Normal and abnormal heme biosynthesis. Part 7. Synthesis and metabolism of coproporphyrinogen-III analogues with acetate or butyrate side chains on rings C and D. Development of a modified model for the active site of coproporphyrinogen oxidase

Analogues of coproporphyrinogen-III have been prepared with acetate or butyrate groups attached to the C and D pyrrolic subunits. The corresponding porphyrin methyl esters were synthesized by first generating a,c-biladienes by reacting a dipyrrylmethane with pyrrole aldehydes in the presence of HBr. Cyclization with copper(II) chloride in DMF, followed by demetalation with 15% H(2)SO(4)-TFA and reesterification, gave the required porphyrins in excellent yields. Hydrolysis with 25% hydrochloric acid and reduction with sodium-amalgam gave novel diacetate and dibutyrate porphyrinogens 9. Diacetate 9a was incubated with chicken red cell hemolysates (CRH), but gave complex results due to the combined action of two of the enzymes present in these preparations. Separation of uroporphyrinogen decarboxylase (URO-D) from coproporphyrinogen oxidase (CPO) allowed the effects of both enzymes on the diacetate substrate to be assessed. Porphyrinogen 9a proved to be a relatively poor substrate for CPO compared to the natural substrate coproporphyrinogen-III, and only the A ring propionate moiety was processed to a significant extent. Similar results were obtained for incubations of 9a with purified human recombinant CPO. Diacetate 9a was also a substrate for URO-D and a porphyrinogen monoacetate was the major product in this case; however, some conversion of a second acetate unit was also evident. The dibutyrate porphyrinogen 9b was only recognized by the enzyme CPO, but proved to be a modest substrate for incubations with CRH. However, 9b was an excellent substrate for purified human recombinant CPO. The major product for these incubations was a monovinylporphyrinogen, but some divinyl product was also generated in incubations using purified recombinant human CPO. The incubation products were converted into the corresponding porphyrin methyl esters, and these were characterized by proton NMR spectroscopy and mass spectrometry. The results extend our understanding of substrate recognition and catalysis for this intriguing enzyme and have allowed us to extend the active site model for CPO. In addition, the competitive action of both URO-D and CPO on the same diacetate porphyrinogen substrate provides additional perspectives on the potential existence of abnormal pathways for heme biosynthesis.     

Ionizable side chains at catalytic active sites of enzymes

Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1,072 Å³. The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes.     

Racemic and optically active alpha-tocopherol analogues with a modified side chain: synthesis and biological activity

Information on the synthesis and biological activity of natural and synthetic analogues of alpha-tocopherol with a modified side chain is systematized. These compounds are of interest as vitamin E metabolites, hydrophilic antioxidants, and precursors of drugs with combined pharmacological properties useful in therapy of pathological disorders caused by oxidative stress.     

Synthesis of sterols with modified side chains.

Synthesis of sterols with side chain containing from four to nine carbons are described.     

Secondary enzyme-substrate interactions: kinetic evidence for ionic interactions between substrate side chains and the pepsin active site

The possibility that pig pepsin has a cation binding specificity in its secondary binding subsites has been examined by the pepsin-catalyzed hydrolysis of a series of synthetic octa- to undecapeptide substrates. These chromophoric substrates are cleaved by pepsin in the phenylalanyl-p-nitrophenylalanyl (Phe-Nph) bond. Lys and Arg residues were placed into seven different positions in the substrates, and their effect on kcat and Km was examined between pH 2.8 and pH 5.8 (I = 0.1 M, 37 degrees C). Kinetic evidence indicates the existence in the enzyme binding subsites S4, S3, S2, S3', S4', and S5' of a group(s) which become(s) negatively charged at higher pH. For most substrates, the magnitude as well as the pH dependence of kcat was unaffected by the presence of Lys or Arg in these peptides. In contrast, changes up to 5 orders of magnitude were observed for Km, depending on the number of basic residues and on their positions in the sequence. Km for a group of substrates at pH greater than 5.5 was lower than 50 nM. Values for kcat/Km for some substrates exceed the level of 10(8) M-1 s-1. Therefore, the free energy derived from ionic interactions in secondary binding sites influences mostly the binding step on the reaction pathway. This result is in contrast to the previous observations that the length and the hydrophobic character of the substrate residues in some positions influence kcat with little effect on Km toward shorter substrates of pepsin [Fruton, J. (1976) Adv. Enzymol. Relat. Areas Mol. Biol. 44, 1-36].     

Ca2+ binding in the active site of HincII: implications for the catalytic mechanism

The 2.8 A crystal structure of the type II restriction endonuclease HincII bound to Ca(2+) and cognate DNA containing GTCGAC is presented. The DNA is uncleaved, and one calcium ion is bound per active site, in a position previously described as site I in the related blunt cutting type II restriction endonuclease EcoRV [Horton, N. C., Newberry, K. J., and Perona, J. J. (1998) Proc. Natl. Acad. Sci. U.S.A. 95 (23), 13489-13494], as well as that found in other related enzymes. Unlike the site I metal in EcoRV, but similar to that of PvuII, NgoMIV, BamHI, BglII, and BglI, the observed calcium cation is directly ligated to the pro-S(p) oxygen of the scissile phosphate. A calcium ion-ligated water molecule is well positioned to act as the nucleophile in the phosphodiester bond cleavage reaction, and is within hydrogen bonding distance of the conserved active site lysine (Lys 129), as well as the pro-R(p) oxygen of the phosphate group 3' of the scissile phosphate, suggesting possible roles for these groups in the catalytic mechanism. Kinetic data consistent with an important role for the 3'-phosphate group in DNA cleavage by HincII are presented. The previously observed sodium ion [Horton, N. C., Dorner, L. F., and Perona, J. J. (2002) Nat. Struct. Biol. 9, 42-47] persists in the active sites of the Ca(2+)-bound structure; however, kinetic data show little effect on the single-turnover rate of DNA cleavage in the absence of Na(+) ions.     

Slow solvation dynamics at the active site of an enzyme: implications for catalysis

Solvation dynamics at the active site of an enzyme, glutaminyl-tRNA synthetase (GlnRS), was studied using a fluorescence probe, acrylodan, site-specifically attached at cysteine residue C229, near the active site. The picosecond time-dependent fluorescence Stokes shift indicates slow solvation dynamics at the active site of the enzyme, in the absence of any substrate. The solvation dynamics becomes still slower when the substrate (glutamine or tRNA(Gln)) binds to the enzyme. A mutant Y211H-GlnRS was constructed in which the glutamine binding site is disrupted. The mutant Y211H-GlnRS labeled at C229 with acrylodan exhibited significantly different solvent relaxation, thus demonstrating that the slow dynamics is indeed associated with the active site. Implications for catalysis and specificity have been discussed.     

Monomeric sarcosine oxidase: 1. Flavin reactivity and active site binding determinants

Monomeric sarcosine oxidase (MSOX) is an inducible bacterial flavoenzyme that catalyzes the oxidative demethylation of sarcosine (N-methylglycine) and contains covalently bound FAD [8alpha-(S-cysteinyl)FAD]. This paper describes the spectroscopic and thermodynamic properties of MSOX as well as the X-ray crystallographic characterization of three new enzyme.inhibitor complexes. MSOX stabilizes the anionic form of the oxidized flavin (pK(a) = 8.3 versus 10.4 with free FAD), forms a thermodynamically stable flavin radical, and stabilizes the anionic form of the radical (pK(a) < 6 versus pK(a) = 8.3 with free FAD). MSOX forms a covalent flavin.sulfite complex, but there appears to be a significant kinetic barrier against complex formation. Active site binding determinants were probed in thermodynamic studies with various substrate analogues whose binding was found to perturb the flavin absorption spectrum and inhibit MSOX activity. The carboxyl group of sarcosine is essential for binding since none is observed with simple amines. The amino group of sarcosine is not essential, but binding affinity depends on the nature of the substitution (CH(3)XCH(2)CO(2)(-), X = CH(2) < O < S < Se < Te), an effect which has been attributed to differences in the strength of donor-pi interactions. MSOX probably binds the zwitterionic form of sarcosine, as judged by the spectrally similar complexes formed with dimethylthioacetate [(CH(3))(2)S(+)CH(2)CO(2)(-)] and dimethylglycine (K(d) = 20.5 and 17.4 mM, respectively) and by the crystal structure of the latter. The methyl group of sarcosine is not essential but does contribute to binding affinity. The methyl group contribution varied from -3.79 to -0.65 kcal/mol with CH(3)XCH(2)CO(2)(-) depending on the nature of the heteroatom (NH(2)(+) > O > S) and appeared to be inversely correlated with heteroatom electron density. Charge-transfer complexes are formed with MSOX and CH(3)XCH(2)CO(2)(-) when X = S, Se, or Te. An excellent linear correlation is observed between the energy of the charge transfer bands and the one-electron reduction potentials of the ligands. The presence of a sulfur, selenium, or telurium atom identically positioned with respect to the flavin ring is confirmed by X-ray crystallography, although the increased atomic radius of S < Se < Te appears to simultaneously favor an alternate binding position for the heavier atoms. Although L-proline is a poor substrate, aromatic heterocyclic carboxylates containing a five-membered ring and various heteroatoms (X = NH, O, S) are good ligands (K(d, X=NH) = 1.37 mM) and form charge-transfer complexes with MSOX. The energy of the charge-transfer bands (S > O > NH) is linearly correlated with the one-electron ionization potentials of the corresponding heterocyclic rings.     

Phosphatidylcholine liposomes containing cholesterol analogues with side chains of various lengths

The effect of the length of the side chain of sterols on their interaction with phosphatidylcholine was studied by measuring the permeability properties of liposomes constituted with sterol analogues with side chains of various lengths. The sensitivities of liposomes constituted with these sterol analogues toward digitonin and polyene antibiotics were also examined.The effects of sterols on phase transition of phosphatidylcholine were examined by measuring their effects on permeability increase due to perturbation of phase equilibrium and by differential scanning calorimetry. An analogue with a short side chain, isopropyl (C-22), had a very similar effect to cholesterol in suppressing the permeability increase, suggesting that the full length of the side chain is not necessary for this effect.The permeability of egg yolk phosphatidylcholine at 42°C was suppressed as much by the analogue C-22 as by cholesterol. Androstene-3-β-ol, an analogue without a side chain, however, had little suppressive effect. Thus it is concluded that the condensing effect of sterol requires a side chain, but not the full length of side chain.Liposomes constituted with analogues having a side chain with more than 5 carbon atoms showed maximum reactivity with a polyene antibiotic, amphotericin B, whereas those constituted with analogues having a side chain with less than 4 carbon atoms showed weaker reactivity. These findings indicate that a side chain with more than 5 carbon atoms is essential for the maximum interaction of liposomes with amphotericin B. Unlike amphotericin B, filipin reacted almost equally well with liposomes containing C-22 and with those containing cholesterol. Thus the chain length of the side chain of sterol is less important for interaction of liposomes with filipin than for their interaction with amphotericin B.Liposomes containing analogues having a side chain with more than 6 carbon atoms showed maximum reactivity with digitonin. Thus for the maximum interaction of liposomes with digitonin, the side chain of sterol should be longer than 6 carbon atoms.     

Oxidative chemistry in the GFP active site leads to covalent cross-linking of a modified leucine side chain with a histidine imidazole: implications for the mechanism of chromophore formation

The mechanism of chromophore biosynthesis in green fluorescent protein (GFP) is triggered by a spontaneous main chain cyclization reaction of residues 65-67. Here, we demonstrate that the initially colorless Y66L variant, designed to trap chromophore precursor states, is oxidatively modified to generate yellow chromophores that absorb at 412 and 374 nm. High- and low-pH crystal structures determined to 2.0 and 1.5 A resolution, respectively, are consistent with pi-orbital conjugation of a planar Leu66-derived adduct with the imidazolinone ring, which is approximately 90 and 100% dehydrated, respectively. Time-, base-, and oxygen-dependent optical properties suggest that the yellow chromophores are generated from a 338 nm-absorbing intermediate, interpreted to be the Y66L analogue of the wild-type GFP chromophore. Generation of this species is catalyzed by a general base such as formate, and proceeds via a cyclization-oxidation-dehydration mechanism. The data suggest that a hydration-dehydration equilibrium exists in the cyclic form of the peptide, and that dehydration is favored upon extensive conjugation with the modified side chain. We conclude that the mechanism of GFP chromophore biosynthesis is not driven by the aromatic character of residue 66. In the low-pH X-ray structure, a highly unusual cross-link is observed between His148 and the oxidized Leu66 side chain, suggesting a conjugate addition reaction of the imidazole nitrogen to the highly electrophilic diene group of the yellow chromophore. The reactivity described here further expands the chemical diversity observed in the active site of GFP-like proteins, and may allow for covalent attachment of functional groups to the protein scaffold for catalytic purposes.     

Symmetry at the active site of the ribosome: structural and functional implications

The sizable symmetrical region, comprising 180 ribosomal RNA nucleotides, which has been identified in and around the peptidyl transferase center (PTC) in crystal structures of eubacterial and archaeal large ribosomal subunits, indicates its universality, confirms that the ribosome is a ribozyme and evokes the suggestion that the PTC evolved by gene fusion. The symmetrical region can act as a center that coordinates amino acid polymerization by transferring intra-ribosomal signals between remote functional locations, as it connects, directly or through its extensions, the PTC, the three tRNA sites, the tunnel entrance, and the regions hosting elongation factors. Significant deviations from the overall symmetry stabilize the entire region and can be correlated with the shaping and guiding of the motion of the tRNA 3'-end from the A- into the P-site. The linkage between the elaborate PTC architecture and the spatial arrangements of the tRNA 3'-ends revealed the rotatory mechanism that integrates peptide bond formation, translocation within the PTC and nascent protein entrance into the exit tunnel. The positional catalysis exerted by the ribosome places the reactants in stereochemistry close to the intermediate state and facilitates the catalytic contribution of the P-site tRNA 2'-hydroxyl.     

Probing the transducin nucleotide binding site with GDP analogues

An affinity study between the G protein of the visual photoreceptor, transducin, and eight different non-hydrolyzable GDP analogues is described. Imidodiphosphate derivatives have been shown to exhibit good affinities to transducin. This very important heterotrimeric G protein is shown to be highly restrictive with regard to structural modifications of the nucleotide at the pyrophosphate moiety, at the 3' position on ribose, as well as at the N1 position of the purine.     

Racemic and optically active tocopherol analogues with a modified side chain: Synthesis and biological activity (Review)

Information on the synthesis and biological activity of natural and synthetic analogues of α-tocopherol with a modified side chain is systematized. These compounds are of interest as vitamin E metabolites, hydrophilic antioxidants, and precursors of drugs with combined pharmacological properties useful in therapy of pathological disorders caused by oxidative stress.     

Burst kinetics and redox transformations of the active site manganese ion in oxalate oxidase: implications for the catalytic mechanism

Oxalate oxidase (EC 1.2.3.4) catalyzes the oxidative cleavage of oxalate to carbon dioxide and hydrogen peroxide. In this study, unusual nonstoichiometric burst kinetics of the steady state reaction were observed and analyzed in detail, revealing that a reversible inactivation process occurs during turnover, associated with a slow isomerization of the substrate complex. We have investigated the underlying molecular mechanism of this kinetic behavior by preparing recombinant barley oxalate oxidase in three distinct oxidation states (Mn(II), Mn(III), and Mn(IV)) and producing a nonglycosylated variant for detailed biochemical and spectroscopic characterization. Surprisingly, the fully reduced Mn(II) form, which represents the majority of the as-isolated native enzyme, lacks oxalate oxidase activity, but the activity is restored by oxidation of the metal center to either Mn(III) or Mn(IV) forms. All three oxidation states appear to interconvert under turnover conditions, and the steady state activity of the enzyme is determined by a balance between activation and inactivation processes. In O(2)-saturated buffer, a turnover-based redox modification of the enzyme forms a novel superoxidized mononuclear Mn(IV) biological complex. An oxalate activation role for the catalytic metal ion is proposed based on these results.     

The aromatase active site: the C-6 "front" side of the androgen molecule is required for binding

Solubilized microsomes from human placenta were partially purified by anion-exchange chromatography on a quaternary methylamino-silica column. The aromatase-active fractions were then used for measuring optical difference spectra with various androgens, substituted at positions C-1, 2, 6, 11, 16, 17, and 19. Of these, only androgens substituted at the C-6, or frontside of the steroid backbone induce a strong type I spectrum, as well as competitive inhibition with the natural substrate, 4-androstene-3,17-dione. An in-house made affinity support, synthesized by reacting 6 beta-bromo-androstenedione with aminohexyl-Sepharose at pH 10-11 via a SN2-mechanism, was unable to trap the aromatase cytochrome P-450 component of the enzyme. A 3-dimensional model of the active site accounting for these interactions is proposed.     

DNA binding and anticancer activity of naphthalimides with 4-hydroxyl-alkylamine side chains at different lengths

A series of novel naphthalimide derivatives modified with various hydroxyl-alkylamines at 4-position have been synthesized. Their DNA binding properties were investigated by UV-Vis, fluoescence, and circular dichroism (CD) spectroscopies and thermal denaturation. The results showed that compounds 3a-e as the DNA intercalator exhibited middle binding affinities with Ct-DNA. The anticancer activities of 3a-e were preliminarily evaluated, compounds 3c and 3e exhibited potent anticancer activities against Bel-7402 cell line with IC(50) values of 5.57 and 9.17μM, respectively. More interestingly, enhancement of the fluorescence emission was found in the complexes of 3a-e with Ct-DNA, especially for 3c. This would make these compounds as potential DNA staining agents.     

Metal binding to angiotensin converting enzyme: implications for the metal binding site

Angiotensin converting enzyme interacts with the chelator, 1,10-phenanthroline (OP) to form an OP-Zn-ACE ternary complex, which subsequently dissociates to OP-Zn and apoenzyme. The association and dissociation rate constants for the reaction OP + Zn-ACE in equilibrium OP-Zn-ACE have been determined and compared with those of known OP-metal complexes. Such constants were also used to calculate the rate constant for formation of the OP-Zn complex from OP-Zn-ACE. The rate of dissociation of zinc from ACE has been measured in the presence of EDTA (which acts only as a metal scavenger) as a function of chelator concentration, at different pH values, and with different buffers. The stability constant for the binding of zinc to apoACE log Kc = 8.2, determined by equilibrium dialysis using atomic absorption spectroscopy to assess metal concentration, is much smaller than that for Zn-carboxypeptidase A. Zn-thermolysin, or Zn-carbonic anhydrase. This weak binding is attributable to the zinc dissociation rate constant of ACE, 7.5 X 10(-3) sec-1 at pH 7.0, which is much greater than that of the other zinc metalloenzymes. These results lead to inferences regarding the metal binding site of ACE.     

Metabolism of pentacarboxylate porphyrinogens by highly purified human coproporphyrinogen oxidase: further evidence for the existence of an abnormal pathway for heme biosynthesis

An abnormal series of porphyrin tetracarboxylic acids known as the isocoproporphyrins, are commonly excreted by patients suffering from the disease porphyria cutanea tarda (PCT). These porphyrins appear to arise by bacterial degradation of dehydroisocoproporphyrinogen that is generated by the premature metabolism of the normal pentacarboxylate intermediate (5dab) by coproporphyrinogen oxidase (copro'gen oxidase). This porphyrinogen can be further metabolized by uroporphyrinogen decarboxylase to give harderoporphyrinogen, one of the usual intermediates in heme biosynthesis. Therefore, it is possible that some of the heme formed under abnormal conditions may originate from the 'isocopro-type' porphyrinogen intermediate. In order to investigate the feasibility of alternative pathways for heme biosynthesis, the four type III pentacarboxylate isomeric porphyrinogens were incubated with purified, cloned human copro'gen oxidase at 37 degrees C with various substrate concentrations under initial velocity conditions. Of the four isomers, only 5dab was a substrate for copro'gen oxidase and this gave dehydroisocoproporphyrin. The structure of the related porphyrin tetramethyl ester was confirmed by proton NMR spectroscopy and mass spectrometry. The K(m) value for proto'gen-IX formation from copro'gen, an indicator of molecular recognition, was similar to the K(m) value for monovinyl product formation with 5dab, although copro'gen-III has an approximately twofold higher K(cat) value. Although 5dab is a slightly poorer substrate than copro'gen-III, these results support the hypothesis that an abnormal route for heme biosynthesis is possible in humans suffering from PCT or related syndromes such as hexachlorobenzene poisoning.