Isosteric Substitution Research Articles

Probing the Structure of a Putative Intermediate in Homologous Recombination: The Third Strand in the Parallel DNA Triplex is in Contact with the Major Groove of the Duplex

A three-stranded DNA that is a putative intermediate of homologous recombination is a novel DNA triplex,R-form DNA. InR- form DNA the third strand includes both purines and pyrimidines and is parallel to the identical strand of the duplex. To text and refine our previously proposedR-form base triplets we have used two approaches: (1) dimethyl sulfate protection ofR-form DNA; and (2) thermal dissociation ofR-form DNAs in which the duplex strands were substituted in a strand-specific manner with either 7-deaza-guanine or 7-deaza-adenine. Together, the footprinting and isosteric substitution results demonstrate that the third strand inR-form DNA is in contact with the purines in the N7position in the major groove of the Watson–Crick duplex in three ((GC):G, (AT):A and (TA):T out of the four possile triplets. Furthermore, these reults suggest that the N7positions of the duplex play a significant role in stabilizing the DNA–DNA contacts during the homology recognition process.

Evidence for the interaction of valine-10 in cystatin C with the S2 subsite of cathepsin B.

The interactions between wild-type or mutant recombinant forms of human cystatin C and rat cathepsin B were characterized by measuring progress curves for substrate hydrolysis in the presence of inhibitor. The investigation was guided by the use of computer modeling and explores the possibility that amino acid residues in the N-terminal region of cystatin C interact with substrate-binding regions in the target enzyme. With cystatin C that has Val-10 replaced by an Arg residue (Val10Arg cystatin C), the inhibition constant, K(i), increased 31-fold if the isosteric substitution Glu-245 to Gln was made in cathepsin B. When the wild-type form of the inhibitor was used, the corresponding effect on K(i) was less than 2-fold. In a similar study, using cathepsin B in which the substitution to Gln is instead at Glu-171, no such difference in how K(i) is affected was observed. Both Glu-245 and Glu-171 are located in the S2 subsite of cathepsin B. The observed effects on K(i) indicate that the additional positive charge introduced in Val10Arg cystatin C is interacting with the negative charge on Glu-245 in cathepsin B when these two proteins form a complex; the cystatin variant is thus binding in a substratelike manner with this region of the enzyme. Indirectly, these results suggest that when native cystatin C and cathepsin B form a complex, Val-10 in the inhibitor interacts with the S2 subsite of the enzyme. A K(i) value of 0.13 nM was obtained for the interaction of Val10Arg cystatin C with papain.(ABSTRACT TRUNCATED AT 250 WORDS)

Two-dimensional crystallization of Escherichia coli-expressed bacteriorhodopsin and its D96N variant: high resolution structural studies in projection

Highly ordered two-dimensional (2-D) crystals of Escherichia coli-expressed bacteriorhodopsin analog (e-bR) and its D96N variant (e-D96N) reconstituted in Halobacterium halobium lipids have been obtained by starting with the opsin protein purified in the denaturing detergent sodium dodecyl sulfate. These crystals embedded in glucose show electron diffraction in projection to better than 3.0 A at room temperature. This is the first instance that expressed bR or a variant has been crystallized in 2-D arrays showing such high order. The crystal lattice is homologous to that in wild-type bR (w-bR) in purple membranes (PM) and permit high resolution analyses of the structure of the functionally impaired D96N variant. The e-bR crystal is isomorphous to that in PM with an overall averaged fractional change of 12.7% (26-3.6-A resolution) in the projection structure factors. The projection difference Fourier map e-bR-PM at 3.6-A resolution indicates small conformational changes equivalent to movement of approximately < 7 C-atoms distributed within and in the neighborhood of the protein envelope. This result shows that relative to w-bR there are no global structural rearrangements in e-bR at this 3.6 A resolution level. The e-D96N crystal is isomorphous to the e-bR crystal with a smaller (9.2%) overall averaged fractional change in the structure factors. The significant structural differences between e-D96N and e-bR are concentrated at high resolution (5-3.6 A); however, these changes are small as quantified from the 3.6 A resolution e-D96N-e-bR Fourier difference map. The difference map showed no statistically significant peaks or valleys within 5 A in projection from the site of D96 substitution on helix C. Elsewhere within the protein envelope the integrated measure of peaks or valleys was < approximately 3 C-atom equivalents. Thus, our results show that for the isosteric substitution of Asp96 by Asn, the molecular conformation of bR in its ground state is essentially unaltered. Therefore, the known effect of D96N on the slowed M412 decay is not due to ground-state structural perturbations.

Differential scanning calorimetry studies of phosphonolipid analogs of lung surfactant glycerophospholipids

A series of synthetic analogs of naturally occurring glycerophospholipids has been studied for gel-to-liquid transition temperature ( T c) by using differential scanning calorimetry. The compounds investigated were dihexadecyl phosphatidylcholine (DHPC) plus 11 newly synthesized phosphonolipids. Six were racemates and have ether-ether fatty chains and 5 were chiral (R) with ether-amide fatty chains bonded to the glycerol moiety; each series also contained a methylene isosteric substitution for one of the oxygen atoms in naturally occurring glycerophospholipid phosphate esters. All analogs have fatty chains of equivalent length in saturated carbon atoms C 2C 16, but varied in N-headgroup structures. The thermal behaviors of these novel molecules were found to depend upon the degree of alkylation of the headgroups and upon whether the hydrocarbon chains are bonded as ethers or amides. The ether-ether (diether) phosphonolipid (DEPN) analogs of dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylethanolamine (DPPE) had increased gel-to-liquid crystal transition temperatures ( T c) compared to their naturally occurring (fatty acyl ester) glycerophospholipid counterparts. In general, DEPN analogs had T c values 3.7−5.8°C higher than ether-amide phosophonolipid (EAPN) compounds of equivalent headgroup, although EAPN-13 was an exception with the highest T c of any compound studied. In both the DEPN and EAPN series, analogs with hydrogen atom(s) covalently bonded to the headgroup nitrogen atom showed higher T c values, possibly related to their increased potential for hydrogen bonding. Interfacial property studies (adsorption, Wilhelmy balance, oscillating bubble) showed that several analogs with higher transitions than DPPC, the principal lung surfactant glycerophospholipid, had improved adsorption and respreading, with equivalent surface tension lowering in compressed spread films on the balance and with minimum surface tensions <1 mN/m in pulsated dispersions on the bubble. Surface activity did not correlate simply with T c and results suggested that other factors, such as molecular shape (wedge shaped as in phosphatidylethanolamine, PE) or bilayer structure (interdigitated as in DHPC) may be important in understanding dynamic respreading differences in interfacial films compressed dynamically past collapse.

Recognition of substrates by tyrosylprotein sulfotransferase. Determination of affinity by acidic amino acids near the target sites.

The sulfation of proteins by tyrosylprotein sulfotransferase (TPST) is highly site-specific. In this study, we examined the sequence specificity of the target site for TPST by determining the kinetics of rat liver TPST with peptides related to the sequence of the C4 component of complement. The data obtained from this study demonstrate that selective elimination of negative charges from the -5 to +5 region of the acceptor tyrosine, either by removal or by isosteric substitution or the acidic amino acids in the region, produced a substantial change in the Km value, with relatively little effect on Vmax. Substitutions at -1 and +1 positions increase the Km value by 22- and 4-fold, respectively, whereas removal of the acidic amino acids from the -5, -4, or +4 positions increased the Km values by a factor of 2-4. The effect of elimination of an acidic amino acid on the Km value was constant and specific for its particular position in relation to tyrosine, and the effect of modification of more than one amino acid was multiplicative. This study provides evidence that: 1) acidic residues near tyrosines promote sulfation by increasing the affinity of enzyme-substrate binding and have little effect on catalytic rate; 2) the contribution of each acidic residue to affinity for TPST is independent and varies according to position relative to the acceptor tyrosine; and 3) the enzyme interacts with a segment of at least 4-5 residues on each side of the tyrosine, with the residues on the -1 and +1 positions being the most important determinants. In general, residues on the NH2-terminal side of the tyrosine have a greater effect on affinity for TPST.

A unified approach to systematic isosteric substitution for acidic groups and application to NMDA antagonists related to 2-amino-7-phosphonoheptanoate

A systematic approach to the replacement of acidic groups with potential bioisosteres is described. The strategy involves simple nucleophilic displacement of a common alkyl halide precursor with a variety of mercaptoazoles and related molecules. The mercaptoazoles and their oxidized derivatives (sulfinyl- and sulfonylazoles) represent a series of possible surrogates for acidic groups which span a pKa range from about 4.5-11.5. This simple strategy was extended to include 2-hydroxy- or 2-aminothiophenyl groups which function as relatively nonacidic isosteres for a phosphonic acid. By replacing the phosphonic acid of 2-amino-7-phosphonoheptanoate (AP-7) with these groups, we have synthesized novel N-methyl-d-aspartate (NMDA) antagonists.

Role of the beta 93-100 determinant loop sequence in receptor binding and biological activity of human luteinizing hormone and chorionic gonadotropin.

The intercysteine loop sequence (93-100) in the beta-subunit has been postulated to be important for receptor binding and specificity in the glycoprotein hormones, LH and human CG (hCG). To demonstrate this directly, and to characterize the structural features essential for activity, we prepared a series of synthetic peptides and analogs incorporating this determinant loop region. Peptides were assayed for inhibition of labeled hCG binding to ovarian membrane receptors and stimulation of testosterone production in Leydig cells. Peptides with the native (93-100) sequence from hCG and hLH inhibited hCG binding half maximally at 2.18 and 2.62 x 10(-4) M, respectively, while the sequence from FSH was inactive. Isosteric substitution of Ala for Cys resulted in an inactive peptide, indicating that the (93-100) disulfide bridge is essential for activity. Optimal binding activity requires at least one net positive charge among the side chains, as shown by loss of activity in hybrid analogs with neutral or negative charges conferred by progressive replacement of Arg by Asp at 94 and 95 or by introduction of Asp at 96 and 97. Despite binding to receptors, the native sequence did not promote testosterone production at doses up to 10(-2) M. This contrasts with a second receptor binding sequence, beta (38-57) that activates testosterone production. There are differences between the (93-100) and (38-57) loop sequences in their chemical and physical properties, biological activity and antigenicity. While the cumulative evidence suggests that they associate with counterpart sites in alpha-subunit to form a topographical binding domain in the whole hormone, our results suggest that each sequence may contribute in different ways to activation of postreceptor events.

Diastereoselektivität der Geruchswahrnehmung von Alkoholen der Iononreihe

Diastereoselective Odor Perception of Alcohols in the Ionone SeriesThe characteristic odor of the diastereoisomers 1 and 2 of 1‐(2,2,6‐trimethylcyclohexyl)‐3‐hexanol is configuration dependent, the trans‐alcohol 1 being identified as the sensorily active component. Structure modification of model 1/2, for example substitution on C(2), C(13), and C(14) (ionon numbering) by CH3 groups, introduction of double bonds in the 3‐ or 4‐position, and isosteric substitution of C(7) by an O‐atom, leads to analogues revealing an unequivocal relation between stereochemistry and odor. The specific odor of alcohol 1 is generally released when all substituents are in an equatorial position; the resulting analogy with the molecular size and shape of odoriferous steroids suggests that the release of the particular scent can be correlated with a steroid‐resembling receptor event.

Adenosine deaminase inhibitors. Synthesis of deaza analogues of erythro-9-(2-hydroxy-3-nonyl)adenine.

Structural analogues of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), in which the adenine moiety of the molecule was modified, were prepared in order to investigate the structural requirement of EHNA as an inhibitor of adenosine deaminase (ADA). Thus, 1- and 3-deaza-EHNA and their 6-deamino analogues were synthesized and evaluated as inhibitors of ADA from calf intestine. Inhibition studies indicated that isosteric substitution of pyrimidine nitrogens by carbons could be tolerated at the enzymatic binding site. In fact, 3-deaza-EHNA was found to have an inhibitory activity comparable to EHNA itself, and 1-deaza-EHNA, though less potent, is a good inhibitor. The 6-amino group gives an important contribution to the enzymatic binding if the N1 nitrogen is also present, conferring on the compound the characteristic of a semitight inhibitor.

Photoaffinity labeling of lactate dehydrogenase by the carbene derived from the 3-diazirino analog of nicotinamide adenine dinucleotide

The 3-diazirino analogue of NAD+, DAD+, in which the diazirine group replaces the carboxamide in an almost isosteric substitution, has been synthesized as a photoaffinity reagent for dehydrogenases. With lactate dehydrogenase, the Kdiss is 4 mM (compare the NAD+ Kdiss of 0.6 mM). On photolysis, three types of interaction can be discerned and separately quantitiated by using [3H]DAD+: noncovalent binding of the photoproducts of DAD+, which is removed by protein denaturation; nonspecific covalent labeling, which is eliminated by the presence of a scavenger such as glutathione; specific covalent labeling at the active site, which is prevented by competition from the natural ligands. The photolabeling efficiency (sites covalently labeled/sites initially occupied) is approximately 0.3. It is evident that the carbene generated from DAD+ is more effective than that from the more bulky 3-diazoacetate or than the less reactive nitrene derived from the 3-azido-NAD+ analogue.

Syntheses and activities of sulfur and selenium isosteric substitution analogs of retinol

The syntheses of sulfur and selenium isosteric substitution analogues of retinol, namely, retinyl phenyl thioether (2b), retinyl phenyl selenoether (2c), and retinyl thioacetate (2e) are described. These retinoid derivatives were examined for activity in terms of "chemoprevention" of cancer by measuring the reverse keratinization of epithelial cells in vitro. Retinoid analogues 2b, 2c, and 2e were found to be active in 20, 80, and 33.3% of the cultures, respectively, as compared to 72.7% activity for trans-retinol.

Inhibition of hypoxanthine-guanine phosphoribosyl transferase

The affinities of eighteen purines or purine analogs for human erythrocytic hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8; HGPRTase) were compared to assess the feasibility of obtaining active inhibitors of the enzyme. Three compounds appeared to inhibit the utilization of hypoxanthine by L5178Y cells in vitro due to inhibition of the enzyme rather than depletion of the intracellular 5-phosphoribosyl-1-pyrophosphate pool. The three competitive inhibitors and their affinity constants ( K i ) using 6-mercaptopurine as substrate were: 6-mercapto-9-(tetrahydro-2-furyl)-purine, 37 μM; 2,6-bis-(hydroxyamino)-9-β- d-ribofuranosyl-purine, 12 μM; and 6-iodo-9-(tetrahydro-2-furyl)-purine, 108 μM. The KIn m for 6-mercaptopurine was 9 μM. Thus, the enzyme tolerates bulky substitution at N 9. 6-Mercapto-9-(tetrahydro-2-furyl)-purine also potentiated the chemotherapeutic effect of azaserine, an inhibitor of de novo purine biosynthesis, in L5178Y ascites tumor-bearing mice. Four 2-substituted, oxazolo-[5, 4- d]-pyrimidine-7-ones and 2-methylthiazolo-[5, 4- d]-pyrimidine-7-one had K i values in the range of 84–173 μM. Consequently, isosteric substitution at N 9 may also be a fruitful and logical course to pursue in the design and synthesis of more potent inhibitors of this important enzyme.

Enzymatic incorporation of ATP and CTP analogues into the 3' end of tRNA.

Structural analogues of adenosine 5'-triphosphate and cytidine 5'-triphosphate were investigated as substrates for ATP(CTP):tRNA nucleotidyl transferase. Eight out of 26 ATP analogues and six out of nine CTP analogues were incorporated into the 3' terminus of tRNA. In general, for the recognition of the substrates the modification of the cytidine is less critical than is the modification of adenosine. An isosteric substitution on the ribose residue is possible in both CTP and ATP. The free hydroxyls of these triphosphates can be replaced by an amino group or hydrogen atom without loss of substrate properties. Modifications of positions 1, 2, 6, and 8 on the adenine ring of ATP are not allowed whereas modification on positions 2, 4 and 5 on the cytosine ring of CTP are tolerated by the enzyme. No differences can be observed in the substrate properties of ATP(CTP):tRNA nucleotidyl transferase isolated from different sources. Methods for preparation of tRNA species, which are shortened at their 3' end by one or more nucleotides, and analytical procedures for characterisation of these modified tRNAs are described.