Mechanism of action of thrombin on fibrinogen: Reaction of the N-terminal CNBr fragment from the Bβ chain of bovine fibrinogen with bovine thrombin

The N-terminal cyanogen bromide fragment from the Bβ chain of bovine fibrinogen was isolated, and its molecular weight was estimated to be approximately 14,000–15,500. The ratio of the Michaelis-Menten constants, $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$, for its hydrolysis by bovine thrombin was found to be 3 × 10^?7 [(NIH unit/liter)s]^?1, indicating that the Bβ fragment is a poor substrate for thrombin compared to the corresponding Aα chain fragment. This value of $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ is too small to account for the rate of release of fibrinopeptide B from fibrinogen by thrombin. It is suggested that, while the Aα chain contains all of the amino acid residues necessary to interact with thrombin, the Bβ chain does not; i.e., some of the binding sites that are used in the hydrolysis of the Bβ chain are assumed to be located on either the α or γ chains of fibrinogen. An alternative hypothesis is that, after the Bβ chain fragment is removed from the fibrinogen molecule, it does not have the proper conformation to be hydrolyzed by thrombin.     

Active site studies of human thrombin and bovine trypsin: peptide substrates.

Twenty-four oligopeptides modeled after the N-terminal portion of the α(A)-chain of human fibrinogen were synthesized and tested as substrates for human thrombin and bovine trypsin. The peptides contained either an Arg-Gly bond, or an Arg-Val bond, or both. Glycine and glutamic acid were substituted at various positions within the peptides, and from the $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ for each peptide with thrombin and with trypsin, the importance of residues on both sides of the Arg-Gly bond was evaluated. The trypsincatalyzed hydrolysis is faster than the thrombin-catalyzed hydrolysis of the peptides with glutamic acid at various distances N-terminal to the Arg-Gly bond. The ratio of the thrombic rate to the tryptic rate is fastest when glutamic acid is six residues away. Thrombin selectively cleaves the Arg-Gly bond in Ac-Val-Arg-Gly-Pro-Arg-Val-OH but cleaves both arginyl bonds rapidly in Ac-Val-Arg-Gly-Pro-Arg-Val-OMe. Trypsin selectively cleaves an Arg-Val bond in Ac-Arg-Gly-Pro-Arg-Val-OMe. The results are discussed in the light of thrombic cleavages of proteins. Most of these are seen to occur at highly polar sequences that frequently contain a proline residue.     

Hydrolysis of a thiopeptide by cadmium carboxypeptidase A

Substitution of the active site zinc ion of carboxypeptidase A by cadmium yields an enzyme inactive towards ordinary peptide substrates. However, a substrate analog (BzGlyNHCH₂CSPheOH) containing a thioamide linkage at the scissile position is cleaved to the thioacid. The kinetic parameters and their pH dependencies are $\text{k}{}_{\mathrm{cat}}\text{K}{}_{\mathrm{m}}\text{= 5.04 × 10}{}^4\text{min}{}^{\mathrm{?1}}\text{M}{}^{\mathrm{?1}}$, decreasing with either acid or base (PK_E1 = 5.64, pK_E2 = 9.55), and k_cat = 1.02 × 10² min^?1, decreasing with acid (pK_ES = 6.61). The thiopeptide is less efficiently cleaved by native (zinc) carboxypeptidase A. This cadmium-sulfur synergism supports a mechanism wherein the substrate amide is activated by metal ion coordination to its (thio) carbonyl.     

Comparative studies on human carboxypeptidases B and N

A series of dicarboxylic acid bi-product analogs of lysine and arginine have been tested as competitive inhibitors of human pancreatic carboxypeptidase B and human plasma carboxypeptidase N. The most effective derivative was guanidinoethylmercaptosuccinic acid with K_is of 0.5 and 1.0 × 10^?6m for Carboxypeptidases B and N, respectively. Values for the all-carbon guanidinopropylsuccinic acid were similar. In addition the kinetic parameters, K_m and $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$, have been determined for the hydrolysis of benzoyl-alanyl-lysine and benzoylalanyl-arginine by human Carboxypeptidases B and N. These substrates have been proposed for use in improved spectrophotometric assays. An enhanced affinity of these substrates versus benzoyl-glycyl-lysine or benzoyl-glycyl-arginine indicates a significant participation of the penultimate amino acid in catalysis of substrate.     

Reaction of peptide thiobenzyl esters with mammalian chymotrypsinlike enzymes: A sensitive assay method

Sensitive methods for the determination of rat mast cell protease I, rat mast cell protease II, human skin chymotrypsinlike enzyme, dog skin chymotrypsinlike enzyme, human leukocyte cathepsin G, and bovine chymotrypsin A_α with peptide thiobenzyl ester substrates are reported. Kinetic constants as well as the maximum sensitivity for the hydrolysis of the peptide substrates succinyl-phenylalanyl-leucyl-phenylalanine thiobenzyl ester and succinyl-alanyl-alanyl-prolyl-phenylalanine thiobenzyl ester were determined. Hydrolysis rates were followed spectrophotometrically at 324 nm by the formation of 4-thiopyridone (? = 19,800 m^?1 cm^?1), the product of the reaction between benzylthiol, released during hydrolysis of the peptide thiobenzyl esters, and 4,4′-dithiodipyridine present in the assay mixture. Peptide thiobenzyl ester substrates were shown to be very sensitive substrates, predominantly because of the large extinction coefficient of 4-thiopyridone and the high $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ values for these compounds.     

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.     

Mechanism of action of thrombin on fibrinogen. Reaction of the N-terminal CNBr fragment from the Aalpha chain of human fibrinogen with bovine thrombin

The 51-residue N-terminal cyanogen bromide fragment from the Aα chain of human fibrinogen was isolated, and the Michaelis-Menten constants, K_m and k_cat, for its hydrolysis by bovine thrombin were determined. The measured values of K_m and k_cat are 4.7 × 10^?5m and 4.8 × 10^?10m [(NIH U/liter) sec]^?1, respectively. Since these values are similar to those for fibrinogen, it appears that the N-terminal CNBr fragment contains all amino acid residues whose interactions with thrombin account for the high specificity of this enzyme for fibrinogen.     

Bacillus subtilis aminopeptidase: specificity toward amino acyl-beta-naphthylamides.

The kinetic parameters for the hydrolyses of different l-α-amino acid-β-naphthylamides by Bacillus subtilis aminopeptidase have been measured for the native enzyme and for the enzyme activated in 5 mm Co(NO₃)₂. In most cases Co²⁺ activation decreased K_m(app) values and increased k_cat values, in other cases k_m(app) and k_cat values were increased; for the remainder of the substrates tested k_m(app) values and k_cat values were decreased. In all cases tested the ratios of $\text{(}\text{k}\text{cat}\text{K}{}_{\mathrm{<mtext>m(</mtext><mtext>app</mtext><mtext>)</mtext>}}\text{)}\text{CO}{}^{\mathrm{2+}}\text{/(}\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{<mtext>m(</mtext><mtext>app</mtext><mtext>)nativ</mtext>}}\text{)}$ were increased (2- to 108-fold). For the native enzyme the order of specificity toward the l-amino acid-β-naphthylamides was Arg > Met > Trp > Lys > Leu and for the Co²⁺ activated enzyme the order of specificity was Lys > Arg > Met > Trp > Leu. The native enzyme hydrolyzed Pro-β-naphthylamide, but not α-Glu-β-naphthylamide; Co²⁺ activation of the enzyme affected an appreciable rate of hydrolysis of the latter substrate.     

The pH dependence of peptide hydrolysis by nitrocarboxypeptidase A

Hydrolysis of benzyloxycarbonyl-GlyGlyPhe by nitro(Tyr 248)carboxypeptidase A over the pH range 4.88–8.04 has been examined. The nitroenzyme retains appreciable activity near pH 6.5, and the limiting value of K_m is scarcely affected. The peptidase activity has a pH dependence characterized by the following parameters: pK_E1 of 6.37 ± 0.19 and pK_E2 of 6.60 ± 0.17 in $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$, and apparent pK of 5.59 ± 0.06 in K_cat. A spectroscopic pK of 6.75 ± 0.01, attributable to the nitro-Tyr 248 residue, has been determined. This correlates with the base-limb pK_E2 in the $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ profile, which appears to be shifted from a higher value, pK_E2 of 9.0, for the native enzyme. The single (acid-limb) pK which characterizes the k_cat profile of the native enzyme is also found to be perturbed to a lesser extent by nitration. A kinetically competent reverse protonation mechanism, based on chemical modification and crystallographic evidence for the enzyme, is described.     

Esterase activity of rabbit pulmonary angiotensin converting enzyme

A series of depsipeptides have been synthesized and used to demonstrate the esterase activity of rabbit pulmonary angiotensin converting enzyme. Among the esters studied, Bz-Phe-OPhe-Ala was found to have the highest $\text{k}{}_{\mathrm{cat}}\text{K}{}_{\mathrm{m}}$ which is about $\text{1}\text{5}$ that of its exact peptide analog, Bz-Phe-Phe-Ala. Esters such as Bz-Gly-OGly-Phe, Bz-Gly-OPhe-Phe and Bz-Gly-OLeu-Ala were also hydrolyzed but at much lower rates. Normal Michaelis-Menten behavior is observed and the kinetic parameters obtained indicate that the esters and their peptide analogs bind to the enzyme equally well, but that peptides are hydrolyzed at much higher rates. Studies on the pH-rate profiles, chloride ion effect, inhibition and chemical modifications detect no mechanistic differences between ester and peptide hydrolysis.     

Base-group specificity at the primary recognition site of ribonuclease T1 for minimal RNA substrates

Kinetic studies on the RNase T₁-catalyzed transesterification of 12 dinucleoside monophosphates, N_p¹N² (N¹ = A, C, and U; N² = A, C, G, and U) at pH 5, 25 °C, and 0.2 m ionic strength, revealed that the catalytic efficiency ($\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$) for GpN substrates (H. L. Osterman, and F. G. Walz, Jr., 1978, Biochemistry, 17, 4142) was ~10⁶-fold greater than corresponding ApNs and at least 10⁸-fold greater than corresponding CpNs and UpNs. The catalytic activity with ApN substrates survives phenol extraction which indicates (along with other criteria) that it is intrinsic to RNase T₁ and is not due to trace contamination by other nucleases. Circumstantial evidence is presented which suggests that homologous GpN and ApN substrates bind productively at different sites on the enzyme. The results of steady-state kinetic studies of RNase T₁ with IpNs (N = C and U) were compared with those for GpNs and indicated that the primary effect of the guanine 2-NH₂ group is to enhance substrate binding at the primary recognition site by ~2.6 kcal/mol. Values of ($\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$) showed the order NpC > NpU (N = A, G, and I) which evidences the existence of a subsite for the leaving nucleoside group that prefers cytidine: interactions at this subsite are reflected in k_cat rather than K_m.     

Structural dynamics of bacterial ribosomes: V. Magnesium-dependent dissociation of tight couples into subunits: Measurements of dissociation constants and exchange rates

The magnesium ion-dependent equilibrium of vacant ribosome couples with their subunits

$\text{70}\text{S}\text{?}\text{k}{}_{\mathrm{?1}}\text{k}{}_1\text{50}\text{S}\text{+30}\text{S}$

has been studied quantitatively with a novel equilibrium displacement labeling method which is more sensitive and precise than light-scattering. At a concentration of 10^?7m, tight couples (ribosomes most active in protein synthesis) dissociate between 1 and 3 mm-Mg²⁺ at 37 °C with a 50% point at 1.9 mm. The corresponding association constants K_a′ are 5.1 × 10⁵m^?1 (1 mm-Mg²⁺), 3.5 × 10⁷m^?1 (2 mm), and 1.2 × 10⁹m^?1 (3 mm), about five orders of magnitude higher than the K_a′ value of loose couples studied by Spirin et al. (1971) and Zitomer & Flaks (1972).In this range of Mg²⁺ concentrations ($\text{37 °C, 50 mm-}\text{NH}{}_4{}^{\mathrm{+}}$) the rate constants depend exponentially and in opposite ways on the Mg²⁺ concentration: k₁ = 2.2 × 10^?3s^?1, k_?1 = 7.7 × 10⁴m^?1s^?1 (2mm-Mg²⁺); k₁ = 1.5 × 10^?4s^?1, k_?1 = 1.7 × 10⁷m^?1s^?1 (5 mm-Mg²⁺). Under physiological conditions (Mg²⁺ ～- 4 mm, ribosome concn ～- 10^?7m), the equilibrium strongly favors association and the rate of exchange is slow ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～- 10}\text{min}$). In the range of dissociation (2 mm-Mg²⁺), association of subunits proceeds without measurable entropy change and hence ΔG^O = ΔH^O. The negative enthalpy change of ΔH^O = ? 10 kcal suggests that association of subunits involves a shape change.Below a critical Mg²⁺ concentration (～- 2 mm), the 50 S subunits are converted irreversibly into the b-form responsible for the transition to loose couples. The results are compatible with two classes of binding sites, one class binding Mg²⁺ non-co-operatively and contributing to the free energy of association by reduction of electrostatic repulsion, and another class probably consisting of hydrogen bonds between components at opposite interfaces whose critical spatial alignment rapidly denatures in the absence of stabilizing magnesium ions.     

Carbonic anhydrase catalyzed oxygen-18 exchange between bicarbonate and water

Oxygen-18 exchange techniques were applied to the dehydration of bicarbonate catalyzed by human carbonic anhydrase C. The rates of depletion of oxygen-18 from labeled bicarbonate were measured for both the catalyzed and uncatalyzed reactions at pH 9.4 and 25 °C. The equilibrium dissociation constant of the enzyme-substrate complex K is 0.321 ± 0.040 m and $\text{k}{}_{\mathrm{<mtext>enz</mtext>}}\text{=}\text{k}{}_2\text{K}{}_{\mathrm{m}}$ is (8.3 ± 1.9) × 10⁵m^?1 sec^?1 under these conditions. On the basis of these results it is demonstrated that the oxygen-18 exchange technique is capable of measuring K and k_enz for the carbonic anhydrase catalyzed dehydration of bicarbonate at a high pH range in which other kinetic techniques are not effective.It was also shown that the oxygen-18 exchange technique is an effective micromethod for the determination of carbonic anhydrase. Rates of isotopic depletion of labeled bicarbonate (in solutions of the enzyme) which fall outside the limits of error for the uncatalyzed rate of depletion demonstrate that this technique can detect concentrations of human carbonic anhydrase C as low as 5 × 10^?11m.     

Kinetic parameters for the binding of p-nitrophenyl alpha-d-mannopyranoside to concanavalin A.

Binding of the chromogenic ligand p-nitrophenyl α-d-mannopyranoside to concanavalin A was studied in a stopped-flow spectrometer. Formation of the protein-ligand complex could be represented as a simple one-step process. No kinetic evidence could be obtained for a ligand-induced change in the conformation of concanavalin A, although the existence of such a conformational change was not excluded. The entire change in absorbance produced on ligand binding occurred in the monophasic process monitored in the stopped-flow spectrometer. The value of the apparent second-order rate constant (k_a) for complex formation (k_a = 54,000 s^?1m^? at 25 °C, pH 5.0, Γ/2 0.5) was independent of the protein concentration when the protein was in the range of 233–831 μm in combining sites and in excess of the ligand. The apparent first-order rate constant (k_?a) for dissociation of the complex was obtained from the rate constant for the decomposition of the complex upon the addition of excess methyl α-d-mannopyranoside (k_?a = 6.2 s^?1 at 25 °C, pH 5.0, Γ/2 0.5). The ratio $\text{k}{}_{\mathrm{<mtext>a</mtext>}}{}_{\mathrm{<mtext>?</mtext><mtext>a</mtext>}}$ (0.9 × 10₄m^?1) was in reasonable agreement with value of 1.1 ± 0.1 × 10⁴m^?1 determined for the equilibrium constant for complex formation by ultraviolet difference spectrometry. Plots of ln($\text{k}{}_{\mathrm{<mtext>a</mtext>}}\text{T}$) and ln($\text{k}{}_{\mathrm{<mtext>a</mtext>}}\text{T}$) vs $\text{1}\text{T}$ were linear (T is temperature) and were used to evaluate activation parameters. The enthalpies of activation for formation and dissociation of the complex are 9.5 ± 0.3 and 16.8 ± 0.2 kcal/mol, respectively. The unitary entropies of activation for formation and dissociation of the complex are 2.8 ± 1.1 and 1.3 ± 0.7 entropy units, respectively. These entropy changes are much less than those usually associated with substantial changes in the conformation of proteins.     

Bacillus subtilis aminopeptidase: Specificity toward amino acid amides,dipeptides, and oligopeptides

Bacillus subtilis aminopeptidase hydrolyzed amino acid amides with a specificity similar to that determined using amino acyl-β-naphthylamides, but at much greater catalytic rates. Neutral and basic amino acid amides were the best substrates. A series of Leu and Lys NH₂-terminal dipeptides hydrolyzed by Co²⁺-activated aminopeptidase showed that the $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ ratios for the Lys substrates were fourfold greater than the corresponding Leu substrates and that catalytic differences reflected the identity of COOH terminal residues. Greatest catalytic rates were obtained when aromatic residues were in the COOH terminal position of the substrate (Trp, Tyr, Phe); but, significant hydrolysis was achieved when aliphatic residues were COOH-terminal in the dipeptide. The Co²⁺-activated enzyme would not hydrolyze peptide bonds composed of the imide nitrogen of Pro, thus, bradykinin was not a substrate. However, the Co²⁺-activated enzyme removed sequentially the first four residues from eledoisin-related peptide and the A chain of bovine insulin.     

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.     

Ontogeny of the mammalian insulin receptor: Studies of human and rat fetal liver plasma membranes

Insulin binding to human fetal plasma liver membranes was studied in preparations segregated into three pools according to length of gestation: 15–18 weeks (Pool A), 19–25 weeks (Pool B), and 26–31 weeks (Pool C). Receptor numbers, calculated by extrapolation of Scatchard plots to the X axis, increased from 25 × 10¹⁰ sites per 100 μg protein in the youngest group (Pool A) to 46 × 10¹⁰ sites per 100 μg protein in Pool B. No further increase in receptor number was seen in Pool C. The affinity constant for insulin at tracer concentrations, $\text{K}{}_{\mathrm{<mtext>e</mtext>}}$ (“empty site”), was 1.53 × 10⁸M^?1 in Pool A and was only slightly higher than $\text{K}{}_{\mathrm{<mtext>f</mtext>}}$ (“filled site”). $\text{K}{}_{\mathrm{<mtext>e</mtext>}}$ was higher in Pool B, 1.75 × 10⁸M^?1, and in Pool C reached a value of 5.63 × 10⁸M^?1. In Pool C $\text{K}{}_{\mathrm{<mtext>f</mtext>}}$ was 2.3 × 10⁸M^?1. Insulin binding of liver plasma membranes from rat fetuses aged 14, 16, 18, and 21 (term) days and adults was also studied. Maximum binding capacity tended to increase with gestational age and was 130 × 10¹⁰ sites per 100 μg protein at term, which was in excess of that found in adult rats (89–90 × 10¹⁰). In addition, $\text{K}{}_{\mathrm{<mtext>e</mtext>}}$ increased from 0.75 × 10⁸M^?1 at 14 days to 3.02 × 10⁸M^?1 at term, a value higher than that found in pregnant and nonpregnant adults. Dissociation of insulin in the presence of high concentrations of insulin was significantly enhanced in tissues from 18-day and term fetuses and adults, but not in membranes from fetal rats aged 14 and 16 days. These data appear to indicate that site-site interactions are not present in early fetal existence. These changes in insulin binding with increased length of gestation are not ascribable to changes in relative proportions of hematopoietic and parenchymal tissue. Human fetal plasma liver membranes demonstrated elevated insulin binding with increased gestational age, but comparison of fetal and adult liver could not be done. However, newborn human infants have been shown to have a higher capacity for binding insulin to circulating monocytes than adults. Also, human fetuses apparently lack the capability to diminish monocyte receptors in the presence of hyperinsulinemia. These experiments show that an increase in insulin receptor binding capacity and affinity also occurs in the liver of the rat fetus at term as compared to the adult rat. The reasons and mechanisms underlying enhanced capacity for insulin binding by fetal and newborn members of human and rodent species are not known.     

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.     

Thiophosphorylation of (Na + K+)-ATPase yields an ADP-sensitive phosphointermediate

(1) Treatment of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ from rabbit kidney outer medulla with the $\text{γ-}{}^{35}\text{S}$ labeled thio-analogue of ATP in the presence of $\text{Na}{}^{\mathrm{+}}\text{+ Mg}{}^{\mathrm{2+}}$ and the absence of K⁺ leads to thiophosphorylation of the enzyme. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for [γ-S]ATP is 2.2 μM and for Na⁺ 4.2 mM at 22°C. Thiophosphorylation is a sigmoidal function of the Na⁺ concentration, yielding a Hill coefficient $\text{n}\text{H}\text{= 2.6}$. (2) The thio-analogue ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 35\; \mu M</mtext>}}$) can also support overall $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity, but $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ at 37°C is only $\text{1.3 γ}\text{mol}\text{· (mg protein)}{}^{\mathrm{?}}\text{·}$ h^?1 or 0.09% of the specific activity for ATP ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 0.43\; mM</mtext>}}$). (3) The thiophosphoenzyme intermediate, like the natural phosphoenzyme, is sensitive to hydroxylamine, indicating that it also is an acylphosphate. However, the thiophosphoenzyme, unlike the phosphoenzyme, is acid labile at temperatures as low as 0°C. The acid-denatured thiophosphoenzyme has optimal stability at pH 5–6. (4) The thiophosphorylation capacity of the enzyme is equal to its phosphorylation capacity, indicating the same number of sites. Phosphorylation by ATP excludes thiophosphorylation, suggesting that the two substrates compete for the same phosphorylation site. (5) The (apparent) rate constants of thiophosphorylation (0.4 s^?1 vs. 180 s^?1), spontaneous dethiophosphorylation (0.04 s^?1 vs. 0.5 s^?1) and K⁺-stimulated dethiophosphorylation (0.54 s^?1 vs. 230 s^?1) are much lower than those for the corresponding reactions based on ATP. (6) In contrast to the phosphoenzyme, the thiophosphoenzyme is ADP-sensitive (with an apparent rate constant in ADP-induced dethiophosphorylation of 0.35 s^?1, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$$\text{ADP = 48 μM at 0.1 mM ATP}$) and is relatively K⁺-insensitve. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for K⁺ in dethiophosphorylation is 0.9 mM and in dephosphorylation 0.09 mM. The thiophosphoenzyme appears to be for 75–90% in the ADP-sensitive E₁-conformation.