Preparation and some properties of 6-substituted flavins as active site probes for flavin enzymes

6-Azidoflavins, 6-thiocyanatoflavins, and 6-mercaptoflavins at the lumiflavin, riboflavin, FMN, and FAD level were prepared from the corresponding 6-aminoflavins and some of their properties investigated. They are bound tightly by apoflavin enzymes which bind either riboflavin, FMN, or FAD. 6-Azidoflavins undergo facile photolysis. One major product was identified as 6-aminoflavin. A further product, which was formed also during acid decomposition of the azide, results from opening of the flavin benzene ring and is proposed to have a lumazine structure. 6-Thiocyanatoflavins are easily converted by dithiothreitol to 6-mercaptoflavins. The latter are stabilized against dimerization in the presence of reducing thiols. 6-Mercaptoflavins have a pK of 5.9, which corresponds to ionization of the 6-SH function. The neutral form is yellow, while the anion is green, due to a long-wavelength band (lambda max approximately 600 nm) extending beyond 700 nm. These properties suggest the use of these 6-substituted flavins for probing the active site of flavin enzymes. Because their reactive substituents are in close proximity to the flavin N(5)-position, these 6-substituted derivatives should also serve as useful probes of the environment around the flavin N(5), a position known to be involved in all flavin-mediated redox processes.     

8-Mercaptoflavins as active site probes of flavoenzymes.

Representative examples of the various classes of flavoproteins have been converted to their apoprotein forms and the native flavin replaced by 8-mercapto-FMN or 8-mercapto-FAD. The spectral and catalytic properties of the modified enzymes are characteristically different from one group to another; the results suggest that flavin interactions at positions N(1) or N(5) of the flavin chromophore have profound influences on the properties of the flavoprotein. 1. The 8-thiolate anion form of 8-mercaptoflavin has an absorption maximum in the region 520 to 550 nm epsilon approximately 30 mM-1 cm-1). This form is retained on binding to flavoproteins whose physiological reactions involve obligatory one-electron transfers (e.g. flavodoxin, NADPH-cytochrome P-450 reductase). In the native form these enzymes stabilize the blue neutral radical of the flavin. A radical form of 8-mercaptoflavin is also stabilized by these proteins. 2. The p-quinoid form of 8-mercaptoflavin has an absorption maximum in the range 560 to 600 nm (epsilon approximately 30 mM-1 cm-1). This form is stabilized on binding to flavoproteins of the dehydrogenase-oxidase class (e.g. glucose oxidase, D-amino acid oxidase, lactate oxidase, Old Yellow Enzyme). These same enzymes in their native flavin form stabilize the red semiquinone, and have a pronounced reactivity with sulfite to form flavin N(5)-sulfite adducts. These properties of the native enzyme, including the ability to react with nitroalkane carbanions, are not exhibited by the 8-mercaptoflavoproteins. 3. A group of flavoenzymes fails to conform strictly to the above classification, exhibiting some properties of both classes. These include the examples of flavoprotein hydroxylases and transhydrogenases studied. 4. The riboflavin-binding protein of hen egg whites binds 8-mercaptoriboflavin preferentially in the unionized state, resulting in a shift in pK from 3.8 with free 8-mercaptoriboflavin to greater than or equal to 9.0 with the protein-bound form.     

Design and synthesis of chromogenic thiopeptolide substrates as MetAPs active site probes

Twenty one chromogenic thiopeptolide substrates were designed and synthesized as the active site probes and analyzed with each S1 site of mutant residues and enzymes of wild-type MetAP1s. The preliminary enzymatic experiments indicate that cysteine 70 or 202, at either Escherichia coli or human MetAP1, played a crucial role in the methionine hydrolysis.     

Steroidal inhibitors as chemical probes of the active site of aromatase

Androstenedione analogs containing 7-substituents have proven to be potent inhibitors of aromatase in human placental microsomes, in MCF-7 mammary cell cultures, and in JAr choriocarcinoma cells. Recent investigations have focused on the use of mechanism-based inhibitors, such as 7-substituted 1,4-androstadienediones, to biochemically probe the active site of aromatase. Inhibition kinetics were determined under initial velocity conditions using purified human placental cytochrome P450_arom protein in a reconstituted system. Derivatives of 1,4-androstadiene-3,17-dione and 1,4,6-androstatriene-3,17-dione exhibited high affinity in the purified enzyme system. 7-(4′-Amino)phenylthio-1,4-androstadiene-3,17-dione, abbreviated 7-APTADD, demonstrated rapid time-dependent, first-order inactivation of reconstituted aromatase activity only in the presence of NADPH. The apparent K_inact for 7-APTADD is 11.8 nM, the first-order rate of inactivation is 2.72 × 10⁻³ sec⁻¹, and the half-time of inactivation at infinite inhibitor concentration is 4.25 min. The values for the rate constant and half-time of inactivation are similar to those observed in the placental microsomal assay system. Further studies were performed with radioiodinated 7-(4′-iodo)phenylthio-1,4-androstadienedione, 7-IPTADD, and the reconstituted aromatase system. Incubations with [¹²⁵I]7-IPTADD were followed by protein precipitation, solvent extraction, and column chromatography. Analysis of the isolated cytochrome P450_arom by gel elctrophoresis and autoradiography demonstrated the presence of only one radioactive band, which corresponded to the protein staining band for cytochrome P450_arom. HPLC radiochromatographic analysis of the isolated cytochrome P50_arom confirmed the presence of only one radioactive peak coeluting with the u.v. peak for cytochrome P50_arom. Peptide mapping analysis by reverse-phase HPLC of digested inhibitor-cytochrome P450_arom complex demonstrates that the radioactive inhibitor is covalently bound to a lipophilic fragment. In summary, these inhibitors produced enzyme-catalyzed inactivation of reconstituted aromatase activity, and radioiodinated 7-IPTAPP binds covalently to the cytochrome P450_arom.     

4-Thioflavins as active site probes of flavoproteins. General properties

4-Thioflavins (oxygen at position 4 replaced by sulfur) have been studied as potential active site probes of flavoproteins. They react readily with thiol reagents, with large spectral changes, which should be useful for testing the accessibility of the flavin 4-position in flavoproteins. They have an oxidation-reduction potential at pH 7 of -0.055 V, approximately 0.15 V higher than that of native flavins. The spectral characteristics in the fully reduced state show two clear absorption bands, dependent on the ionization state (pK = 4.5). The lowest energy band of the neutral dihydroflavin has a maximum at approximately 485 nm while that of the anion is approximately 425 nm. This should be useful in defining the ionization state of the reduced flavin in flavoproteins. The spectral characteristics of the semiquinoid forms of 4-thioflavins have been determined bound to the apoproteins of flavodoxin and D-amino acid oxidase. The neutral radical has an absorption maximum at 730 nm, while the anion radical has an unusually sharp peak at 415 nm. The reduced forms of 4-thioflavins, free and enzyme bound, react with O2 to regenerate oxidized 4-thioflavin. Reduced 4-thio-FAD p-hydroxybenzoate hydroxylase, however, in its reaction with O2, undergoes a substantial conversion to the native FAD-enzyme. 4-Thioflavins are unusually susceptible to attack by nucleophiles such as hydroxylamine and amines to form the respective 4-hydroxyimino- and 4-aminoflavins, offering the possibility of forming stable covalent flavin-protein linkages with suitably positioned protein residues. Thiols also react with 4-thioflavins, promoting their conversion to the normal (4-oxo) flavin coenzymes. Such reactivity has been found with the apoenzymes of glucose oxidase and lactate oxidase, providing evidence for a thiol residue in the active site of these enzymes.     

Human glycinamide ribonucleotide transformylase: active site mutants as mechanistic probes

Human glycinamide ribonucleotide transformylase (GART) (EC 2.1.2.2) is a validated target for cancer chemotherapy, but mechanistic studies of this therapeutically important enzyme are limited. Site-directed mutagenesis, initial velocity studies, pH-rate studies, and substrate binding studies have been employed to probe the role of the strictly conserved active site residues, N106, H108, and D144, and the semiconserved K170 in substrate binding and catalysis. Only two conservative substitutions, N106Q and K170R, resulted in catalytically active enzymes, and these active mutant enzymes gave pH-rate profiles and a steady-state kinetic mechanism essentially identical to those of the native enzyme. All inactive mutants were able to bind both substrates, ruling out disrupted formation of the ternary complex as the source of inactivity. Differences between human and Escherichia coli GART, previously used as a model for the human enzyme, were evident.     

4-Thioflavins as active site probes of flavoproteins. Reactions with sulfite

4-Thioflavins react with sulfite under aerobic conditions to yield highly fluorescent products with absorption maxima around 410 nm. These products have been identified as 4-hydroxy-4-sulfonylflavins, and have been shown to arise from a series of reactions following the O2-dependent reoxidation of an intermediate with absorption maxima at 363 and 465 nm. Under anaerobic conditions, the same intermediate is formed, but decays to a 350 nm absorbing species, which is probably the N(5)-sulfite adduct of 4-thioflavin. A plausible mechanism is described for the formation of the derivatives, and several of their chemical and physical properties are described. Distinctly different results between different proteins are obtained when sulfite reacts with enzyme-bound 4-thioflavins. 4-Thio-FAD-D-amino acid oxidase and 4-thio-FMN-lactate oxidase react rapidly to yield the N(5)-sulfite adducts, as occurs with the native enzymes. 4-Thio-FAD-p-hydroxybenzoate hydroxylase reacts slowly in a manner paralleling the reaction with the free 4-thioflavins.     

Serine transhydroxymethylase. Equilibrium binding of folate analogs as active site probes

Formation of a quinoid-like structure within the glycyl-pyridoxal phosphate moiety of serine transhydroxymethylase (5,10-methylenetetrahydrofolate: glycine hydroxymethyltransferase, EC 2.1.2.1) is dependent upon the dissociation of the 2-S hydrogen of glycine which in turn requires the presence of tetrahydrofolate or analogs thereof. Equilibrium binding studies with the series folate, dihydrofolate, and tetrahydrofolate showed that reduction of the pteridine ring enhances both quinoid formation and binding. A 5,8-deazafolate series showed that modifications in the 4 position, 10 position and the glutamyl position yield interrelated alterations of quinoid formation which could not be correlated with binding.     

Tetracyanonickelate probes the active site of sulfur-free rhodanese

Tetracyanonickelate (Ni(CN)4(2-)) was used as a probe for the active site of sulfur-free rhodanese (E) in physical and kinetic studies. Ni(CN)4(2-) quenches the intrinsic fluorescence as well as the fluorescence of enzyme-bound 2-anilinonaphthalene-8-sulfonic acid (2,8-ANS), an inhibitor that is competitive with respect to thiosulfate. A facile binding method based on centrifugation was developed to study Ni(CN)4(2-) binding to E. Binding studies performed using either of the electrophoretic variants A and B, fractionated by DE52 column chromatography, showed one high affinity Ni(CN)4(2-)-binding site in each species and additional weak sites on the more electropositive form A. The high affinity Ni(CN)4(2-) binding was corroborated by ultrafiltration binding (Kd = 3.95 +/- 0.35 microM), titration of intrinsic fluorescence (Kd = 1.8 +/- 0.11 microM), and displacement of enzyme-bound 2,8-ANS (Kd = 1.9 +/- 1.1 microM). A nonlinear least squares analysis of kinetic data collected under conditions used for the binding studies gave a Ni(CN)4(2-) inhibition constant of 21 microM. It is concluded that Ni(CN)4(2-) binds to sulfur-free rhodanese in solution near the active site as has been shown in x-ray crystal studies (Lijk, L. J., Kalk, K. H., Brandenburger, N. P., and Hol, W. G. J. (1983) Biochemistry 22, 2952-2957). In keeping with recent suggestions that the conformational state of the enzyme is dynamically determined, the discrepancy between Ni(CN)4(2-) affinity as determined by physical methods and that by kinetic methods suggests that Ni(CN)4(2-) may be able to distinguish the conformation of the working enzyme from those of the idle forms.     

Aminoalcohols as probes of the two-subsite active site of beta-D-xylosidase from Selenomonas ruminantium

Catalysis and inhibitor binding by the GH43 beta-xylosidase are governed by the protonation states of catalytic base (D14, pK(a) 5.0) and catalytic acid (E186, pK(a) 7.2) which reside in subsite -1 of the two-subsite active site. Cationic aminoalcohols are shown to bind exclusively to subsite -1 of the catalytically-inactive, dianionic enzyme (D14(-)E186(-)). Enzyme (E) and aminoalcohols (A) form E-A with the affinity progression: triethanolamine>diethanolamine>ethanolamine. E186A mutation raises the K(i)(triethanolamine) 1000-fold. By occupying subsite -1 with aminoalcohols, affinity of monosaccharide inhibitors (I) for subsite +1 is demonstrated. The single access route for ligands into the active site dictates ordered formation of E-A followed by E-A-I. E-A-I forms with the affinity progression: ethanolamine>diethanolamine>triethanolamine. The latter affinity progression is seen in formation of E-A-substrate complexes with substrate 4-nitrophenyl-beta-d-xylopyranoside (4NPX). Inhibition patterns of aminoalcohols versus 4NPX appear competitive, noncompetitive, and uncompetitive depending on the strength of E-A-4NPX. E-A-substrate complexes form weakly with substrate 4-nitrophenyl-alpha-l-arabinofuranoside (4NPA), and inhibition patterns appear competitive. Biphasic inhibition by triethanolamine reveals minor (<0.03%) contamination of E186A preparations (including a His-Tagged form) by wild-type-like enzyme, likely originating from translational misreading. Aminoalcohols are useful in probing glycoside hydrolases; they cause artifacts when used unwarily as buffer components.     

Characterization of active and inactive forms of the phenol hydroxylase stimulatory protein DmpM.

The stimulatory protein DmpM of phenol hydroxylase from methylphenol-degrading Pseudomonas sp. strain CF600 has been found to exist in two forms. DmpM purified from the native strain was mostly active in stimulating phenol hydroxylase activity, whereas an inactive form accumulated in a recombinant strain. Both forms exhibited a molecular mass of 10 361.3 +/- 1.3 Da by electrospray mass spectrometry, but nondenaturing gel filtration showed molecular masses of 31 600 Da for the inactive form and 11 500 Da for the active form. Cross-linking and sedimentation velocity results were consistent with the inactive form being a dimer. Partial thermal or chemical denaturation, or treatment with trifluoroethanol, readily activated dimeric DmpM. A combination of circular dichroism and fluorescence spectroscopies, activity assays, and native and urea gel electrophoresis were used to further characterize reactivation with urea. These results showed that dissociation of the dimeric form of DmpM precedes denaturation at low protein concentrations and results in activation. The same concentration of urea that effects dissociation also converts the monomeric form to a different conformation.     

Characterization of active site variants of xanthine hydroxylase from Aspergillus nidulans

Xanthine/α-ketoglutarate (αKG) dioxygenase (XanA) is a non-heme mononuclear Fe^II enzyme that decarboxylates αKG to succinate and CO₂ while hydroxylating xanthine to generate uric acid. In the absence of a XanA crystal structure, a homology model was used to target several putative active site residues for mutagenesis. Wild-type XanA and ten enzyme variants were purified from recombinant Escherichia coli cells and characterized. The H149A and D151A variants were inactive and the H340A variant exhibited only 0.17% of the wild-type enzyme activity, consistent with the proposed role of His149, Asp151, and His340 as Fe ligands. The K122A variant led to a 2-fold increase in the K_d of αKG as measured by fluorescence quenching analysis, in agreement with Lys122 acting to stabilize the binding of αKG. The N358A variant exhibited a 23-fold decrease in k_cat/K_m compared to wild-type XanA, pointing to a key role of Asn358 in catalysis. 9-Methylxanthine was exploited as an alternate substrate, and the C357A, E137A, and D138A variants were found to exhibit relatively enhanced activity consistent with Cys357, Glu137, and Asp138 being proximal to N-9 or involved in its proper positioning. 6,8-Dihydroxypurine was identified as a slow-binding competitive inhibitor of XanA, and significant decreases (E137A and D138A) or increases (Q356A and N358A) in of the variants were interpreted in terms of distinct interactions between this compound and the corresponding active site side chains. Further support for Cys357 residing at the active site was obtained using thiol-specific reagents that inactivated wild-type enzyme (with partial protection by substrate), whereas the C357A variant was resistant to these reagents. The Q101A, Q356A, and C357A variants showed elevated ferroxidase activity in the absence of substrates, pointing to the presence of the corresponding side chains at the active site. These results confirm most aspects of the homology model and provide additional insight into the enzyme reactivity.     

Detection of allosteric kinase inhibitors by displacement of active site probes

Non-adenosine triphosphate (ATP) competitive, allosteric inhibitors provide a promising avenue to develop highly selective small-molecule kinase inhibitors. Although this class of compounds is growing, detection of such inhibitors can be challenging as standard kinase activity assays preferentially detect compounds that bind to active kinases in an ATP competitive manner. We have previously described a time-resolved fluorescence resonance energy transfer (TR-FRET)-based kinase binding assay using the competitive displacement of ATP competitive active site fluorescent probes ("tracers"). Although this format has gained acceptance, published data with this and related formats are almost entirely without examples of non-ATP competitive compounds. Thus, this study addresses whether this format is useful for non-ATP competitive inhibitors. To this end, 15 commercially available non-ATP competitive inhibitors were tested for their ability to displace ATP competitive probes. Despite the diversity of both compound structures and their respective targets, 14 of the 15 compounds displaced the tracers with IC(50) values comparable to literature values. We conclude that such binding assays are well suited for the study of non-ATP competitive inhibitors. In addition, we demonstrate that allosteric inhibitors of BCR-Abl and MEK bind preferentially to the nonphosphorylated (i.e., inactive) form of the kinase, indicating that binding assays may be a preferred format in some cases.     

Site-directed mutants of charged residues in the active site of tyrosine hydroxylase

The active site of tyrosine hydroxylase consists of a hydrophobic cleft with an iron atom near the bottom. Within the cleft are several charged residues which are conserved across the family of pterin-dependent hydroxylases. We have studied four of these residues, glutamates 326 and 332, aspartate 328, and arginine 316 in tyrosine hydroxylase, by site-directed substitution with alternate amino acid residues. Replacement of arginine 316 with lysine results in a protein with a Ktyr value that is at least 400-fold greater and a V/Ktyr value that is 4000-fold lower than those found in the wild-type enzyme; substitution with alanine, serine, or glutamine yields insoluble enzyme. Arginine 316 is therefore critical for the binding of tyrosine. Replacement of glutamate 326 with alanine has no effect on the KM value for tyrosine and results in a 2-fold increase in the KM value for tetrahydropterin. The Vmax for DOPA production is reduced 9-fold, and the Vmax for dihydropterin formation is reduced 4-fold. These data suggest that glutamate 326 is not directly involved in catalysis. Replacement of aspartate 328 with serine results in a 26-fold higher KM value for tyrosine, a 8-fold lower Vmax for dihydropterin formation, and a 13-fold lower Vmax for DOPA formation. These data suggest that aspartate 328 has a role in tyrosine binding. Replacement of glutamate 332 with alanine results in a 10-fold higher KM value for 6-methyltetrahydropterin with no change in the KM value for tyrosine, a 125-fold lower Vmax for DOPA formation, and an only 3.3-fold lower Vmax for tetrahydropterin oxidation. These data suggest that glutamate 332 is required for productive tetrahydropterin binding.     

Chromophoric cinnamic acid substrates as resonance Raman probes of the active site environment in native and unfolded alpha-chymotrypsin

Chromophoric [4-(dimethylamino)cinnamoyl]imidazole reacts with the serine protease alpha-chymotrypsin to form an acyl enzyme. At pHs below 4.0, the acyl enzyme turns over very slowly to yield the free acid. During this slow deacylation it is possible to obtain a very good resonance Raman spectrum of the acyl intermediate by using the 350.7-nm line of the krypton laser. The resonance Raman carbonyl frequency of the covalently bonded substrate and its wavelength at maximum intensity in the absorption spectrum of the acyl enzyme have been taken and used to monitor the active site environment. A comparison has been made of the absorption and Raman spectra of the acyl enzyme and those of the corresponding chromophoric methyl ester, aldehyde, and imidazole model compounds. A linear correlation is found between the wavelength of maximum absorption and the Raman frequency of the carbonyl group over a wide range of solvent conditions for each of the model compounds. By combining the Raman carbonyl frequency with the absorption maximum, we can determine that the bond order changes in the carbonyl bond of the bound substrate are not due to changes in the solvent, since the carbonyl frequency and the absorption maximum of the acyl enzyme do not fall on any of the linear correlations for the model compounds. The unusual spectroscopic properties of the bound substrate appear to be due to some specific enzyme-induced change in the substrate when it is bound at the active site. Thermal unfolding of the acyl enzymes changes both the carbonyl frequency of the acyl enzyme and its absorption maximum to completely different values.(ABSTRACT TRUNCATED AT 250 WORDS)     

6-Thiocyanatoflavins and 6-mercaptoflavins as active-site probes of flavoproteins

6-Thiocyanatoflavins have been found to be susceptible to nucleophilic displacement reactions with sulfite and thiols, yielding respectively the 6-S-SO3--flavin and 6-mercaptoflavin, with rate constants at pH 7.0, 20 degrees C, of 55 M-1 min-1 for sulfite and 1000 M-1 min-1 for dithiothreitol. The 6-SCN-flavin binds tightly to riboflavin-binding protein as the riboflavin derivative, to apoflavodoxin, apo-lactate oxidase, and apo-Old Yellow Enzyme as the FMN derivative, and to apo-D-amino acid oxidase as the FAD derivative. The riboflavin-binding protein derivative is inaccessible to dithiothreitol attack, and the lactate oxidase and D-amino acid oxidase derivatives show only limited accessibility. However, the flavodoxin and Old Yellow Enzyme derivatives react readily with dithiothreitol, indicating that the flavin 6-position is exposed to solvent in these proteins. The lactate oxidase and D-amino acid oxidase derivatives convert slowly but spontaneously to the 6-mercaptoflavin enzyme forms in the absence of any added thiol, indicating the presence of a thiol residue in the flavin binding site of these proteins. The reaction rates have been investigated of 6-mercaptoflavins with iodoacetamide, N-ethylmaleimide, methyl methanethiosulfonate, H2O2, and m-chloroperbenzoate, in both the free and protein-bound state. The results confirm the conclusions drawn from the studies with 6-SCN-flavins described above and from 6-N3-flavins [Massey, V., Ghisla, S., & Yagi, K. (1986) Biochemistry (preceding paper in this issue)]. The spectral properties of the protein-bound 6-mercaptoflavin vary widely among the five proteins studied and show stabilization of the neutral flavin with flavodoxin and riboflavin-binding protein and of the anionic species by Old Yellow Enzyme, lactate oxidase, and D-amino acid oxidase. In the case of the latter two enzymes, the stabilization appears to be due to interaction of the negatively charged flavin with a positively charged protein residue located near the flavin pyrimidine ring. This positively charged residue appears to be responsible also for the strong stabilization of the two-electron oxidation state of the mercaptoflavin as the 6-S-oxide. With the other flavoproteins studied this oxidation level is stabilized as the 6-sulfenic acid or 6-sulfenate.     

Poly-L-proline type II peptide mimics as probes of the active site occupancy requirements of cGMP-dependent protein kinase.

Based on the X-ray crystal structure of cAMP-dependent protein kinase (PKA) with the endogenous inhibitor PKI and the X-ray crystal structure of cyclin-dependent kinase 2 (CDK2) with a substrate peptide, a proposal is put forth that some protein kinases bind peptide substrates in their active sites in the poly-L-proline type II (PPII) conformation. In this work, PPII peptide mimics are evaluated as pseudosubstrate inhibitors of cGMP-dependent protein kinase (PKG) to explore if PKG also binds peptide substrates in the PPII conformation. Inhibition data of our PPII mimetics provide evidence that the P-1, P-2, and P-3 residues of substrate peptides bind in the PPII conformation (phi approximately -75 degrees, psi approximately 145 degrees). In addition, the inhibition data also suggest that the P-1, P-2, and P-3 residues in substrate peptides bind with a gauche(-) chi1 angle.