Formation Of Noncovalent Complexes Research Articles

Spectroscopic properties and photoreactivity with DNA of new monofunctional pyridopsoralens.

Abstract— New psoralen derivatives have been synthesized in order to enhance their affinity towards DNA. The spectral properties (absorption, fluorescence emission, fluorescence quantum yield) and the photostability of pyrido[3,4‐c]psoralen are first reported. The drastic changes observed in the solubility and in the fluorescence emission when these compounds are added to native DNA give evidence of the formation of non covalent dark complexes. Upon UV irradiation (365 nm) of the complexes, a photobinding occurs. Heat denaturation and renaturation experiments of modified DNA show that only monoadducts are formed. From the analysis of their fluorescence properties the involvement of the 4′, 5′ double bond is assumed. The monofunctional character has also been established for psoralens having a fused pyridine ring in the 4′, 5′ site. On the opposite, a fused tetrahydropyrido group in the 4′, 5′ site is inefficient to inactivate this reactive site.

Effect of carbohydrate and metal ion binding on the reactivity of the essential thiol groups of lima bean lectin.

A free sulfhydryl group previously has been shown to be required for carbohydrate binding to the lectin from lima bean (Phaseolus lunatus) (Gould, N. R. and Scheinberg, S. L. (1970) Arch. Biochem. Biophys. 141, 607-613). Modification of this group by sulfhydryl reagents was specifically inhibited by D-GalNAc. We have further examined the reactivity of sulfhydryl groups in lima bean lectin with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) as a probe for carbohydrate and metal ion binding. The 4 thiol groups in tetrameric lima bean lectin component III gave identical kinetics for reaction with Nbs2 involving formation of a weak noncovalent complex between Nbs2 and the lectin. The pH-independent reactivity of the thiol groups at neutral pH suggested that the thiols may exist as ion pairs with a nearby ionized group. Carbohydrate ligands were competitive inhibitors of thiol modification. The thiol groups on all 4 subunits of lima bean lectin were completely and reversibly protected by carbohydrate binding. The ability of carbohydrates to inhibit thiol modification correlated with their potency as inhibitors in a precipitin inhibition assay. The best inhibitors were the oligosaccharides alpha-D-GalNAc-(1 leads to 3)[alpha-L-fucose-(1 leads to 2)]beta-D-Gal(1 leads to R) and alpha-D-GalNAc-(1 leads to 2)beta-D-Gal(1 leads to R). Apparent thermodynamic parameters for binding of several carbohydrates were determined by measuring the temperature dependence of thiol protection. Removal of the bound metal ions Ca2+ and Mn2+ following dialysis into EDTA inactivated the lectin and increased the reactivity of the thiol groups 60-fold. This conversion was temperature-dependent and could be reversed upon addition of metal ions. The fast-reacting thiol groups were not protected by haptenic sugars from modifications by Nbs2.

Computer modeling of the recombination reaction of rhodopsin

Various mechanistic schemes for the recombination reaction of rhodopsin were designed and tested using computer modeling and simulation with data from kinetics experiments. The reaction schemes were mathematically modeled by systems of nonlinear first-order ordinary differential equations (ODEs) with unknown rate constants. Each model was fitted to the experimental data by using a modified simplex algorithm for parameter (rate constant) estimation and Gear's method for solving stiff systems of ODEs. The recombination reaction of rhodopin was best modeled by branched, multistep reaction schemes which included formation of noncovalent complexes, acid-base equilibria, and acid and base-catalyzed dehydration of a Schiff base intermediate. The biochemical bases for these models are discussed.

Reversible inhibition of butyrylcholinesterase with aromatic hydrocarbons

Reversible inhibition of butyrylcholinesterase (acylcboline acylhydrolase, EC 3.1.1.8) with several aromatic hydrocarbons was investigated and the results obtained were analyzed in terms of the hydrophobic interaction, making use of the solvent-water partition coefficients for the parameterization of the hydrophobic character of the ligand molecule. To obtain purely hydrophobic effects, the compounds incapable of specific solvation (hydrogen-bond formation) in water as well as in the hydrophobic phase were specially selected for the reaction series. Determination of the hydrophobic properties of the enzyme binding site allows further investigation into the other specificity-determining factors of the non-covalent complex formation step of butyrylcholinesterase-catalyzed reactions.

Interactions of retroviruses with chemical carcinogens: I. Noncovalent binding of unactivated polycyclic aromatic hydrocarbons

The noncovalent binding of carcinogenic polycyclic aromatic hydrocarbons (PAH), e.g., benzo( a)pyrene, to retroviruses was quantitated using a rate zonal centrifugation assay, and the effects of the binding on a retrovirus specific function, reverse transcription, were determined. The level of binding for the enveloped retroviruses was much higher than that found for nonenveloped viruses (2.5 to 40-fold greater), such as bacteriophage T4 and adenovirus type 5; there was no special affinity of PAH compounds for retroviruses as compared with another enveloped virus, Sindbis virus; and there was no binding to the viral glycoproteins (type specific antigens). These results suggest that the binding is best interpreted as partitioning of the hydrophobic PAH compounds between viral envelope lipids and the surrounding aqueous buffer, and this interpretation is supported by the temperature and salt dependence of the binding. Using isolated retroviral cores we also found that there is a relatively small, but significant, level of binding of benzo( a)pyrene to retroviral cores. Further, we observed that the noncovalent binding of benzo( a)pyrene to Rauscher leukemia virus inhibits the RNA-dependent DNA polymerase activity. The inhibition requires preincubation of the virus and PAH, i.e., the formation of noncovalent virus-PAH complexes, and is consistent with a noncompetitive model of enzyme inhibition with an inhibition constant, K i of about 40 μ M.

Affinity labelling of tryptophanyl-transfer RNA synthetase

A double affinity-labelling approach has been developed in order to convert an oligomeric enzyme with multiple active centres into a single-site enzyme. Tryptophanyl-transfer RNA synthetase (EC 6.1.1.2) from beef pancreas is a symmetric dimer, α 2 An ATP analogue, γ-( p-azidoanilide)-ATP does not serve as a substrate for enzymatic aminoacylation of tRNA Trp but acts as an effective competitive inhibitor in the absence of photochemical reaction, with K 1 = 1 × 10 −3m ( K mfor ATP = 2 × 10 −4m). The covalent photoaddition of azido-ATP ‡ ‡ Abbriviations used: azido-ATP, γ-( p-azidoanilide)adenosine triphosphate; TRSase, tryptophanyl-tRNA synthetase (EC 6.1.1.2); R-Trp-tRNA, a residue of ambucilyl-tryptophanyl-tRNA covalently bound to the protein; Cl-R-Trp-tRNA, N-chlorambucilyl-tryptophanyl-tRNA; chlorambucil, γ- p-bis(2-chloroethyl)-aminophenyl-butyrie acid; -R-tryptophan, a residue of ambucilyl-tryptophan covalently bound to the protein; BDC, benzoylated diethylaminoethyl cellulose. results in complete loss of enzymatic activity in both the ATP-[ 32P]pyrophosphate exchange reaction and tRNA aminoacylation. ATP completely protects the enzyme against inactivation. However, covalent binding of azido-ATP is also observed outside the active centres. The difference between covalent binding of the azido-ATP in the absence and presence of ATP corresponds to 2 moles of the ATP analogue per mole of the enzyme. Two binding sites for tRNA Trp have been found from complex formation at pH 5.8 in the presence of Mg 2+. The two tRNA molecules bind, with K dis = 3.6 × 10 −8m and K dis = 0.9 × 10 −6m, respectively, pointing to a strong negative co-operativity between the binding sites for tRNA. N-chlorambucilyl-tryptophanyl-tRNA Trp and TRSase form a complex with K dis = 5.5 × 10 −8m at pH 5.8 in the presence of 10 m m-Mg 2+. This value is similar to the value of K dis for tryptophanyl-tRNA of 4.8 × 10 −8 m. Under the same conditions a 1:1 complex (in mol) is formed between the enzyme and Trp-tRNA or N-chlorambucilyl-Trp-tRNA. On incubation, a covalent bond is formed between N-chlorambucilyl-Trp-tRNA and TRSase; 1 mole of affinity reagent alkylates 1 mole of enzyme independently of the concentration of the modifier. The alkylation reaction is completely inhibited by the presence of tRNA Trp whereas the tRNA devoid of tRNA Trp does not affect the rate of alkylation. In the presence of either ATP or tryptophan, or a mixture of the two, the alkylation reaction is inhibited even though these ligands have no effect on the complex formation between TRSase and the tRNA analogue. Photoaddition of the azido-ATP completely prevents the reaction of the enzyme with the tRNA analogue, although the non-covalent complex formation is not affected. Exhaustive alkylation of TRSase partially inhibits the reaction of ATP [ 32P]pyrophosphate exchange and completely blocks the aminoacylation of tRNA Trp. Cleavage of the tRNA which is covalently bound to TRSase restores both the ATP-[ 32P]pyrophosphate exchange and aminoacylation activity. The TRSase which is covalently-bound to R-Trp-tRNA is able to incorporate only one ATP molecule per dimeric enzyme into the active centre. This doubly modified enzyme is completely enzymatically inactive. Removal of the tRNA residue from the doubly modified enzyme results in the formation of the derivative with one blocked ATP site. Therefore, a “single-site” TRSase may be generated either by alkylation of the enzyme with Cl-R-Trp-tRNA or after the removal of covalently bound tRNA from the doubly labelled protein. Tryptophanyl-tRNA synthetase containing blocked ATP and/or tRNA binding site(s) seems to bo a useful tool for investigation of negative co-operativity and may help in the elucidation of the structure function relationships between the active centres.

Étude de l'inhibition de l'activité estérasique de la trypsine par les dérivés acides et amides des esters spécifiques

Abstract The inhibition of the esterase activity of trypsin (EC 3.4.4.4) by acid and amide derivatives of specific ester substrates has been determined at several pH values between 5 and 9 and at 25°. With either specific or non-specific ester substrates, the inhibition by the acid derivatives is stronger at low pH. The reverse is the case for the amide derivatives. The sigmoid curve of p K i dependency versus pH for both series of inhibitors suggests a very close interaction between the —COX group of the acid or amide derivatives and the histidine at the active center of the enzyme during the formation of the first non-covalent complex. The affinities of these different specific derivatives for the enzyme have been compared at the optimal pH value of the enzymatic activity.