Release of p-nitrophenyl phosphorylcholine-hydrolyzing phosphodiesterase from mouse brain membrane by phospholipase-C

p-Nitrophenyl phosphorylcholine-hydrolyzing phosphodiesterase activity, which is present more predominantly in brain than liver, kidney or small intestine, was released from the homogenate of brain membrane by Bacillus cereus phospholipase-C. The release of the enzyme was time-dependently induced selectively by phospholipase-C, but not by phospholipases A₂ or D, or protease. The released phosphodiesterase was partially purified by DEAE-sephacel and HPLC gel chromatographies with the specific activities of 400 and 1500 nmol/mg · h, respectively. The optimum pH and K_m values of the partially purified phosphodiesterase, possessing a mol. wt of 100,000, were observed to be pH 11 and 10 μM, respectively, quite similar to the values of the membrane-bound enzyme, except thermostability and temperature dependency of activity. Interestingly, among phosphorylcholine-containing compounds only glycero-phosphorylcholine exhibited the competitive inhibition of the phosphodiesterase activity.     

A hexaimino cryptand containing naphthyl groups; structural characterization of mononuclear complexes and catalysis in hydrolysis of p-nitrophenyl phosphate

Mononuclear metal complexes of a hexaimino cryptand containing three naphthyl groups have been prepared and the structure of the copper(I) complex was determined by X-ray diffraction. The complexes show acceleration of hydrolysis of p-nitrophenyl phosphate ester.     

The expression in E. coli of a polymeric gene coding for an esterase mimic catalyzing the hydrolysis of p-nitrophenyl esters

We have prepared a hybrid protein consisting of seven esterase units, Glu-Ala-His-Ala-Ser-Phe-Phe-Phe, fused to the N-terminal of galactokinase (E. coli). The structural gene for this bifunctional protein was obtained by cloning a polymer made up of three chemically synthesized oligonucleotides to which the galactokinase gene was fused in frame. The hybrid protein was purified to homogeneity with the aid of the galactokinase moiety and showed an M_r of 51 000-53 000. The preparation could catalyze the hydrolysis of p-nitrophenyl esters and, due to the inbuilt hydrophobic spacers, Phe-Phe-Phe, improved catalysis of more hydrophobic substrates was obtained.     

Kinetic studies of the alkaline mesentericopeptidase. Study on the active centre topography of alkaline mesentericopeptidase by means of bifunctional reversible inhibition.

The inhibitory effect of alkylboronic acids H(CH2)nB(OH)2(n=2-8) and Ph(CH2)n-B(OH)2, (n=0-4), on the alkaline mesentericopeptidase-catalysed hydrolysis of synthetic substrates was studied. It was shown that alkylboronic acids act as bifunctional reversible inhibitors. The borate group interacts with an ionogenic group of the enzyme with a pKa of about 6.9-7.0. The latter is probably the catalytically active imidazole of the active centre. The hydrocarbon part of the molecule also takes part in the formation of the enzyme-inhibitor complex. The dependence of the degree of the enzyme-inhibitor complex formation upon the length of the side-chain of the inhibitor indicates the presence of two binding sites on the enzyme molecule.     

Extent of enthalpy-entropy compensation in protein-ligand interactions

The extent of enthalpy-entropy compensation in protein-ligand interactions has long been disputed because negatively correlated enthalpy (ΔH) and entropy (TΔS) changes can arise from constraints imposed by experimental and analytical procedures as well as through a physical compensation mechanism. To distinguish these possibilities, we have created quantitative models of the effects of experimental constraints on isothermal titration calorimetry (ITC) measurements. These constraints are found to obscure any compensation that may be present in common data representations and regression analyses (e.g., in ΔH vs. -TΔS plots). However, transforming the thermodynamic data into ΔΔ-plots of the differences between all pairs of ligands that bind each protein diminishes the influence of experimental constraints and representational bias. Statistical analysis of data from 32 diverse proteins shows a significant and widespread tendency to compensation. ΔΔH versus ΔΔG plots reveal a wide variation in the extent of compensation for different ligand modifications. While strong compensation (ΔΔH and -TΔΔS opposed and differing by < 20% in magnitude) is observed for 22% of modifications (twice that expected without compensation), 15% of modifications result in reinforcement (ΔΔH and -TΔΔS of the same sign). Because both enthalpy and entropy changes arise from changes to the distribution of energy states on binding, there is a general theoretical expectation of compensated behavior. However, prior theoretical studies have focussed on explaining a stronger tendency to compensation than actually found here. These results, showing strong but imperfect compensation, will act as a benchmark for future theoretical models of the thermodynamic consequences of ligand modification.     

Effect of the sugar moiety on complex formation of cycloamyloses with p-nitrophenyl glycosides

Circular diochroism (CD) spectra of four p-nitrophenyl glycosides and their cycloamylose complexes were investigated at various concentrations of cycloamylose and at temperatures ranging from 20 to 60°C. The CD spectra of p-nitrophenyl glycosides changed remarkably in the presence of cycloamyloses, and significant differences in spectral shape and intensity were observed between the cyclohexaamylose complex and the cycloheptaamylose complex. The difference CD spectra between the free guest and its complex indicates that the nitrophenyl group is included in the cycloamylose cavity but its disposition is different between the complexes with cyclohexaamylose and cycloheptaamylose. Values of enthalpy and entropy of the cyclohexaamylose complex are considerably larger than those of the corresponding cycloheptaamylose complex, although the free energy differs only slightly. It is suggested that the nitrophenyl group is more loosely bound to the cycloheptaamylose cavity than to the cyclohexaamylose cavity, and has much more flexibility in its disposition.     

A study on the enthalpy-entropy compensation in protein unfolding

A large number of thermodynamic data including the free energy, enthalpy, entropy, and heat capacity changes were collected for the denaturation of various proteins. Regression indicated that remarkable enthalpy-entropy compensation occurred in protein unfolding, which meant that the change in enthalpy was almost compensated by a corresponding change in entropy resulting in a smaller net free energy change. This behavior was proposed to result from the water molecule reorganization, which contributed significantly to the enthalpy and entropy changes but little to the free energy change in protein unfolding. It turned out that the enthalpy-entropy compensation could provide novel insights into the problem of enthalpy and entropy convergence in protein unfolding.     

Obfuscation of allosteric structure-function relationships by enthalpy-entropy compensation.

The pH and temperature dependence of the allosteric properties of phosphofructokinase (PFK) from Bacillus stearothermophilus have been studied from 5 to 9 and 6 to 40 degrees C, respectively. Throughout this pH and temperature range the allosteric ligands MgADP and phospho(enol)pyruvate (PEP) have no effect on kcat. The dissociation constants of the substrate, fructose 6-phosphate, and the allosteric ligands, as well as the absolute value of the coupling free energies between these ligands, all increase when the pH is raised, indicating that the inhibition by PEP and the activation by MgADP increase despite each ligand's somewhat lower affinity. However, the constituent coupling enthalpies and entropies substantially diminish in absolute value as pH is increased, suggesting that the magnitudes of molecular perturbations engendered by the binding of allosteric ligands do not correlate with the magnitudes of the functional consequences of those perturbations. Temperature and pH exert their influence on the observed allosteric behavior by changing the relative contributions made by the largely compensating DeltaH and TDeltaS terms to the coupling free energy.     

Spectrophotometric titration of tyrosyl residues in alkaline mesentericopeptidase.

At pH 7.0 the alkaline mesentericopeptidase has ultraviolet absorption spectrum with a minimum at 251 nm and a maximum at 280 nm and no visible absorption. From the tyrosine to tryptophan ratio a value of 3 tryptophyl residues per mole of protein is obtained. The molar extinction coefficient at 280 nm is 3.55 X 10(4)M-1cm-1. Spectrophotometric titration studies show that the molecule of mesentericopeptidase contains seven phenolic groups with a pKapp - 9.92 and four to five groups with a pKapp = 11.96. Denaturing agents, such as 5 M guanidine hydrochloride or alkali, normalize the ionization of the tyrosyl residues. There is a good correlation between the spectrophotometric titration data and the results for the reactivities of the tyrosines in mesentericopeptidase towards tetranitromethane. The correlation is explained by the mechanism of nitration. Conclusions about the state of the tyrosyl residues and the three-dimensional structure of mesentericopeptidase are made.     

Reactivity with p-nitrophenyl acetate and interaction between the amino and imidazole groups of histidine and related compounds

The study of the reaction of p-nitrophenyl acetate (PNPA) with histidine and certain derivatives showed that the species in which the amino group is unprotonated (R(NH₂)Im) react with second-order rate constants ( ) that are higher than predicted by a Brønsted relation for a series of neutral amino acids. The reason for this behavior was investigated through an analysis of the kinetics of the reaction of PNPA with these compounds in order to assess the reactivities of the amino and imidazole groups in the two species . The rate constant for the reaction with the imidazole group ( ) of N^π-methyl histidine agrees with the value predicted by a Brønsted relation obtained from a series of model imidazole compounds. N^τ-Methyl histidine, however, is unreactive, indicating that N^τ is the reactive nitrogen in the imidazole ring of histidine. The values found for histidine, histidine methyl ester, and N^α-dimethyl histidine are lower than predicted by the Brønsted relation. This behavior was found to be due to low reactivity of the

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. The evidence presented suggests that the lower reactivity of is due to an ion-dipole interaction between the protonated amino group and the unprotonated imidazole ring, which displaces the tautomeric equilibrium toward the unreactive N^τ-H form. The higher reactivity of the imidazole group in the species R(NH₂)Im, relative to that in , is responsible for the observed high values for histidine, for histidine methyl ester, for N^τ-methyl histidine, and for N^α-dimethyl histidine, in contrast with the normal value found for N^τ-methyl histidine. The conclusions from this study of histidine and its derivatives support the proposal of an interaction between the protonated N-terminal amino group and the imidazole ring of His⁶ in the octapeptide hormone angiotensin.     

Effects of cationic micelles on the rate of reaction of p-nitrophenyl esters with dihydrolipoic acid and dihydrolipoylamido derivatives

Dihydrolipoic acid (DHLA) 2a and its surfactant derivatives, trialkyl(2-lipoylamidoethyl) ammonium salts 2b-c, have been investigated, mainly in micellar solutions of cetyltrimethyl-ammonium bromide (CTABr), as esterolytic reagents toward p-nitrophenyl esters. The origins of the observed kinetic effects are discussed, and the reactivity of these reagents are compared with that of other thiolytic systems. The results indicate that DHLA, although not a surfactant, is effectively comicellized by CTABr, and micelles of CTABr and DHLA are among the most effective esterolytic systems, at moderately alkaline pHs, so far reported.     

Kinetics of the alkaline phosphatase catalyzed hydrolysis of disodium p-nitrophenyl phosphate in frozen model systems

The alkaline phosphatase catalyzed hydrolysis of disodium-p-nitrophenyl phosphate was studied in four model systems comprising sucrose, maltodextrin, carboxymethylcellulose (CMC), and CMC-lactose in a temperature range of -28 to 20 degrees C. In the maltodextrin and CMC-lactose model systems, the reaction rate decreased to a very low value as the glass transition temperature was approached. In the CMC and CMC-lactose systems with low initial solute concentration, as a consequence of freeze-concentration, a rate maximum around the initial freezing temperature was observed. The Arrhenius equation described the temperature dependence of the reaction rate both in the liquid and the glassy states in all systems studied, while a slightly curved Arrhenius plot was observed in the "rubbery" state of the CMC and CMC-lactose systems. The WLF equation with system-dependent coefficients described the kinetics in the rubbery state of all the model systems except sucrose, excluding the short temperature range where reaction rate enhancement with decreasing temperature was observed.     

Photochemical evidence for the presence of histidyl residue in the active site of alkaline mesentericopeptidase.

The photosensitized oxidation of alkaline mesentericopeptidase in the presence of methylene blue results in a first-order rate of inactivation. The loss of enzymatic activity towards casein and N-acetyl-L-tyrosine ethyl ester closely correlates with the destruction of one histidyl residue. A pK value of 6.8 is determined from the sigmoid pH-dependence of the photoinactivation rate. This suggests the involvement of a normal titrating imidazole group in the active site of mesentericopeptidase. The competitive inhibitor Na-benzoyl-L-arginine protects the enzyme from photoinactivation. A conclusion is made that the active site histidyl residue is modified. Circular dichroism spectra show no change in the protein conformation during the photodynamic treatment.     

Limited hydrolysis of bovine plasma albumin at neutral and alkaline pH catalyzed by associated proteinases.

Proteinase contaminants in some plasma albumin samples have previously been shown to produce cleavage of the albumin molecule at acid pH. The F conformer, existing at pH 3.8, is cleaved near erisidue number 400 to yield a large N-terminal fragment of approximately 46,000 daltons. No cleavage was found at pH above approximately 4.4. It is shown in this paper that the proteinase contaminants are active over a broad pH range from 2.5 to 11.4 provided conditions are such as to induce some breakdown of the native conformation of the albumin molecule. Addition of Tris-borate buffer (0.1 M) at pH 7.5-9 is sufficient to permit cleavage. At pH near 9 this occurs predominantly 42,000 and 27,000 daltons. Near neutral pH substantial cleavage occurs in 4-8 M urea solution or in the presence of sodium dodecyl sulfate (AD110 complex). Under these conditions there are two large fragments (42,000 and 47,000 daltons) and essentially two small ones (20,000-27,000 daltons). Under conditions where there is no cleavage at 38-40 degrees, substantial cleavage results at 50-65 degrees but enzyme inactivation also occurs toward the top of this range. The alkaline activity is inhibited by soybean trypsin inhibitor but not by pepstatin; the reverse is true of the low pH activity. Cleavage at neutral or alkaline pH under the various conditions occurs primarily at X-Leu bonds while the low pH activity was already shown to occur at X-Phe. These facts suggest the presence of at least two enzymes. There is surprisingly little pH dependence over the range 7.5-9 in any of the media examined, even though albumin is known to undergo a significant conformational change in this range, the N leads to B transition. This transition is thought to be essentially a tertiary change with little loss of helix content. It is suggested that loss of native secondary structure, especially uncoiling of helical regions, is crucial to permit attack by these enzymes.