Kinetic characteristics of the ATP-pyrophosphate isotope exchange catalyzed by RNA ligase from T4 bacteriophage

The dependence of initial rate v0 of ATP--PPi exchange reaction catalyzed by RNA-ligase of bacteriophage T4 on the concentration of ATP(s), pyrophosphate (z) and Mgcl2 has been determined. The dependence of v0 on s and z described by the equation v0 = k-1k2E0/(k-1 + K2) (1 + K1/s + k2/z) has been obtained for the reaction of E + S in equilibrium ES in equilibrium E1 + Z, where E--enzyme, E1--adenylylenzyme, S--ATP, Z--pyrophosphate, K1 and K2--constants of equilibrium, k-1, k2--velocity constants of transition of ES to E + S and E1 + Z, E0--complete concentration of enzyme. The low inhibition of the ATP--PPi exchange by the acceptor A(pA)2 and donors pAp, p(Ap)3, pCp has been shown. The dependence of v0 on the concentration of MgCl2 is consent with the incorporation of only dimagnesium salts of substrates in the isotope-exchange reaction.     

Elucidation of the chemical nature of the steady-state intermediates in the mechanism of carboxypeptidase A

Cryospectrokinetic studies of zinc and cobalt carboxypeptidase A disclosed two intermediates in the hydrolysis of both peptides and depsipeptides and furnished all the rate and equilibrium constants for the reaction scheme E + S in equilibrium ES1 in equilibrium ES2---E + P [Auld, D. S., Galdes, A., Geoghegan, K. F., Holmquist, B., Martinelli, R. A., & Vallee, B. L. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5041-5045]. Since the ES2 intermediate is the predominate enzyme species present at steady state, its chemical nature is deducible from subzero chemical quench studies done after steady state is established. Extrapolation of the product concentration to zero time, [P0], measures the concentration of the enzyme species in which bond cleavage has occurred. For peptides, the [P0]values are zero, indicating that no product is generated prior to turnover and therefore the ES2 intermediate involves a complex between enzyme and intact peptide substrate. For depsipeptides, [P0] values are 1 mol of produce per mole of enzyme over the entire temperature range -20 to -50 degrees C, indicating cleavage of the ester bond occurs prior to the rate-limiting step so that ES2 is more properly denoted by EP1P2, where P1 and P2 are the substrates for the reverse reaction. The rate-limiting step for depsipeptides thus involves release of the products which may occur directly or through a mandatory conformational change followed by rapid product release.     

PH-dependence of the steady-state rate of a two-step enzymic reaction.

1. The pH-dependence is considered of a reaction between E and S that proceeds through an intermediate ES under "Briggs-Haldane' conditions, i.e. there is a steady state in ES and [S]o greater than [E]T, where [S]o is the initial concentration of S and [E]T is the total concentration of all forms of E. Reactants and intermediates are assumed to interconvert in three protonic states (E equilibrium ES; EH equilibrium EHS; EH2 equilibrium EH2S), but only EHS provides products by an irreversible reaction whose rate constant is kcat. Protonations are assumed to be so fast that they are all at equilibrium. 2. The rate equation for this model is shown to be v = d[P]/dt = (kcat.[E]T[S]o/A)/[(KmBC/DA) + [S]o], where Km is the usual assembly of rate constants around EHS and A-D are functions of the form (1 + [H]/K1 + K2/[H]), in which K1 and K2 are: in A, the molecular ionization constants of ES; in B, the analogous constants of E; in C and D, apparent ionization constants composed of molecular ionization constants (of E or ES) and assemblies of rate constants. 3. As in earlier treatments of this type of reaction which involve either the assumption that the reactants and intermediate are in equilibrium or the assumption of Peller & Alberty [(1959) J. Am. Chem. Soc. 81, 5907-5914] that only EH and EHS interconvert directly, the pH-dependence of kcat. is determined only by A. 4. The pH-dependence of Km is determined in general by B-C/A-D, but when reactants and intermediate are in equilibrium, C identical to D and this expression simplifies to B/A. 5. The pH-dependence of kcat./Km, i.e. of the rate when [S]o less than Km, is not necessarily a simple bell-shaped curve characterized only by the ionization constants of B, but is a complex curve characterized by D/B-C. 6. Various situations are discussed in which the pH-dependence of kcat./Km is determined by assemblies simpler than D/B-C. The special situation in which a kcat./Km-pH profile provides the molecular pKa values of the intermediate ES complex is delineated.     

Sodium ion discharge from pig kidney Na+, K+-ATPase Na+-dependency of the E1P-E2P equilibrium in the absence of KCl

The two phosphoenzymes (E1P and E2P) of Na+,K+-ATPase were measured as ADP-sensitive and K+-sensitive fractions. The sum of these fractions was nearly 1 in the range of 50 to 1,200 mM NaCl. The effects of Na+ on the levels of E1P and E2P, on the rate constant of E2P leads to E1P transition (k2), on the rate constant of E2P dephosphorylation (k3), on the rate constant of E1P leads to E2P transition (k1) and on the apparent equilibrium constant between E1P and E2P (Kapp) were examined. k1 was found to decrease with increasing Na+ concentration, whereas k2 increased. Kapp was found to be directly proportional to the third power of Na+ concentration. k3 increased with increasing Na+ concentration and saturated at about 1 M NaCl. These results are consistent with a simple model in which ATP hydrolysis occurs through effectively only two phosphoenzyme intermediates in the absence of K+ and three sodium ions are discharged cooperatively from the enzyme during the E1P leads to E2P conversion.     

Protein-lipid interactions in cytochrome oxidase from Saccharomyces cerevisiae. Effects of detergents and reconstitution of enzyme activity by phospholipids by using cholate-mediated exchange.

Cytochrome oxidase, purified from the yeast Saccharomyces cerevisiae, was shown to have associated phospholipid, cholate or detergent, which could be varied by dialysis or (NH4)2SO4 precipitation of the protein. Cholate and the detergents Triton X-100 and Tween 80 were shown to differ in their ability to support enzyme activity. Changes in the Vmax, but not the Km, for ferrocytochrome c as the cholate concentration was varied indicate that cholate increases the number of exposed active sites of the enzyme. Cholate was used to introduce chosen phospholipids into the lipid environment of yeast cytochrome oxidase. Kinetic studies clearly showed that cholate can mediate exchange of exogenous for endogenous phospholipid. All phospholipids screened supported activity up to the basal value for the unsubstituted enzyme, whereas mitochondrial phosphatidylethanolamine and various phosphatidlycholines (except 1,2-dipalmitoyl-sn-glycero-3-phosphocholine) produced enhanced activity. A detailed kinetic examination revealed that the major effect of phosphatidylethanolamine is to increase k+1, whereas the major effect of phosphatidylcholine is to increase K+2 in the minimal kinetic scheme E + S k+1 in equilibrium k-1 ES k+2 leads to E + P Cardiolipin, although supporting activity, does not give any enhancement of k+1 or k+2 over the values for the cholate control. The relevance of these observations to protein-lipid interactions in cytochrome oxidase is discussed.     

The peptidoglycan crosslinking enzyme system in Streptomyces strains R61, K15 and rimosus. Kinetic coefficients involved in the interactions of the membrane-bound transpeptidase with peptide substrates and beta-lactam antibiotics

The transpeptidation reaction performed by the membranes of Streptomyces strain R61 fits the general rate equation for an enzyme-catalysed bimolecular reaction. The same membranes (E) interact with beta-lactams (I) to form inactive penicillin-enzyme-membrane complexes (EI) of rather high stability, which subsequently break down (E + I leads to EI leads to E + degradation products). The enzyme is regenerated and the antibiotic is released in the form of an inactive metabolite. With benzylpenicillin, the degradation product is benzylpenicilloic acid. The reaction is heat-labile. The first step of the reaction (E + I leads to EI) is characterized by a second-order rate constant (kformation in M-1 s-1) and the second step (EI leads to E + degradation products) by a first-order rate constant (kbreakdown in s-1). The effects in vitro of various beta-lactams on the membrane-bound transpeptidase, as expressed by the relevant kformation and kbreakdown values, parallel the effects in vivo of the same antibiotics as expressed by their ability to prevent the germination and growth of conidiospores. The kinetic parameters of the transpeptidase that was solubilized with N-cetyl-N,N,N-trimethylammonium bromide with respect to its interaction with both peptide substrates and beta-lactam antibiotics are quantitatively different from those of the membrane-bound enzyme. Moreover, the solubilized enzyme fragments benzylpenicillin with formation of phenylacetylglycine, a reaction which is similar to that catalysed by the exocellular R61 enzyme. The membranes of Streptomyces strains rimosus and K15 possess an active 'classic' penicillinase. They were not studied but the kinetic coefficients of the corresponding solubilized transpeptidases were determined and compared with those of the solubilized enzyme from strain R61.     

Specific association of bromocresol purple anions with a magnesium complex of a phosphorylated intermediate during steady-state hydrolysis of ATP by the Mg2+ + Ca2+-dependent ATPase of sarcoplasmic reticulum

The mechanism of dimeric binding of bromocresol purple (BCP) anions to Mg2+ + Ca2+-ATPase of the sarcoplasmic reticulum (SR) and the resulting partial inhibition of the ATPase activity were studied. BCP anions in three states, free monomer, bound monomer, and bound dimer, were spectrophotometrically calculated by solving simultaneous equations, delta A lambda 1-lambda 2 = sigma delta ai (epsilon i lambda 1-epsilon i lambda 2), and concentration changes of these states were analyzed. The addition of ATP caused an increase in the bound dimer and a decrease in the free monomer, but the change of the bound monomer was slight. The decrease in delta A (decrease phase) on the addition of ATP on dual-wavelength spectrophotometry at 585-610 nm was related to an increase in the amount of dimer bound to the SR membranes. The magnitude of the decrease phase increased with an increase in Mg2+ concentration and decreased with an increase in the concentration of Ca2+. BCP anions at the probe concentration partially inhibited the ATPase activity, and brought about a decrease in the ADP-sensitive E-P (E1P) and an increase in the ADP-insensitive E-P (E2P), though BCP anions did not affect the amount of total E-P. On elimination of Mg2+ at the steady-state E-P level both E2P and E2P . (BCP)2 were decomposed, suggesting that the enzyme form binding the BCP dimer was Mg . E-P. An increase in Mg2+ concentration increased E2P but an increase in Ca2+ concentration decreased E2P. Decomposition of E2P to P1 was inhibited by BCP anions. The following simple scheme was suggested to explain the partial inhibition of the ATPase activity, (Formula: see text). Application of BCP anions was discussed for use as a probe for Mg . E-P in the steady-state ATP hydrolysis.     

Fructose 1,6-bisphosphate aldolase from rabbit muscle. The isomerization of the enzyme-dihydroxyacetone phosphate complex.

The formation and dissociation of the aldolase-dihydroxyacetone phosphate complex were studied by following changes in A240 [Topper, Mehler & Bloom (1957), Science 126, 1287-1289]. It was shown that the enzyme-substrate complex (ES) slowly isomerizes according to the following reaction: (formula: see text) the two first-order rate constants for the isomerization step being k+2 = 1.3s-1 and k-2 = 0.7s-1 at 20 degrees C and pH 7.5. The dissociation of the ES complex was provoked by the addition of the competitive inhibitor hexitol 1,6-bisphosphate. At 20 degrees C and pH 7.5, k+1 was 4.7 X 10(6)M-1-S-1 and k-1 was 30s-1. Both the ES and the ES* complexes react rapidly with 1.7 mM-glyceraldehyde 3-phosphate, the reaction being practically complete in 40 ms. This shows that the ES* complex is not a dead-end complex. Evidence was also provided that aldolase binds and utilizes only the keto form of dihydroxyacetone phosphate.     

Functional consequences of mutations in the beta-strand sector of the Ca2(+)-ATPase of sarcoplasmic reticulum

Kinetic studies of the phosphoenzyme intermediates of site-specific mutants were used to examine the role of Gly233 in the reaction mechanism of the sarcoplasmic reticulum Ca2(+)-ATPase. When this glycine residue, which is highly conserved among cation-transporting ATPases, was replaced by valine, arginine, or glutamic acid, a complete loss of the ability to pump Ca2+ was observed. The mutant enzymes were able to form an ADP-sensitive phosphoenzyme intermediate (E1P) by reaction with ATP in the presence of Ca2+, but this intermediate decayed to the ADP-insensitive form (E2P) very slowly, relative to the wild-type enzyme. The mutant phosphoenzyme intermediate remained ADP-sensitive, even when phosphorylation from ATP was performed under conditions which permitted accumulation of the ADP-insensitive phosphoenzyme intermediate in the wild type. The mutants were also defective in their ability to form the ADP-insensitive phosphoenzyme intermediate by phosphorylation from inorganic phosphate. In addition, they displayed a higher affinity for Ca2+ and a lower cooperativity in Ca2+ binding than did the wild-type enzyme, as measured through the phosphorylation reaction with ATP. These findings can be rationalized either in terms of a parallel shift of E1 to E2 and E1P to E2P conformational equilibria toward the E1 and E1P forms, respectively, or in terms of destabilization of the phosphoryl-protein interaction in the E2P form. The roles of 7 other residues located in the vicinity of Gly233 were also examined by mutation. Although the side chains of these residues are potential Ca2+ ligands, their replacement did not affect the Ca2+ affinity of the enzyme, suggesting the lack of a role of this region of the peptide in formation of Ca2(+)-binding sites.     

Ligand-dependent reactivity of (Na+ + K+)-ATPase with showdomycin

Showdomycin inhibited pig brain (Na+ + K+)-ATPase with pseudo first-order kinetics. The rate of inhibition by showdomycin was examined in the presence of 16 combinations of four ligands, i.e., Na+, K+, Mg2+ and ATP, and was found to depend on the ligands added. Combinations of ligands were divided into five groups in terms of the magnitude of the rate constant; in the order of decreasing rate constants these were: (1) Na+ + Mg2+ + ATP, (2) Mg2+, Mg2+ + K+, K+ and none, (3) Na+ + Mg2+, Na+, K+ + Na+ and Na+ + K+ + Mg2+, (4) Mg2+ + K+ + ATP, K+ + ATP and Mg2+ + ATP, (5) K+ + Na + + ATP, Na+ + ATP, Na+ + K+ + Mg2+ + ATP and ATP. The highest rate was obtained in the presence of Na+, Mg2+ and ATP. The apparent concentrations of Na+, Mg2+ and ATP for half-maximum stimulation of inhibition (KS0.5) were 3 mM, 0.13 mM and 4 MicroM, respectively. The rate was unchanged upon further increase in Na+ concentration from 140 to 1000 mM. The rates of inhibition could be explained on the basis of the enzyme forms present, including E1, E2, ES, E1-P and E2-P, i. e., E2 has higher reactivity with showdomycin than E1, while E2-P has almost the same reactivity as E1-P. We conclude that the reaction of (Na+ + K+)- ATPase proceeds via at least four kinds of enzyme form (E1, E2, E1 . nucleotide and EP), which all have different conformations.     

The use of intrinsic protein fluorescence to quantitate enzyme-bound persulfide and to measure equilibria between intermediates in rhodanese catalysis

The intrinsic fluorescence of the enzyme rhodanese is quenched by as much as 30% when sulfur is transferred to the free enzyme form, E, giving the sulfur-substituted enzyme, ES. This fluorescence change (lambda ex = 295 nm and lambda em = 335 nm) has been used to quantitate the E and ES forms which are isolatable, obligatory intermediates in rhodanese catalysis. Fluorescence titration was performed using cyanide to irreversibly remove sulfur from ES. The results show a stoichiometry corresponding to 1 bound sulfur/molecule of the ES form of rhodanese (Mr = 33,000). The fluorescence changes were used to measure the concentrations of E and ES when these were in reversible equilibria induced by interactions with the substrates S2O3(2-) and SO3(2-). These results were compared with an equilibrium constant derived from published kinetic studies for the reaction (formula; see text) The very close agreement between the physical and kinetic methods indicate that there are no significant concentrations of intermediates other than E and ES. Overall, the results are compatible with the formation of a persulfide intermediate in rhodanese catalysis and are consistent with conclusions from x-ray crystallography and absorption spectroscopy. In addition, these procedures offer a facile method to measure equilibria between catalytic intermediates in the rhodanese reaction using functionally relevant concentrations.     

Cytoplasmic K+ effects on phosphoenzyme of Na,K-ATPase proteoliposomes and on the Na+-pump activity

Three phosphorylated reaction intermediates (EP) of Na,K-ATPase, and ADP-sensitive K+-insensitive EP (E1P), an ADP- and K+-sensitive EP (E*P), and a K+-sensitive ADP-insensitive EP (E2P), have been discovered at present. By using Na,K-ATPase proteoliposomes (PL) prepared from the electric eel enzyme, we found in this study that E*P existed even in the presence of K+ on both sides of the PL and that there was a sidedness difference in K+ sites between E*P and E2P. Cytoplasmic K+ (K+cyt) accelerated the conversion of E*P to E2P but did not dephosphorylate the E2P. Although the extracellular K+ accelerated the dephosphorylation of E2P, it did not interact with E*P directly. This K+cyt effect was also verified by the activation of Na+-pump in the Na+-K+ exchange mode. In the presence of K+cyt, both the ATP hydrolysis and Na+ uptake rates of the PL containing K+ inside vesicles increased sigmoidally with the concentrations of ATP and cytoplasmic Na+ (Na+cyt). However, in the absence of K+cyt, these Na+-pump reactions in PL containing K+ inside vesicles had only a hyperbolic curve. These results imply that the E*P to E2P conversion is one of the rate-limiting steps of the Na+-pump in the presence of a high concentration of ATP and that K+cyt may control this reaction step by enhancing the conversion rate of E*P to E2P.     

Tryptophan fluorescence of (Na+ + K+)-ATPase as a tool for study of the enzyme mechanism.

1. The protein fluorescence intensity of (Na+ + K+)-ATPase is enhanced following binding of K+ at low concentrations. The properties of the response suggest that one or a few tryptophan residues are affected by a conformational transition between the K-bound form E2 . (K) and a Na-bound form E1 . Na. 2. The rate of the conformational transition E2 . (K) leads to E . Na has been measured with a stopped-flow fluorimeter by exploiting the difference in fluorescence of the two states. In the absence of ATP the rate is very slow, but it is greatly accelerated by binding of ATP to a low affinity site. 3. Transient changes in tryptophan fluorescence accompany hydrolysis of ATP at low concentrations, in media containing Mg2+, Na+ and K+. The fluorescence response reflects interconversion between the initial enzyme conformation, E1 . Na and the steady-state turnover intermediate E2 . (K). 4. The phosphorylated intermediate, E2P can be detected by a fluorescence increase accompanying hydrolysis of ATP in media containing Mg2+ and Na+ but no K+. 5. The conformational states and reaction mechanism of the (Na+ + K+)-ATPase are discussed in the light of this work. The results permit a comparison of the behaviour of the enzyme at both low and high nucleotide concentrations.     

Kinetic analysis of the acid-alkaline conversion of horseradish peroxidases.

The nature of the acid-alkaline conversion of horseradish peroxidases was studied by measuring four rate constants in reactions, E + H+ (k1) in equilibrium (k2) EH+ and E + H2O (k3) in equilibrium (k4) EH+ + OH-, where EH+ and E denote the acid and alkaline forms of the enzymes. The values of k1, (k2 + k3), and k4 were obtained by measuring the relaxation rates of the acid leads to alkaline and alkaline leads to acid conversions by means of th pH jump method with a stopped-flow apparatus. The value of k3 could also be obtained by measuring the rate of reactions between hydrogen peroxide and peroxidases at alkaline pH. The measurements were conducted with four peroxidases having different pKa values: peroxidase A )pKa = 9.3), peroxidase C (pKa = 11.1), diacetyldeuteroperoxidase A (pKa = 7.7), and diacetyldeuteroperoxidase C (pKa = 9.1). The value of k1 was about 10(10) M-1 s-1 in the reaction of the four enzymes while k4 was quite different between the enzymes. The pKa was determined by k3 and k4 for the natural peroxidases and by k1 and k2 for the diacetyldeuteroperoxidases. The mechanism of the acid-alkaline conversion was discussed in comparison with that of metmyoglobin.     

The general modifier ("allosteric") unireactant enzyme mechanism: redundant conditions for reduction of the steady state velocity equation to one that is first degree in substrate and effector

The general unireactant modifier mechanism in the absence of product can be described by the following linked reactions: E + S k1 in equilibrium k-1 ES k3----E + P; E + I k5 in equilibrium k-5 EI; EI + S k2 in equilibrium k-2 ESI k4----EI + P; and ES + I k6 in equilibrium k-6 ESI where S is a substrate and I is an effector. A full steady state treatment yields a velocity equation that is second degree in both [S] and [I]. Two different conditions (or assumptions) permit reduction of the velocity equation to one that is first degree in [S] and [I]. These are (a) that k-2k3 = k-1k4 (Frieden, C., J. Biol. Chem. 239, pp. 3522-3531, (1964)) and (b) that the I-binding reactions are at equilibrium (Reinhart, G. D., Arch. Biochem. Biophys. 224, pp. 389-401 (1983)). It is shown that each condition gives rise to the other (i.e., if the I-binding reactions are at equilibrium, then k-2k3 must equal k-1k4 and vice-versa). If one assumes equilibrium for the I-binding steps, the velocity equation derived by the method of Cha (J. Biol. Chem. 243, pp. 820-825 (1968)) is apparently second degree in [I] (Segel, I. H., Enzyme Kinetics, p. 838, Wiley-Interscience (1975)), but reduces to a first degree equation when the relationship derived by Frieden is inserted. If one starts by assuming a single equilibrium condition for I binding, e.g., k-5[EI] = k5[E][I] or k-6[ESI] = k6[ES][I], then a traditional algebraic manipulation of the remaining steady state equations provides first degree expressions for the concentrations of all enzyme species and also discloses the Frieden relationship.     

The effect of monovalent and divalent cations on the ATP-dependent Ca2+-binding and phosphorylation during the reaction cycle of the sarcoplasmic reticulum Ca2+-transport ATPase

The coupling of Ca2+ movements and phosphate fluxes as well as the time-dependent occurrence of sequential reaction intermediates in the forward mode of the Ca,Mg-dependent ATPase reaction have been investigated using leaky vesicles (A23187) in the presence of varying Ca2+, Mg2+, and K+ concentrations. The employed ATP concentration of 2 microM does not allow more than one reaction cycle to occur. The respective fractions of ADP-sensitive and ADP-insensitive phosphoenzyme have been determined. The chosen experimental conditions (0-1 degree C, pH 6.0, absence of solubilizers) allow a prolonged time of observation and exclude interfering alterations of coupling and binding parameters, respectively. It is shown that under the experimental conditions K+ interacts with at least four different reaction steps (phosphoenzyme formation, E1P----E2P transition, E2P hydrolysis, and E2----E1 transformation). Mg2+ represents the sole ionic co-factor for the formation of the substrate MgATP if it is present in high concentrations (5 mM). Additional Ca2+ is bound to the substrate as well as to unspecific sites otherwise occupied by Mg2+ if Mg2+ is reduced to 0.1 mM. In this case the E1P----E2P transition rate (including Ca2+ translocation and Ca2+ release from low-affinity sites) is little diminished. If, in the absence of K+, both Mg2+ and Ca2+ are deficient E2P hydrolysis is vastly retarded. We find Ca2+ release to occur time-coincidently with E1P formation and not concomitantly with the comparably slow appearance of E2P; the molar amount of Ca2+ released, however, rather agreed with that of E2P formed. This suggests that under the prevailing conditions of a high proton concentration, phosphoenzyme states containing occluded Ca2+ or Ca2+ bound to low-affinity sites are transitional and not detectable. Preliminary findings on this subject have been published by us and colleagues from this laboratory [Hasselbach, W., Agostini, B., Medda, P., Migala, A. & Waas, W. (1985) in The sarcoplasmic reticulum calcium pump: Early and recent developments critically overviewed (Fleischer, S. & Tonomura, Y., eds) pp. 19-49, Academic Press, Orlando].     

Properties of the conversion of an enzyme-ATP complex to a phosphorylated intermediate in the reaction of Na+-K+-dependent ATPase1.

Previously, we proposed the following reaction machanism for the transport ATPase (EC 3.6.1.3) reaction in the presence of high concentrations of Mg2+ and Na+:(see article). Some kinetic and thermodynamic properties of steps 3 and 4 were investigated, and the following results were obtained. 1. When the reaction was started by adding ATP to the enzyme in the presence of 50 mM Na+ and 0.5 mM K+ or in the presence of 50mM Na+ and 0.5mM Rb+, the amount of E ADP P increased with time and maintained a constant level after reaching a maximum. We could not observe the initial burst of EP formation, which was observed by Post er al. in the presence of 8 mM Na+ and 0.01 mM Rb+. 2. The existence of quasi-equilibrium between E2ATP and E ADP P in the presence of low concentrations of Na+ was suggested by the fact that the values of the reciprocal of the equilibrium constant, K3 of step 3 obtained by the following three methods were almost the same. a) The value of 1+K3 was estimated from the ratio of vo/[EP] to kd, where vo is the rate of ATP hydrolysis in the steady state, [EP] the concentration of EP, and kd the first-order rate constant of EP disappearance after stopping EP formation. b) This value was also calculated from the ratio of the amount of P1 liberated to that of decrease in EP after stopping EP formation. c) The value of K3 was also calculated from the initial rapid decrease in EP on adding K+ and EDTA, assuming that the rapid decrease was due to a shift of the equilibrium toward E2ATP on adding K+. For example, the value of K3 with 10mM NaCL and 0.5mM KCL was 7--11. Although ATP formation due to a shift of the equilibrium toward E2ATP by a K+ jump in the presence of a low concentration of Na+ was observed at 0 degrees, the amount of ATP formed by a K+ jump at 15 degrees was less than the value expected from the shift of the equilibrium. 3. The values of delta H degrees and delta S degrees of step 3 were estimated in the presence of a sufficient amount of Na+ and in the absence of K+. They were +4--+5 kcal mole minus 1 and +15--+16 entropy units mole minus1, respectively. On the basis of kinetic studies of the elementary steps and the overall reaction of Na+-K+-dependent ATPase [EC 3.6.1.3], we (1--4) showed that a phosphorylated intermediate, EP, is formed via two kinds of enzyme-substrate complex, E1ATP and E2ATP, that the EP is in K+-dependent quasi-equilibrium with E2ATP, and that in the presence of high concentration of Mg2+, EP is in a high-energy state and contains bound ADP, E ADP P.(see article).     

A transient-kinetic study of the nitrogenase of Klebsiella pneumoniae by stopped-flow calorimetry. Comparison with the myosin ATPase.

The pre-steady-state kinetics of MgATP hydrolysis by nitrogenase from Klebsiella pneumoniae were studied by stopped-flow calorimetry at 6 degrees C and at pH 7.0. An endothermic reaction (delta Hobs. = +36 kJ.mol of ATP-1; kobs. = 9.4 s-1) in which 0.5 proton.mol of ATP-1 was released, has been assigned to the on-enzyme cleavage of MgATP to yield bound MgADP + Pi. The assignment is based on the similarity of these parameters to those of the corresponding reaction that occurs with rabbit muscle myosin subfragment-1 (delta Hobs. = +32 kJ.mol of ATP-1; kobs. = 7.1 s-1; 0.2 proton released.mol of ATP-1) [Millar, Howarth & Gutfreund (1987) Biochem. J. 248, 683-690]. MgATP-dependent electron transfer from the nitrogenase Fe-protein to the MoFe-protein was monitored by stopped-flow spectrophotometry at 430 nm and occurred with kobs. value of 3.0 s-1 at 6 degrees C. Thus, under these conditions, hydrolysis of MgATP precedes electron transfer within the protein complex. Evidence is presented that suggests that MgATP cleavage and subsequent electron transfer are reversible at 6 degrees C with an overall equilibrium constant close to unity, but that, at 23 degrees C, the reactions are essentially irreversible, with an overall equilibrium constant greater than or equal to 10.     

Regulation of exercise carbohydrate metabolism by estrogen and progesterone in women

To assess the roles of endogenous estrogen (E2) and progesterone (P4) in regulating exercise carbohydrate use, we used pharmacological suppression and replacement to create three distinct hormonal environments: baseline (B), with E2 and P4 low; estrogen only (E), with E2 high and P4 low; and estrogen/progesterone (E + P), with E2 and P4 high. Blood glucose uptake (R(d)), total carbohydrate oxidation (CHO(ox)), and estimated muscle glycogen utilization (EMGU) were assessed during 60 min of submaximal exercise by use of stable isotope dilution and indirect calorimetry in eight eumenorrheic women. Compared with B (1.26 +/- 0.04 g/min) and E + P (1.27 +/- 0.04 g/min), CHO(ox) was lower with E (1.05 +/- 0.02 g/min). Glucose R(d) tended to be lower with E and E + P relative to B. EMGU was 25% lower with E than with B or E + P. Plasma free fatty acids (FFA) were inversely related to EMGU (r(2) = 0.49). The data suggest that estrogen lowers CHO(ox) by reducing EMGU and glucose R(d). Progesterone increases EMGU but not glucose R(d). The opposing actions of E(2) and P(4) on EMGU may be mediated by their impact on FFA availability or vice versa.