pH and with substrate beta-GAR

Glycinamide ribonucleotide transformylase (GAR Tfase) is a key folate-dependent enzyme in the de novo purine biosynthesis pathway and, as such, has been the target for antitumor drug design. Here, we describe the crystal structures of the human GAR Tfase (purN) component of the human trifunctional protein (purD-purM-purN) at various pH values and in complex with its substrate. Human GAR Tfase exhibits pH-dependent enzyme activity with its maximum around pH 7.5-8. Comparison of unliganded human GAR Tfase structures at pH 4.2 and pH 8.5 reveals conformational differences in the substrate binding loop, which at pH 4.2 occupies the binding cleft and prohibits substrate binding, while at pH 8.5 is permissive for substrate binding. The crystal structure of GAR Tfase with its natural substrate, beta-glycinamide ribonucleotide (beta-GAR), at pH 8.5 confirms this conformational isomerism. Surprisingly, several important structural differences are found between human GAR Tfase and previously reported E. coli GAR Tfase structures, which have been used as the primary template for drug design studies. While the E. coli structure gave valuable insights into the active site and formyl transfer mechanism, differences in structure and inhibition between the bacterial and mammalian enzymes suggest that the human GAR Tfase structure is now the appropriate template for the design of anti-cancer agents.     

Mechanistic studies of choline oxidase with betaine aldehyde and its isosteric analogue 3,3-dimethylbutyraldehyde

Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine via two sequential FAD-dependent reactions in which betaine aldehyde is formed as an intermediate. The chemical mechanism for the oxidation of choline catalyzed by choline oxidase was recently elucidated by using kinetic isotope effects [Fan, F., and Gadda, G. (2005) J. Am. Chem. Soc. 127, 2067-2074]. In this study, the oxidation of betaine aldehyde has been investigated by using spectroscopic and kinetic analyses with betaine aldehyde and its isosteric analogue 3,3-dimethylbutyraldehyde. The pH dependence of the kcat/Km and kcat values with betaine aldehyde showed that a catalytic base with a pKa of approximately 6.7 is required for betaine aldehyde oxidation. Complete reduction of the enzyme-bound flavin was observed in a stopped-flow spectrophotometer upon anaerobic mixing with betaine aldehyde or choline at pH 8, with similar k(red) values > or = 48 s(-1). In contrast, only 10-26% of the enzyme-bound flavin was reduced by 3,3-dimethylbutyraldehyde between pH 6 and 10. Furthermore, this compound acted as a competitive inhibitor versus choline. NMR spectroscopic analyses indicated that betaine aldehyde exists predominantly (99%) as a diol form in aqueous solution. In contrast, the thermodynamic equilibrium for 3,3-dimethylbutyraldehyde favors the aldehyde (> or = 65%) over the hydrated form in the pH range from 6 to 10. The keto species of 3,3-dimethylbutyraldehyde is reactive toward enzymic nucleophiles, as suggested by the kinetic data with NAD+-dependent yeast aldehyde dehydrogenase. The data presented suggest that choline oxidase utilizes the hydrated species of the aldehyde as substrate in a mechanism for aldehyde oxidation in which hydride transfer is triggered by an active site base.     

Synthesis and evaluation of a new series of tri-, di-, and mono-N-alkylcarbamylphloroglucinols as conformationally constrained inhibitors of cholesterol esterase

1,3,5-Tri-N-alkylcarbamylphloroglucinols (1-4) are synthesized as conformationally constrained analogs of triacylglycerols (TGs) to probe Jenck's proximity effect in the cholesterol esterase inhibition. For the cholesterol esterase inhibition, inhibitors 1-4 are 220-760-fold more potent than 1,2,3-tri-N-alkylcarbamylglycerols (13-15) that are substrate analogs of TG. Comparison of tridentate inhibitors 1-4, bidentate inhibitors 3,5-di-N-n-alkylcarbamyloxyphenols (5-8) and monodentate inhibitors 5-N-n-alkylcarbamyloxyresorcinols (9-12) indicates that inhibitory potencies are as followed: tridentate inhibitor > bidentate inhibitor > monodentate inhibitor. The log k(i) and pK(i) values of tridentate inhibitors, bidentate inhibitors, and monodentate inhibitors are linearly correlated with the alkyl chain length indicating a common mechanism in each inhibition. Also, positive slopes of these correlations indicate that the longer chain inhibitors bind more tightly to the enzyme than the shorter ones. Molecular dockings of tridentate 1, bidentate 5, and monodentate 9 into the X-ray crystal structure of cholesterol esterase suggest that one carbamyl group in the cis form of the inhibitor binds to the acyl chain-binding site of the enzyme. The second carbamyl groups in the trans forms of inhibitors 1 and 5 bind to the second acyl chain-binding site of the enzyme. The third carbamyl group in the trans form of inhibitor 1 binds to the third acyl chain-binding site of the enzyme. Moreover, the configuration of the inhibitor in the enzyme-inhibitor complex is the (1,3,5)-(cis, trans, trans)-tricarbamate form that mimics the (+gauche, -gauche)-conformation of TG.     

A quantum chemical study on the mechanism of glycinamide ribonucleotide transformylase inhibitor: 10-Formyl-5,8,10-trideazafolic acid

A density functional theory (DFT) study is presented on the reaction mechanism of glycinamide ribonucleotide (GAR) with 10-formyl-5,8,10-trideazafolic acid (10f-TDAF), which is an inhibitor designed for GAR transformylase (GAR Tfase). There are three different paths for this system and the results indicate that inhibitor 10f-TDAF can form a very stable intermediate with the substrate GAR or generate an imine bond with GAR by elimination of water. The results have verified the presumption from available experiments and implied that 10f-TDAF would be an important target for anti-neoplastic intervention.