Spectroscopic studies on the copper(II)—Inosine system

Interactions of inosine derivatives with copper(II) were studied in the pH range 1.4–13 in 50% H₂O-50% DMSO solution. The distinct pH dependence of the optical spectra observed in copper(II)-inosine complexes are correlated to their respective EPR changes as a function of pH. It was concluded that a simple 1:1 complex of copper(II)-inosine is formed in the pH range 1.4–5.0 and bis complexes are present in the pH 5.0–6.2 region solutions of inosine and Cu(II). From pH 6.2 to 7.8 a diamagnetic, hydroxybridged complex dominates. At pH 7.8–9.2 an insoluble, oxybridged species is formed in addition to the soluble paramagnetic Cu(NI)₄ complex. Starting from pH 9.1 the N-polymeric complex is formed which is stable up to pH 12.5, and above pH 12.5 the only species is the Cu(ribose)₂ complex.     

Isolation,primary structure,and synthesis of leucokinins V and VI: Myotropic peptides of Lleucophaea maderae

1. Two additional octapeptides which stimulate the contractile activity of the cockroach hindgut have been isolated from head extracts of the cockroach, Leucophaea maderae.2. A series of four high performance liquid-chromatographic (HPLC) fractionations yielded sufficient quantities of pure peptides for micro amino acid analysis and primary sequence determinations. The sequence determined for two peptides were: Gly-Ser-Gly-Phe-Ser-Ser-Trp-Gly-NH₂ and pGlu-Ser-Ser-Phe-His-Ser-Trp-Gly-NH₂. These octapeptides were designated leucokinins V and VI (L-V, L-VI), respectively.3. Minimum concentrations of natural L—V and L-VI required to produce a response from the isolated cockroach hindgut were 4.5 × and 4.9 × 10⁻¹¹ M, respectively. These concentrations were quite similar to the threshold concentrations observed for the synthetic products.     

Synthesis and conformation of the cyclic octapeptides cyclo(Phe-Pro)4, cyclo(Leu-Pro)4, and cyclo[Lys(Z)-Pro]4

Cyclic octapeptides, cyclo(X-Pro)₄, where X represents Phe, Leu, or Lys(Z), were synthesized and their conformations investigated. A C₂-symmetric conformer containing two cis peptide bonds was found in all of these cyclic octapeptides. The numbers of available conformations due to the cis–trans isomerization of Pro peptide bonds depended on the nature of the solvent and X residue: they decreased in the following order: cyclo[Lys(Z)-Pro]₄ > cyclo(Leu-Pro)₄ > cyclo(Phe-Pro)₄ in CDCl₃. ¹³C spin-lattice relaxation times (T₁) of these cyclic octapeptides were measured, and the contribution of segmental mobility to T₁ was found to vary with the nature of the X residue.     

Kinetics of reduction of ferrioxamine B by chromium(II), vanadium(II), and dithionite

Kinetic studies of the reduction of ferrioxamine B (Fe(Hdesf)⁺) by Cr(H₂O)₆²⁺, V(H₂O)₆²⁺, and dithionite have been performed. For Cr(H₂O)₆²⁺ and V(H₂O)₆²⁺, the rate is ?d[Fe(Hdesf)⁺]/dt = k[Fe(Hdesf)⁺][M²⁺]. For Cr(H₂O)₆²⁺, k = 1.19 × 10⁴ M^?1 sec^?1 at 25°C and μ = 0.4 M, and k is independent of pH from 2.6 to 3.5. For V(H₂O)₆²⁺, k = 6.30 × 10² M^?1 sec^?1 at 25°C, μ = 1.0 M, and pH = 2.2. The rate is nearly independent of pH from 2.2 to 4.0. For Cr(H₂O)₆²⁺ and V(H₂O)₆²⁺, the activation parameters are ΔH^≠ = 8.2 kcal mol^?1, ΔS^≠ ?12 eu and ΔH^≠ = 1.7 kcal mol^?1, ΔS^≠ = ?40 eu (at pH 2.2) respectively. Reduction by Cr(H₂O)₆²⁺ is inner-sphere, while reduction by V(H₂O)₆²⁺ is outer-sphere. Reduction by dithionite follows the rate law ?d[Fe(Hdesf)⁺]/dt =kK^{$\text{1}\text{2}$}[Fe(Hdesf)⁺][S₂O₄^2?]^{$\text{1}\text{2}$} where K is the equilibrium constant for dissociation of S₂O₄^2? into SO₂^? radicals. The value of k at 25°C and μ = 0.5 is 2.7 × 10³ M^?1 sec^?1 at pH 5.8, 3.5 × 10³ M^?1 sec^?1 at pH 6.8, and 4.6 × 10³ M^?1 sec^?1 at pH 7.8, and ΔH^≠ = 6.8 kcal mol^?1 and ΔS^≠ = ?19 eu at pH 7.8.     

Equilibrium constants under physiological conditions for the condensation of acetaldehyde with tetrahydrofolic acid

The nonenzymatic reaction of ethanol-derived CH₃CHO with tissue constituents continues to be of interest as a potential mechanism underlying the toxicity of alcohol. The current study has focused on the spontaneous condensation of CH₃CHO with H₄folate under physiological conditions (38 °C, pH 7.0, I = 0.25 M). Computer analysis of uv spectral changes with increasing CH₃CHO concentrations demonstrated the presence of at least two different adducts. The observed equilibrium constant (K_obs) for the formation of the first adduct is 91 ± 2 m^?1 (121 ± 2 m^?1 at 25 °C), a value which is unaffected by variations in ionic strength (0.06–1.0 m) or by free [Mg²⁺] up to 5 mm. The NMR spectrum is compatible with the structure: 5,10-CH₃CH-H₄folate analogous to the naturally occurring 5,10-CH₂-H₄folate. The formation of the latter compound from HCHO and H₄folate, however, is much more favorable under the same conditions [K_obs = 3.0 ± 0.2 × 10⁴ M^?1 (38 °C), 3.6 ± 0.1 × 10⁴ M^?1 (25 °C)]. At the levels of CH₃CHO which accumulate during ethanol metabolism in vivo only a small fraction of the H₄folate will exist as the CH₃CHO derivative, yet it may ultimately be the ratio of free CH₃CHO to free HCHO in tissue which determines the physiological importance of the CH₃CHO adduct. Other adduct(s) of CH₃CHO with H₄folate are observed at very high levels of CH₃CHO but are unlikely to be of physiological significance.     

Sequences of recently identified adipokinetic peptides: what do they tell us with respect to their hyperlipaemic activity in migratory locusts?

Complete dose-response curves for recently identified members of the AKH/RPCH family (four decapeptides and six octapeptides) have been measured inLocusta migratoria monitoring the lipid-mobilishing activityin vivo. In addition, dose-response curves have been produced for two octapeptide analogues which have a combination of amino acids at position 2 and 3 not occurring in naturally found AKH members. In the decapeptide members changes at position 10 from Thr to Ser are well tolerated, but the combination of Ser at position 5 and 7 around the Pro⁶ residue results in lowered activity, and efficacy of only 70%. In the octapeptides a single Leu/Val exchange at position 2 does not change the potency, however Tyr or Ile at position 2 lead to at least 3-fold loss of activity. The Ser⁵-Pro⁶-Ser⁷ combination in an octapeptide, as in the decapeptide, reduces potency. Octapeptides with 3 aromatic amino acids (Phe², Tyr⁴, Trp⁸) show no typical dose-response curve and have low efficacies. The combination of Val²-Thr³ which has never been found in an octapeptide is tolerated well, but Leu²-Val³ is not. The latter peptide is rather inactive and has a low efficacy; very likely because the hydrophilicity/hydrophobicity pattern at the N-terminus of the peptide is absent.     

Complex formation with alkali and alkaline earth metal ions of cyclic octapeptides,cyclo(Phe-Pro)4, cyclo(Leu-Pro)4, and cyclo[Lys(Z)-Pro]4

Complex formation with alkali and alkaline earth metal ions of cyclic octapeptides, cyclo(Phe-Pro)₄, cyclo(Leu-Pro)₄, and cyclo[Lys(Z)-Pro]₄ was investigated in relation to conformation. In an alcohol solution, cyclo(Phe-Pro)₄ did not form complexes. However, cyclo(Leu-Pro)₄ and cyclo[Lys(Z)-Pro]₄ formed complexes selectively with Ba²⁺ and Ca²⁺ ions. Changing the solvent from alcohol to acetonitrile, the complexation behavior was very different. In acetonitrile, cyclo(Phe-Pro)₄ was found to form a complex with Ba²⁺, and CD spectra of cyclo(Leu-Pro)₄ and cyclo[Lys(Z)-Pro]₄ changed sharply on complexation with K⁺. Rate constants of the complex formation between the cyclic octapeptides and metal salts were in the range of 0.7–12 L mol^?1 min^?1 in an alcohol solution. One of the two types of complex formation in acetonitrile was much faster than that in an alcohol solution.     

Toxins of the sea anemone Epiactis prolifera

Fe³⁺-EDTA chelates react with the superoxide radical at physiological pH values (k = 1.3 × 10⁶M^?1 s^?1 at pH 7 but is lower at more alkaline pH values) but do not appear to catalyze O₂^? dismutation at a significant rate. Complexes of Fe³⁺ with desferrioxamine, bathophenanthroline, or diethylenetriaminepentaacetic acid react much more slowly, if at all. Fe²⁺ complexes of EDTA, ATP, and diethylenetriaminepentaacetic acid also react with O₂^? at alkaline pH values. The significance of these reactions in the mechanism of the “iron-catalyzed Haber-Weiss reaction” is discussed.     

Dinuclear complexes with a μ-cyano ligand. Part X. Synthesis and characterization of several derivatives of cis-diaquaamminecobalt(III) complexes. Influence of pH

Six new dinuclear complexes, derived from cis-[Co(H₂O)₂(NH₃)₄]³⁺, cis-[Co(H₂O)₂(en)₂]³⁺ and [M(CN)₄^2? (M = Ni, Pd, Pt) were prepared and characterized by means of chemical analysis, electronic and IR measurements. The influence of the pH on the rate of the reaction was studied for the two derivatives of [Pd(CN)₄]^2?, showing that the best conditions to obtain the dinuclear compounds are at pH near 6, where the predominant species are cis-[Co(OH)(H₂O)(amine)₂]²⁺. The [Pt(CN)₄]^2? derivatives show PtPt interactions both in the solid state and in solution.     

Vanadyl(IV) conalbumin. II. Mixed metal and anion binding studies

Electron paramagnetic resonance (epr) studies demonstrate that at low levels of conalbumin (CA) saturation with Fe³⁺ or VO²⁺, a ph-dependent preference of the metal exists for different protein binding-site configurations,A, B, and C. The vanadyl ion epr spectra of mixed VO²⁺, Fe³⁺-conalbumin in which Fe³⁺ is preferentially bound to the N- or C-terminal binding site are consistent with all three configurations being formed at both metal sites. At high pH the spectra suggest interaction between binding sites. In the absence of HCO₃^?, VO²⁺ is bound almost exclusively in B configuration; a full binding capacity of 2 VO²⁺ per CA is retained. Stoichiometric amounts of HCO₃^? convert the epr spectrum from B to an A, B, C type. Addition of oxalate to bicarbonate-free preparations converts the B spectrum to an A′, B, C′ type where the B resonances have lost intensity to the A′ and C′ resonances but have not changed position. The data suggest that configuration B is anion independent and that only one equivalent of binding sites at pH 9 responds to the presence of HCO₃^1? or oxalate by changing configuration but not metal binding capability. The form of the bound anion may be HCO₃^? rather than CO₃^2?. The formation rate of the colored ferric conalbumin complex by oxidizing Fe²⁺ to Fe³⁺ in limited HCO₃^? at pH 9 is also consistent with one equivalent of sites having different anion requirements than the remaining sites. Increased NaCl or NaClO₄ concentration or substitution of D₂O for water as solvent affect the environment of bound VO²⁺, but the mechanisms of action are unknown.     

Fructose-1,6-diphosphatase from Mangifera indica

Fructose-1,6-diphosphatase (FDPase) from unripe mango was separated into two components by ammonium sulfate fractionation, one active at pH 6 (acidic FDPase) and the other at pH 8.5 (alkaline FDPase). The alkaline component had a lower K_m. (0.15 × 10^?3 M) than the acidic component (1.7 × 10^?3 M) towards the substrate (FDP) and the allosteric inhibitor AMP. It also showed greater heat stability and higher activation in the presence of EDTA as compared to the acidic FDPase. Both components showed a higher activation with Mn²⁺ ions than with Mg²⁺ ions.     

The solvent extraction of metal ions from aqueous solutions containing NNN′N′-Ethylenediaminetetraacetic and related ccids. Part 1. Uranium(IV)

The uranium(IV) complexes [U(EDTA)(H₂O)₂], [U(HOEDTA)]⁺, and [U(DTPA)]^? are well-formed in the pH fange 2–3 ([DTPA]^5- = diethylenetriaminepentaacetate; [HOEDTA]^3-  N-(2-hydroxyethyl)ethylenediaminetriacetate). Of these, only [U(DTPA)]- is extracted from an aqueous phase at pH 2 by the perchlorate salt of the primary amine, Primene JM-T. As the aqueous phase pH was raised, extraction occurred in all three cases and hydrolysed species may be extracted from EDTA and HOEDTA solutions but [U(DTPA)]^? resists hydrolysis. The addition of sulphate had a marked effect on the extraction of U(IV) from EDTA and HOEDTA through the formation of [U(EDTA)(SO₄)(H₂O)]^2- and [U(HOEDTA)(SO₄)(H₂O)_n]^?. The equilibrium constant, log β₁, for: [(U(EDTA)(H₂O)₂] 2 [SO₄]^2? ? [U(EDTA)(SO₄)(H₂O)]^2- 2 H₂O was found to be 2.43 ± 0.04 (I = 1 mol dm^?3, NaClO₄; pH 2.0; 20 °C) from spectrophotometric data.With tri-n-octylphosphine oxide (TOPO) electronic spectroscopy showed that the same U(IV) complex was extracted at pH 2 for Cs₂UCl₆, U(IV)/ HOEDTA, and U(IV)/DTPA and the aminepoly- carboxylates were aqueous phase masking agents but with [U(EDTA)(H₂O)₂] oxidation gave a uranyl(VI) organic phase species.Uranium(IV) is strongly extracted from aqueous solutions of HOEDTA at pH 2 or 3 by bis(2-ethyl- hexyl)phosphoric acid (HBEHP) but less so from EDTA and DTPA. Since U(IV) is completely extracted from Cs₂UCl₆ it could be that the amine- polycarboxylates were aqueous phase masking agents although spectral evidence did not support this.     

A comparison of the binding of imidazole to valence hybrid and methemoglobin: an assignment of individual heme reactivity

The ferric hemes of valence hybrid hemoglobins combine with imidazole in a manner analogous with the hemes of methemoglobin. Equilibrium studies show that imidazole binding to methemoglobin is minimally described by the sum of two independent processes (K₁ = 200 M^?1 and K₂ = 37 M^?1), both of which contribute equally to the observed difference spectrum. Using valance hybrid hemoglobins, which show single binding processes under similar conditions, it is possible to identify the high affinity sites in methemoglobin with the α chains and the low affinity sites with the β chains.Kinetic studies show that the valance hybrid hemoglobins react in a single exponential fashion with imidazole in contrast with methemoglobin which shows a biphasic reaction (k₁ = 85 M^?1 sec^?1k₂ = 25 M^?1 sec^?1). A comparison of the rates of reaction of the hybrids allows the assignment of the fast phase in methemoglobin to the β chains and the slow phase to the α chains.The heterogeneity of the imidazole reaction with methemoglobin occurs over the pH range 5.5–9.5 within which two ionization processes are discernable at pH 6.9 and 7.5.     

Syntheses of iron(III) aroyl hydrazones containing pyridoxal and salicylaldehyde. The crystal and molecular structure of two iron(III)-pyridoxal isonicotinoyl hydrazone complexes

Iron(III) complexes of three aroyl hydrazones, pyridoxal isonicotinoyl hydrazone (H₂pih), pyridoxal benzoyl hydrazone (H₂pbh), and salicylaldehyde benzoyl hydrazone (H₂sbh), were synthesized and characterized. In aqueous medium at pH 7, [Fe(pih)(Hpih)]·3H₂O is formed. In acidic methanol, a 1:1 ligand-to-metal complex is formed, [FeCl₂(H₂pih)]Cl (1), whereas in aqueous medium at low pH cis-[FeCl₂(H₂pih)(H₂O)]Cl·H₂O (2) is formed. Compounds 1 and 2 are high-spin d⁵ with μ_eff = 5.88 μ_B and 5.93 μ_B (298 K). The crystal structures of 1 and 2 show that H₂pih acts as a tridentate neutral ligand in which the phenolic and hydrazidic protons have shifted to the pyridine nitrogen atoms. The co- ordination polyhedron of 1 is ‘square’ pyramidal, whereas that of 2 is pseudo-octahedral. Compound 1 is triclinic, space group P$\text{l}$, with a = 12.704(2) Å, b = 8.655(2) Å, c = 8.820(2) Å, α = 105.42(1)°, β = 89.87(1)°, γ = 107.60(1)°, V = 888 ų, and Z = 2; 2 is monoclinic, space group P2₁/c, with a = 15.358(4) Å, b = 7.304(3) Å, c = 17.442(4) Å, β = 101.00(2)°, V = 1921 ų, and Z = 4.     

Histidine as an essential residue in the active site of the copper enzyme galactose oxidase.

The pH dependence of the oxidation of β-methyl-d-galactopyranoside by galactose oxidase at 1.33 mm O₂ has been determined. The k_cat exhibits a bell-shaped dependence on the ionization of at least two groups in the enzyme-substrate complex, pK_b' = 6.3 and pK_a' = 7.1, respectively. The pH-independent value for k_cat at 1.33 mm O₂ (nonsaturating) and saturating glycoside is 1435 s^?; the pH optimum is 6.7. Galactose oxidase is inactivated rapidly by iodoacetamide. Although the reaction is much slower, iodoacetate also inactivates the enzyme. The inactivation by iodoacetamide obeys saturation kinetics; at pH 7.0 k₃ = 2.19 min^?1 and K_i = 5.1 mM; k₃ but not K_i exhibits a bell-shaped pH dependence, with pK_a values of 6.3 and 7.6, respectively. Labeling with [¹⁴C]iodoacetamide establishes that one carboxamidomethyl group is incorporated per enzyme molecule. This incorporation parallels the loss of enzymatic activity. Only N-3-carboxymethylhistidine is detected in chromatograms following hydrolysis of the labeled protein. The protein-bound copper is not lost as a consequence of alkylation. Apogalactose oxidase does not react with iodoacetamide. The alkylation is inhibited by the oxidation of an active center tryptophan residue (s) by N-bromosuccinimide. The fraction of residual enzyme activity remaining after tryptophan oxidation corresponds to the extent of labeling by [¹⁴C]iodoacetamide. Although alkylation causes little change in the spin Hamiltonian parameters of the Cu(II) atom, it nearly abolishes both the optical activity and optical absorbance of the metal. The native tryptophan fluorescence of the enzyme, which is a sensitive probe of its active site, is also markedly affected. Since binding of a substrate, β-methyl-d-galactopyranoside, reduces fluorescence as it does in the active enzyme and binding of CN^? at the Cu(II) site as detected by electron spin resonance appears unaffected by the alkylation, the effect of alkylation is on catalysis, per se. Both a catalytic and a subtle conformational role for the active site histidine are inferred from the results.     

Analysis of the 220-MHz,P.M.R. spectra of some products of the amadori and heyns rearrangements

In order to establish whether p.m.r. spectroscopy is useful for identifying Amadori- and Heyns-rearrangement products, the p.m.r. spectra at 220 MHz of 16 rearrangement products derived from d-glucose or d-fructose and amino acids have been investigated. At pH 3, the protons of the NCH₂ group of N-substituted 1-amino-1-deoxy-d-fructose (Amadori-rearrangement products) resonate at δ 3.25–3.60 in D₂O and are shifted upfield by 0.3–0.6 p.p.m. at pH 9. These protons exchange with deuterium. Also, in D₂O there is an equilibrium of the acyclic, furanose, and pyranose structures, the last being favoured. At pH ? 7, the equilibrium is completely shifted to the β-pyranose form, which adopts exclusively the ²C₅ conformation. At pH 3, the equilibrium favours the β-furanose form. At pH 3, H-1e and H-1a of N-substituted 2-amino-2-deoxy-d-glucoses (Heyns-rearrangement products) resonate at δ 5.55 and 5.04, respectively. At pH 9, the signal for H-2 is shifted upfield by 0.2–0.7 p.p.m. In D₂O solution, these compounds exist as an equilibrium of α- and β-pyranose forms in the ⁴C₁ conformation. The α anomer is stabilised by the amino acid group at position 2. At pH 3, the αβ-ratio is 2–4:1, and, at pH 9, 1.0–1.1:1.     

A requirement for chelation in obtaining functional chloroplasts of sunflower and wheat.

In studying conditions for obtaining photosynthetically functional chloroplasts from mesophyll protoplasts of sunflower and wheat, a strong requirement for chelation was found. The concentration of chelator, either EDTA or pyrophosphate (PP_i), required for maximum activation depended on the pH, the concentration of orthophosphate (P_i) in the assay, and the chelator used. Studies with EDTA indicate that including the chelator in the isolation, resuspension, and assay media, in the absence of divalent cations, was most effective. Increased concentration of EDTA from 1 to 10 mm broadened the pH response curve for photosynthesis, inasmuch as a higher concentration of chelator was required for activation of photosynthesis at lower pH.Either EDTA, PP_i, or citrate could activate photosynthesis of sunflower chloroplasts isolated and assayed at pH 8.4. At pH 7.6, PP_i and EDTA were equally effective at low P_i concentrations but PP_i was particularly effective in shortening the induction period at high concentrations of P_i (2.5 mm) in the assay medium. Including 1 mm 3-phosphoglycerate in the assay medium with or without P_i could not replace the need for chelation. However, 3-phosphoglycerate + EDTA in the assay medium with 0.5 mm P_i, pH 7.6, gave a short induction period and rates of photosynthesis similar to those with 10 mm PP_i. The results suggest that PP_i can have a dual effect at the lower pH through chelation and inhibition of the phosphate transporter.Photosynthesis by sunflower chloroplasts isolated and assayed at pH 8.4 with 0.2 mm EDTA (+ 0.5 mm P_i in the assays) was severely inhibited by 2 mM CaCl₂, MgCl₂, or MnCl₂. Wheat chloroplasts isolated and assayed at pH 8.4 without chelation, and assayed with 0.2 mm P_i, had low rates of photosynthesis (25 μmol O₂ evolved mg^?1 chlorophyll h^?1) which were strongly inhibited by 2 to 4 mm MgCl₂, MnCl₂, or CaCl₂. With inclusion of EDTA and P_i at optimum levels, isolated chloroplasts of sunflower and wheat have high rates of photosynthesis and PP_i or divalent cations are not of benefit.     

Ion transport through liquid membrane by cyclic octapeptides

Cation transport through a chloroform liquid membrane by cyclic octapeptides—cyclo(Leu-Pro)₄, cyclo(Phe-Pro)₄, and cyclo[Lys(Z)-Pro]₄—was investigated. All of these cyclic octapeptides transported K⁺ and Ba²⁺, and the rate of cation transport was correlated with the ability to extract cations from the aqueous phase to the chloroform phase. Among them, cyclo (Leu-Pro)₄ was the most efficient and transported K⁺ and Ba²⁺ selectively from other alkali and alkaline earth cations, respectively. The rate of K⁺ transport by cyclo(Leu-Pro)₄ was about one-third as fast as that by dicyclohexyl 18-crown-6. Picrate anion transport against its concentration gradient was observed by cyclo(Leu-Pro)₄, which is conjugated with the selective transport of K⁺. Complex formation in a liposome between cyclo(Leu-Pro)₄ and Ba²⁺ was observed, but the binding constant was low.     

Vibrational circular dichroism of polypeptides, V. A study of 3(10)-helical-octapeptides

We have measured vibrational CD spectra in the 3600–1250 cm^?1 region of two monodisperse, protected octapeptides, which form right-handed 3₁₀-helices in CDC1₃ solution. The spectra are similar in sign pattern to those obtained for right-handed α-helices in solution but are smaller in magnitude and, additionally, provide evidence of some line-shape differences. The delineation of this type of ordered conformation was accomplished by means of ¹H-nmr. Such a solution structure is consistent with the x-ray crystal structure of one of these molecules.     

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.