The addition of bisulfite to 5-fluorouracil. Evidence for a change in rate determining step

The kinetics of bisulfite addition to 5-fluorouracil were studied as a function of increasing concentrations of potential general acids. Values of $\text{k}{}_{\mathrm{obsd}}\text{[}\text{SO}{}_3{}^{\mathrm{=}}\text{]}$ measured at 25°C and ionic strength 1.0 M increased linearly and then became invariant with increasing concentrations of either HSO₃^? or (OHCH₂CH₂)₂N⁺C(CH₂OH)₃ HCl (BisTris⁺HCl). A small kinetic hydrogen-deuterium isotope effect ($\text{k}{}_{\mathrm{HS}}\text{k}{}_{\mathrm{DS}}\text{= 1.10}$) was observed for the general acid catalysed portion of the addition reaction. The kinetics of bisulfite elimination from 5-fluoro-5,6-dihydrouracil-6-sulfonate were studied in ethanolamine buffers. As previously observed with 1,3-dimethyl-5,6-dihydrouracil-6-sulfonate, this reaction is subject to general base catalysis and exhibits a large kinetic hydrogen-deuterium isotope effect (). The kinetic results for the addition reaction are consistent with a multistep reaction pathway involving the initial formation of an oxyanion sulfite addition intermediate (II) which subsequently adds a proton and undergoes tautomerization to yield the final 5-fluoro-5,6-dihydrouracil-6-sulfonate product. Thus the elimination of bisulfite from 5-fluoro-5,6-dihydrouracil-6-sulfonate probably proceeds by an ElcB mechanism which involves, at relatively low concentrations of general base, rate determining general base catalyzed proton abstraction from carbon 5 to yield intermediate II followed by the rapid elimination of sulfite to yield 5-fluorouracil. These results may be related to both the enzymatically catalyzed dehalogenation of bromoand iodouracil and the methylation of deoxyuridylate by thymidylate synthetase.     

Influence of ionic strength upon the proton inventory for the water-catalyzed hydrolysis of N-acetylbenzotriazole

Proton inventory investigations of the hydrolysis N-acetylbenzotriazole at pH 3.0 (or the equivalent point on the pD rate profile) have been conducted at two different temperatures and at ionic strengths ranging from 0 to 3.0 M. The solvent deuterium isotope effects and proton inventories are remarkably similar over this wide range of conditions. The proton inventories suggest a cyclic transition state involving four protons contributing to the solvent deuterium isotope effect for the water-catalyzed hydrolysis. The hydrolysis data are described by the equation $\text{k}{}_{\mathrm{n}}\text{= k}{}_{\mathrm{<mtext>o</mtext>}}\text{(1 ? n + nπ}{}_{\mathrm{<mtext>a</mtext>}}{}^1\text{)}{}^4$ with $\text{π}{}_{\mathrm{<mtext>a</mtext>}}{}^1\text{～ 0.74}$, where k_o is the observed first-order rate constant in protium oxide, n is the atom fraction of deuterium in the solvent, k_n is the rate constant in a protium oxide-deuterium oxide mixture, and $\text{π}{}_{\mathrm{<mtext>a</mtext>}}{}^1$ is the isotopic fractionation factor.     

Model of singlet oxygen scavenging by α-tocopherol in biomembranes

Quenching of singlet molecular oxygen (${}^1\text{Δ}{}_{\mathrm{g}}\text{O}{}_2$) by α-tocopherol (I) involves the hydroxy function of the chromanol ring of I. In phosphatidylcholine (PC) uni- and multilamellar vesicles this structural element of I is localized at the interface polar headgroup/hydrophobic core. A dielectric constant of ? ～ 25 was determined for this special region of the PC bilayer. The ratio k_Q/k_R of rate constants of quenching processes (k_Q) and irreversible reactions (k_R) of I with ${}^1\text{Δ}{}_{\mathrm{g}}\text{O}{}_2$ increases with decreasing polarity of the solvent. In ethanolic solutions where ? = 25.5, k_Q/k_R is about 40. Extrapolation of these results to phospholipid bilayers suggests that at the nearness of the ester carbonyl oxygen of the PC fatty acid moieties, α-tocopherol can deactivate approximately 40 ${}^1\text{Δ}{}_{\mathrm{g}}\text{O}{}_2$ molecules before being destroyed. It is concluded that in vivo, one may expect to find a higher k_Q/k_R ratio if the chromanol ring of I hides within the more hydrophobic interiors of the membrane surface peptides.     

Nuclear magnetic resonance study of the rate of electron transfer between cytochrome c and iron hexacyanides

The rates of electron exchange between ferricytochrome c (C^III)3 and ferrocytochrome c (C^II) were observed as a function of the concentrations of ferrihexacyanide (Fe^III) and ferrohexacyanide (Fe^II) by monitoring the line widths of several proton resonances of the protein. Addition of Fe^II to C^III homogeneously increased the line widths of the two downfield paramagnetically shifted heme methyl proton resonances to a maximal value. This was interpreted as indicating the formation of a stoichiometric complex, C^III·Fe^II, in the over-all reaction:

$\text{C}{}^{\mathrm{III}}\text{+}\text{Fe}{}^{\mathrm{II}}\text{?}\text{k}{}_{\mathrm{?1}}\text{k}{}_1\text{C}{}^{\mathrm{III}}\text{·}\text{Fe}{}^{\mathrm{II}}\text{?}\text{k}{}_{\mathrm{?2}}\text{k}{}_2\text{C}{}^{\mathrm{II}}\text{·}\text{Fe}{}^{\mathrm{III}}\text{?}\text{k}{}_{\mathrm{?3}}\text{k}{}_3\text{C}{}^{\mathrm{III}}\text{+}\text{Fe}{}^{\mathrm{II}}$

Values for $\text{k}{}_1\text{k}{}_{\mathrm{?1}}\text{= 0.4 × 10}{}^3\text{m}{}^{\mathrm{?1}}\text{and}\text{k}{}_2\text{= 208}\text{s}{}^{\mathrm{?1}}$, respectively, were calculated from the maximal change in line width observed at pH 7.0 and 25 °C. Changes in the line width of C^III in the presence of Fe^II and either KCl or Fe^III suggest that complexation is principally ionic, that Fe^III and Fe^II compete for a common site. Addition of saturating concentrations of Fe^III to C^III produced only minor changes in the nuclear magnetic resonance spectrum of C^III suggesting that complexation occurs on the protein surface.Addition of Fe^III to C^II in the presence of excess Fe^II (to retain most of the protein as C^II) increased the line width of the methyl protons of ligated methionine 80. A value for k_?2 ≈ 2.08 × 10⁴ s^?1 was calculated from the dependence of linewidth on the concentration of Fe^II at 24 °C. These rates are shown to be consistent with the over-all rates of reduction and oxidation previously determined by stopped flow measurements, indicating that k₂ and k_?2 were rate limiting. From the temperature dependence the enthalpies of activation are 7.9 and 15.2 kcal/mol for k₂ and k_?2, respectively.     

Evidence for general catalysis and formation of nitrobenzene in the oxidation of phenylhydroxylamine in aqueous phosphate buffer

N-Phenylhydroxylamine is oxidized in aqueous phosphate buffer to nitrosobenzene, nitrobenzene, and azoxybenzene. Degradation is O₂ dependent and shows general catalysis by $\text{H}{}_2\text{PO}{}_4{}^{\mathrm{?}}$ (k₁ = 2.3 M^?2 sec^?1) and $\text{PO}{}_4{}^{\mathrm{?3}}$ (k₂ = 2.3 × 10⁵M^?2 sec^?1) or kinetically equivalent terms. Evidence is presented suggesting the intermediacy of a highly reactive species leading to these products.     

Inhibition of chymotrypsin by alkyl phosphonates: a quantitative structure-activity analysis.

A quantitative structure-activity relationship has been formulated for 53 alkyl phosphonates [R₂OPO(CH₃)SR₃] inhibiting chymotrypsin: . In this so-called bilinear model, k_i is the bimolecular rate constant (m^?1 s^?1), β is a disposable parameter evaluated by a computerized iterative procedure, MR is the molar refractivity of a substituent, $\text{σ}{}_3{}^1$ is Taft's polar parameter, and I is an indicator variable for substituents containing a sulfonium group. The correlation coefficient for this equation is 0.985. This quantitative structure-activity relationship is compared with those previously formulated for the action of chymotrypsin on acylamino acid ester substrates.     

Kinetic studies on the reaction of ethylenediaminetetraacetatochromium(III) complex with hydrogen peroxide

The rate of reaction of [Cr(III)Y]_aq (Y is EDTA anion) with hydrogen peroxide was studied in aqueous nitrate media [μ = 0.10 M (KNO₃)] at various temperatures. The general rate equation, Rate = $\text{k}{}_1\text{+}\text{k}{}_2\text{K}{}_1\text{[H}{}^{\mathrm{+}}\text{]}{}^{\mathrm{?1}}\text{1 +}\text{K}{}_1\text{[H}{}^{\mathrm{+}}\text{]}{}^{\mathrm{?1}}$ [Cr(III)Y]_aq[H₂O₂] holds over the pH range 5–9. The decomposition reaction of H₂O₂ is believed to proceed via two pathways where both the aquo and hydroxo-quinquedentate EDTA complexes are acting as the catalyst centres. Substitution-controlled mechanisms are suggested and the values of the second-order rate constants k₁ and k₂ were found to be 1.75 × 10^?2 M^?1 s^?1 and 0.174 M^?1 s^?1 at 303 K respectively, where k₂ is the rate constant for the aquo species and k₂ is that for the hydroxo complex. The respective activation enthalpies (ΔH^*₁ = 58.9 and ΔH^*₂ = 66.5 KJ mol^?1) and activation entropies (ΔS^*₁ = ?85 and ΔS^*₂ = ?40 J mol^?1 deg^?1) were calculated from a least-squares fit to the Eyring plot. The ionisation constant pK₁, was inferred from the kinetic data at 303 K to be 7.22. Beyond pH 9, the reaction is markedly retarded and ceases completely at pH ? 11. This inhibition was attributed in part to the continuous loss of the catalyst as a result of the simultaneous oxidation of Cr(III) to Cr(VI).     

Stoichiometry versus coupling ration in the cotransport of Na and different neutral amino acids

The stoichiometry of Na coupling to amino acid movement across the brush border membrane of the rabbit distal ileum has been determined under initial rate conditions.The coupling ratio, defined as the amino acid-dependent Na influx/the Na-dependent amino acid influx, was equal to unity for alanine, measured over a 10-fold range of Na and alanine concentrations. Coupling ratio values determined under a single set of conditions for a number of amino acids varied from 1 for serine to 4.6 for methionine. Reducing the methionine concentration from 12.5 to 1.5 mM caused the coupling ratio value to fall from 4.6 to 1.2.These results are explained by assuming a fixed stoichiometry of 1 : 1 under all conditions, with initial binding of the amino acid (A) to the Na-dependent carrier (E) but with some amino acids being able to cross on the Na-dependent carrier in the absence of Na.The variation in coupling ratio values can be used to calculate K_A, the apparent dissociation constant of amino acid from the Na-dependent carrier in the absence of Na, and the ratio $\text{k}{}_1\text{k}{}_2$, where k₁ and k₂ are first-order rate constants for translocation of the complexes EA and EANa, respectively. This method of processing results has been defined as delta analysis. The value of K_A for methionine is 3.6 ± 1.1 mM and the $\text{k}{}_1\text{k}{}_2$ ratio is 1.01 ± 0.07. The constant coupling ratio value of 1 for alanine indicates that the value for K_A is extremely high or that the k₁ value is extremely low.     

The dehalogenation of iodouracil by cysteine. Intramolecular general-acid catalysis of cysteine addition to 5-iodouracil

The rate-determining step of the cysteine-catalyzed deiodination of 5-iodouracil is the formation of 5-iodo-6-cysteinyl-5,6-dihydrouracil. The rate of the reaction depends upon the concentration of un-ionized 5-iodouracil and the following ionic species of cysteine; ^?OOC(NH₃⁺)CHCH₂S^?. Unlike the reaction of 2-mercapto-ethanol with 5-iodouracil, the cysteine reaction is not subject to catalysis by imidazolium ion and tris(hydroxymethyl)aminomethane hydrochloride. When the rates of cysteine reacting with 5-iodouracil are measured in both H₂O and D₂O, a large kinetic isotope effect is observed ($\text{k}{}_2{}^{\mathrm{<mtext>H</mtext><mtext>20</mtext>}}\text{k}{}_2{}^{\mathrm{<mtext>D</mtext><mtext>20</mtext>}}\text{= 4.10}$), thus implicating the protonated α amino group of cysteine as an intramolecular general acid catalyst for the reaction. These results and possible mechanisms for the actual dehalogenation of the intermediate 5-iodo-6-cysteinyl-5,6-dihydrouracil are discussed in terms of a possible mechanism for enzymatic halopyrimidine dehalogenation.     

On the mechanism of the reaction of tris(hydroxymethyl)aminomethane with activated carbonyl compounds: A model for the serine proteinases

The pH dependence of the reaction of tris(hydroxymethyl)aminomethane (Tris) with the activated carbonyl compound 4-trans-benzylidene-2-phenyloxazolin-5-one (I) is given by the equation $\text{k′}{}_2\text{=}\text{k}{}_{\mathrm{<mtext>b</mtext>}}\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{(K}{}_{\mathrm{<mtext>a</mtext>}}\text{+ [}\text{H}{}^{\mathrm{+}}\text{])}\text{+}\text{k}{}_{\mathrm{<mtext>a</mtext>}}\text{[}\text{OH}{}^{\mathrm{?}}\text{]K}{}_{\mathrm{<mtext>a</mtext>}}\text{(K}{}_{\mathrm{<mtext>a</mtext>}}\text{+ [}\text{H}{}^{\mathrm{+}}\text{])}$, where K_a is the dissociation constant of TrisH⁺. Spectrophotometric experiments show that the Tris ester of α-benzamido-trans-cinnamic acid is formed quantitatively over a range of pH values, regardless of the relative contribution of k_b and k_a terms to k′₂. Hence, both terms refer to alcoholysis. While the mechanism of the reaction is not determined unequivocally in the present work, the magnitude of the k_b term, together with its dependence on the basic form of Tris, suggests that ester formation is occurring by nucleophilic attack of a Tris hydroxyl group on the carbonyl carbon of the oxazolinone, with intramolecular catalysis by the Tris amino group. The rate enhancement due to this group is at least 10² and possibly of the order 10⁶. This system is compared with other model systems for the acylation step of catalysis by serine esterases and proteinases.     

A theory of dynamic and steady responses in chemoreception

A theoretical model which can account for both the dynamic and steady responses is proposed based on the occupation theory. The reaction scheme used is;

Here, S and A are stimulus chemicals and receptor sites unbound, respectively. The binding of S to A leads to an active complex (SA)_active, which is successively transformed into an inactive complex (SA)_active. The response is assumed to be proportional to number of (SA)_active. When a stimulating solution is applied instantaneously at t = 0, the solution to the set of differential equations based on the above scheme is obtained as follows;

where p and C stand for the fraction of (SA)_active to the total number of receptor sites and stimulus concentration, respectively, and α_i, and ω_i (i = 1, 2) are numerical parameters depending on the rate constants and on C. The steady response is expressed as the third term in the above equation, which indicates that the response accords with the Beidler taste equation. Mathematical analysis of the above scheme shows that the dynamic response appears when k₁C > k_?2, and the calculated results for the dynamic response agree approximately with the Hill equation. The Hill coefficient lays within 1·00 and 0·79 and reaches unity with increasing $\text{k}{}_{\mathrm{?1}}\text{k}{}_2$, which implies that the dynamic response under this condition satisfies the Beidler taste equation. For the case of gradual application of stimuli, i.e. the experimental condition, the time course of p is simulated with use of an analogue computer rather than with a numerical solution to the above equation. The results indicate that the dynamic response diminishes with decreasing the application speed of stimulus solution. The present theory accounts consistently for various experimental data observed in the chemoreceptor systems.     

Preferential solvation of methylmercurated calf thymus deoxyribonucleic acid.

The changes in polymer-solvent interactions that occur when native calf thymus DNA is dialyzed against Na₂SO₄ solutions of a given ionic strength and buffer concentration but of varying concentrations in methylmercuric hydroxide have been investigated with the help of solution density measurements at 25 °C and pH 6.8–7.0. From measurements executed under equilibrium dialysis conditions at the three salt levels 5 mm, 0.05 m, and 0.5 m Na₂SO₄ (m refers to molality) and in the presence of 5 mm cacodylic acid buffer, the density increments $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ for native calf thymus DNA were determined as a function of CH₃HgOH concentration. $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ was found not to vary with organomercurial concentration, irrespective of the concentration of supporting electrolyte, until a certain CH₃HgOH concentration level has been reached, viz., , beyond which $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ increases strongly with increasing concentration of CH₃HgOH. As is shown by optical melting, $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ becomes a function of organomercurial concentration the moment DNA undergoes denaturation brought about by the complexing of CH₃HgOH with the various N-binding sites of the base residues in the DNA double helix.Polymer-solvent interactions, expressed in terms of preferential water interactions (“net hydration”) and preferential salt interactions (“salt solvation”), were derived from the $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ data in combination with data obtained on the preferential interaction of CH₃HgOH with denatured DNA and data on the partial specific volumes of all major solution components, gathered from density measurements on solutions with fixed concentrations of diffusible components. Evidence is presented which shows that denaturation in general decreases the net hydration while salt becomes preferentially associated with the polyelectrolyte. This process is further amplified by the interaction of CH₃HgOH with denatured DNA: Methylmercurated DNA alters the redistribution of diffusible components at dialysis equilibrium to such an extent that in a formal sense large amounts of water are rejected from the immediate vicinity of the polymer. The molecular implications of these findings are explored. The results are further discussed in the light of previous findings where the methylmercury-induced denaturation of DNA had been studied with the help of buoyant density measurements in a Cs₂SO₄ density gradient and by velocity-sedimentation in a variety of sulfate media.     

Oxidation of anionic nitroalkanes by flavoenzymes,and participation of superoxide anion in the catalysis

The reactivities of anionic nitroalkanes with 2-nitropropane dioxygenase of Hansenula mrakii, glucose oxidase of Aspergillus niger, and mammalian d-amino acid oxidase have been compared kinetically. 2-Nitropropane dioxygenase is 1200 and 4800 times more active with anionic 2-nitropropane than d-amino acid oxidase and glucose oxidase, respectively. The apparent K_m values for anionic 2-nitropropane are as follows: 2-nitropropane dioxygenase, 1.61 mm; glucose oxidase, 16.7 mm; and d-amino acid oxidase, 11.1 mm. Anionic 2-nitropropane undergoes an oxygenase reaction with 2-nitropropane dioxygenase and glucose oxidase, and an oxidase reaction with d-amino acid oxidase. In contrast, anionic nitroethane is oxidized through an oxygenase reaction by 2-nitropropane dioxygenase, and through an oxidase reaction by glucose oxidase. All nitroalkane oxidations by these three flavoenzymes are inhibited by Cu and Zn-superoxide dismutase of bovine blood, Mn-superoxide dismutases of bacilli, Fe-superoxide dismutase of Serratia marcescens, and other $\text{O}{}_2{}^{\mathrm{?}}$ scavengers such as cytochrome c and NADH, but are not affected by hydroxyl radical scavengers such as mannitol. None of the $\text{O}{}_2{}^{\mathrm{?}}$ scavengers tested affected the inherent substrate oxidation by glucose oxidase and d-amino acid oxidase. Furthermore, the generation of $\text{O}{}_2{}^{\mathrm{?}}$ in the oxidation of anionic 2-nitropropane by 2-nitropropane dioxygenase was revealed by ESR spectroscoy. The ESR spectrum of anionic 2-nitropropane plus 2-nitropropane dioxygenase shows signals at g₁ = 2.007 and g₁₁ = 2.051, which are characteristic of $\text{O}{}_2{}^{\mathrm{?}}$. The $\text{O}{}_2{}^{\mathrm{?}}$ generated is a catalytically essential intermediate in the oxidation of anionic nitroalkanes by the enzymes.     

Quantitative structure-activity relationships of chymotrypsin-ligand interactions: An analysis of interactions in p3 space.

Fourteen derivatives of l-alanine of the type CH₃CH(NHCO-3-C₅H₄N)COOR₃ have been synthesized and their hydrolysis by chymotrypsin was studied with the object of characterizing enzymic space (?₃) to which R₃ binds. The binding of R₃ ($\text{log}\text{1}\text{K}{}_{\mathrm{m}}$) was shown via correlation analysis to correlate with molar refractivity (MR) of R₃ rather than hydrophobicity (π). The results confirmed our earlier predictions. A correlation equation for the hydrolysis of 77 acyl-amino acid esters of the general formula R₂CH(NHCOR₁)COOR₃ relating $\text{log}\text{(k}{}_{\mathrm{<mtext>cat</mtext>}}\text{k}{}_{\mathrm{m}}\text{)}$ to molar refractivity of R₁, R₂, and R₃ and to $\text{σ}{}^1$ (Taft's polar parameter) of R₃ was formulated. The general picture of ligand interactions with chymotrypsin as seen with correlation analysis is discussed.     

Effects of 5-hydroxylysine on acetylene reduction and NH4+-assimilation in the cyanobacterium Anabaena cylindrica.

5-hydroxylysine, an analogue of glutamate and lysine, causes $\text{NH}{}_4{}^{\mathrm{+}}$ production by N₂-fixing A. cylindrica; it also reversibly inhibits GS activity in vitro but has no effect on alanine dehydrogenase or GOGAT. On adding 5-hydroxylysine intracellular pools of glutamine, glutamate and aspartate decrease; those of alanine and serine increase. 5-hydroxylysine alleviates the inhibitory effect of $\text{NH}{}_4{}^{\mathrm{+}}$ on heterocyst production and C₂H₂ reduction and in $\text{NH}{}_4{}^{\mathrm{+}}\text{-grown}$ cultures results in heterocyst synthesis and in C₂H₂ reduction. The data suggest that the GS-GOGAT pathway is the sole route of importance in primary $\text{NH}{}_4{}^{\mathrm{+}}$ assimilation in A. cylindrica, that $\text{NH}{}_4{}^{\mathrm{+}}$ alone does not inhibit nitrogenase and heterocyst production, and that GS and/or a product is involved in regulating the production of both.     

Presteady-state kinetic study of the elementary processes in the chymotrypsin-catalyzed hydrolysis of specific ester substrate. Rate-limiting association process due to the secondary binding.

Presteady-state kinetic studies of α-chymotrypsin-catalyzed hydrolysis of a specific chromophoric substrate, N-(2-furyl)acryloyl-l-tryptophan methyl ester, were performed by using a stopped-flow apparatus both under [E]₀ ? [S]₀ and [S]₀ ? [E]₀ conditions in the pH range of 5–9, at 25 °C. The results were accounted for in terms of the three-step mechanism involving enzyme-substrate complex (E · S) and acylated enzyme (ES′); no other intermediate was observed. This substrate was shown to react very efficiently, i.e., the maximum of the second-order acylation rate constant ($\text{k}{}_2\text{K}{}_{\mathrm{s}}\text{′}\text{)}{}_{\mathrm{<mtext>max</mtext>}}\text{= 4.2 × 10}{}^7\text{M}{}^{\mathrm{?1}}\text{s}{}^{\mathrm{?1}}$. The limiting values of K_s′ (dissociation constant of E · S), K₂ (acylation rate) and k₃ (deacylation rate) were obtained from the pH profiles of these parameters to be 0.6 ± 0.2 × 10^?5 m, 360 ± 15 s^?1 and 29.3 ± 0.8 s^?1, respectively. Likewise small values were observed for K_i of N-(2-furyl)-acryloyl-l-tryptophan and N-(2-furyl)acryloyl-d-tryptophan methyl ester and K_m of N-(2-furyl)acryloyl-l-tryptophan amide. The strong affinities observed may be due to intense interaction of β-(2-furyl)acryloyl group with a secondary binding site of the enzyme. This interaction led to a $\text{k}{}_{\mathrm{?1}}\text{k}{}_2$ value lower than unity, i.e., the rate-limiting process of the acylation was the association, even with the relatively low k₂ value of this methyl ester substrate, compared to those proposed for labile p-nitrophenyl esters.     

Equilibrium constants under physiological conditions for the reactions of L-phosphoserine phosphatase and pyrophosphate: L-serine phosphotransferase

The observed equilibrium constants (K_obs) for the l-phosphoserine phosphatase reaction [EC 3.1.3.3] have been determined under physiological conditions of temperature (38 °C) and ionic strength (0.25 m) and physiological ranges of pH and free [Mg²⁺]. Using Σ and square brackets to indicate total concentrations $\text{K}{}_{\mathrm{<mtext>obs</mtext>}}\text{=}\text{Σ L-serine][Σ P}{}_{\mathrm{i}}\text{]}\text{Σ L-phosphoserine]H}{}_2\text{O]}\text{, K =}\text{L-H · serine}{}^{\mathrm{\pm }}\text{]HPO}{}_4{}^{\mathrm{2?}}\text{]}\text{[L-H · phosphoserine}{}^{\mathrm{2?}}\text{]H}{}_2\text{O]}$. The value of K_obs has been found to be relatively sensitive to pH. At 38 °C, K⁺] = 0.2 m and free [Mg²⁺] = 0; K_obs = 80.6 m at pH 6.5, 52.7 m at pH 7.0 [ΔG_obs⁰ = ?10.2 kJ/mol (?2.45 kcal/mol)], and 44.0 m at pH 8.0 ([H₂O] = 1). The effect of the free [Mg²⁺] on K_obs was relatively slight; at pH 7.0 ([K⁺] = 0.2 m) K_obs = 52.0 m at free [Mg²⁺] = 10^?3, m and 47.8 m at free [Mg²⁺] = 10^?2, m. K_obs was insignificantly affected by variations in ionic strength (0.12–1.0 m) or temperature (4–43 °C) at pH 7.0. The value of K at 38 °C and I = 0.25 m has been calculated to be 34.2 ± 0.5 m [ΔG_obs⁰ = ?9.12 kJ/mol (?2.18 kcal/ mol)]([H₂O] = 1). The K for the phosphoserine phosphatase reaction has been combined with the K for the reaction of inorganic pyrophosphatase [EC 3.6.1.1] previously estimated under the same physiological conditions to calculate a value of 2.04 × 10⁴, m [ΔG_obs⁰ = ?28.0 kJ/mol (?6.69 kcal/mol)] for the K of the pyrophosphate:l-serine phosphotransferase [EC 2.7.1.80] reaction. $\text{K}{}_{\mathrm{<mtext>obs</mtext>}}\text{=}\text{[}\text{Σ L-serine}\text{][}\text{Σ}\text{P}{}_{\mathrm{<mtext>i</mtext>}}\text{]}\text{[}\text{Σ L-phosphoserine}\text{][}\text{H}{}_2\text{O}\text{]}\text{, K =}\text{[L-H · serine}{}^{\mathrm{\pm }}\text{]HPO}{}_4{}^{\mathrm{2?}}\text{]}\text{[L-H · phosphoserine}{}^{\mathrm{2?}}\text{]H}{}_2\text{O}$. Values of K_obs for this reaction at 38 °C, pH 7.0, and I = 0.25 m are very sensitive to the free [Mg²⁺], being calculated to be 668 [ΔG_obs⁰ = ?16.8 kJ/mol (?4.02 kcal/mol)] at free [Mg²⁺] = 0; 111 [ΔG_obs⁰ = ?12.2 kJ/mol (?2.91 kcal/mol)] at free [Mg²⁺] = 10^?3, m; and 9.1 [ΔG_obs⁰ = ?5.7 kJ/mol (?1.4 kcal/mol) at free [Mg²⁺] = 10^?2, m). K_obs for this reaction is also sensitive to pH. At pH 8.0 the corresponding values of K_obs are 4000 [ΔG_obs⁰ = ?21.4 kJ/mol (?5.12 kcal/mol)] at free [Mg²⁺] = 0; and 97.4 [ΔG_obs⁰ = ?11.8 kJ/ mol (?2.83 kcal/mol)] at free [Mg²⁺] = 10^?3, m. Combining K_obs for the l-phosphoserine phosphatase reaction with K_obs for the reactions of d-3-phosphoglycerate dehydrogenase [EC 1.1.1.95] and l-phosphoserine aminotransferase [EC 2.6.1.52] previously determined under the same physiological conditions has allowed the calculation of K_obs for the overall biosynthesis of l-serine from d-3-phosphoglycerate. $\text{K}{}_{\mathrm{<mtext>obs</mtext>}}\text{=}\text{[}\text{Σ L-serine}\text{][}\text{Σ NADH}\text{][}\text{Σ}\text{P}{}_{\mathrm{<mtext>i</mtext>}}\text{][}\text{Σ}\text{α-}\text{ketoglutarate}\text{]}\text{[Σ d-3-}\text{phosphoglycerate}\text{][Σ NAD}{}^{\mathrm{+}}\text{][Σ L-glutamat0]}$ The value of K_obs for these combined reactions at 38 °C, pH 7.0, and I = 0.25 m (K⁺ as the monovalent cation) is 1.34 × 10^?2, m at free [Mg²⁺] = 0 and 1.27 × 10^?2, m at free [Mg²⁺] = 10^?3, m.     

Dipole moments of random coil polypeptides

The Rotational Isomeric States model is applied to calculate dipole moments of polypeptides of the twenty natural α-amino acids in the random coil state. Dipole moments of each repeat unit (μ_i), are evaluated using a quantum mechanics procedure. Dipole moment ratios ($\text{D}{}_{\mathrm{x}}\text{=}\text{〈μ}{}^2\text{〉}\text{x}\text{μ}{}_{\mathrm{i}}{}^2\text{,}\text{x = number of repeat units}$) of homopolypeptides are calculated and extrapolated to x →?. With a few exceptions, D_? = 0.36 ± 0.1. Ten actual proteins and three enzymes are also studied; their dipole ratios (D_x′ =〈μ〉/x) range from 7.34 to 10.57 in 10^?59 C² m² (6.6–9.5 D²). Diffferences in the values of D_x′ are due mainly to the different contributions, μ_i, of the amino acid residues contained in each polymer, whereas the sequence of amino acids has a very minor effect.