Kinetic behaviour and properties of aldehyde dehydrogenase from rat testis mitochondria. Effect of Mg2+ ions.

1. Mitochondrial aldehyde dehydrogenase is purified to near homogeneity by hydroxylapatite-, affinity- and hydrophobic interaction-chromatography. 2. The enzyme is an oligomeric protein and its molecular weight, as determined by gel-filtration, is 117,000 +/- 5000. 3. Active only in the presence of exogenous sulfhydryl compounds and NAD(+)-dependent, aldehyde dehydrogenase works optimally with linear-chain aliphatic aldehydes and is practically inactive with benzaldehyde. The pH-optimum is at about pH 8.5. 4. Km-Values for aliphatic aldehydes (C2-C6) range between 0.17 and 0.32 microM. The Km for NAD+ increases from 16 microM with acetaldehyde to 71 microM with capronaldehyde. 5. Millimolar concentrations of Mg2+ promote high increases of both V and Km for NAD+. At the same time, saturation curves with C4-C6 aldehydes can be simulated with a substrate inhibition model. 6. Inhibition by NADH is competitive: with capronaldehyde, the inhibition constant for NADH is 52 microM in the absence of Mg2+ and 14 microM in the presence of 4 mM Mg2+; with acetaldehyde, the inhibition constant is about three times higher (36 and 159 microM, respectively).     

A model for the kinetics of activation and catalysis of ribulose 1,5-bisphosphate carboxylase.

Further evidence for time-dependent interconversions between active and inactive states of ribulose 1,5-bisphosphate carboxylase is presented. It was found that ribulose bisphosphate oxygenase and ribulose bisphosphate carboxylase could be totally inactivated by excluding CO2 and Mg2+ during dialysis of the enzyme at 4 degrees C. When initially inactive enzyme was assayed, the rate of reaction continually increased with time, and the rate was inversely related to the ribulose bisphosphare concentration. The initial rate of fully activated enzyme showed normal Michaelis-Menten kinetics with respect to ribulose bisphosphate (Km = 10muM). Activation was shown to depend on both CO2 and Mg2+ concentrations, with equilibrium constants for activation of about 100muM and 1 mM respectively. In contrast with activation, catalysis appeared to be independent of Mg2+ concentration, but dependent on CO2 concentration, with a Km(CO2) of about 10muM. By studying activation and de-activation of ribulose bisphosphate carboxylase as a function of CO2 and Mg2+ concentrations, the values of the kinetic constants for these actions have been determined. We propose a model for activation and catalysis of ribulose bisphosphate carboxylase: (see book) where E represents free inactive enzyme; complex in parentheses, activated enzyme; R, ribulose bisphosphate; M, Mg2+; C, CO2; P, the product. We propose that ribulose bisphosphate can bind to both the active and inactive forms of the enzyme, and slow inter-conversion between the two states occurs.     

Two distinct classes of nucleotide binding sites in sarcoplasmic reticulum Ca-ATPase revealed by 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)-ATP

It was previously reported that 2',3'-O-(2,4,6-trinitrocyclohexadienylidene) (TNP)-nucleotides bind with high affinity to the sarcoplasmic reticulum Ca-ATPase (Dupont, Y., Chapron, Y., and Pougeois, R. (1982) Biochem. Biophys. Res. Commun. 106, 1272-1279 and Watanabe, T., and Inesi, G. (1982) J. Biol. Chem. 257, 11510-11516). Here we report a study of the Ca-ATPase nucleotide binding sites using TNP-nucleotides. Competition at equilibrium between TNP-nucleotides and ATP was measured in the absence of calcium; it was found that TNP-nucleotides and ATP competitively bind to two classes of sites of equal concentration (3.5 nmol/mg). The ATP dissociation constants for the two classes of sites were found to be sensitive to H+ and Mg2+ concentrations. In the absence of Mg2+ (independently of pH) or at acid pH (independently of Mg2+ concentration), the nucleotide sites behave like one single family of sites of intermediate affinity (Kd = 20 microM). They split into two classes of sites of high (Kd = 2-4 microM) and low (Kd greater than 1 mM) affinity at pH values higher than neutral and in the presence of Mg2+. The calcium-activated ATP hydrolysis is accelerated by TNP-ATP (or TNP-AMP-PNP) binding on the phosphorylated enzyme. It is concluded 1) that the Ca-ATPase enzyme possesses two classes of ATP binding sites, 2) that the affinity of these two sites and the nature of their interaction is modulated by the H+ and Mg2+ concentrations, and 3) that the hydrolytic activity of the high affinity ATP binding site is activated by ATP or TNP-AMP-PNP (or TNP-ATP) binding in a low affinity ATP binding site.     

On the structure and conformational dynamics of yeast phenylalanine-accepting transfer ribonucleic acid in solution.

The solution structure of yeast tRNAPhe was investigated by using ethidium as a fluorescent probe in the D loop and the anticodon loop. For this purpose the dihydrouracils in position 16/17 and wybutine in position 37 were substituted by ethidium. The lifetimes and the time-dependent anisotropy of ethidium fluorescence were measured by pulsed nanosecond fluorometry. The kinetics of the transitions between different states of the tRNAPheEtd derivatives were determined by chemical relaxation measurements. It was found that the ethidium label irrespective of its position exhibits three different states called T1, T2 and T3 characterized by lifetimes tau 1 = 30 ns, tau 2 = 12 ns, and tau 3 = 3 ns. The lifetime differences are due to different accessibilities of ethidium for solvent quenching in the three states. Thus, there are three different defined structural environments of the ethidium in both the anticodon and the D loop. The distribution of the three states was measured as a function of Mg2+ concentration and temperature; it was found that state T3 is favored over states T2 and T1 by both increasing Mg2+ concentration and increasing temperature. The chemical relaxation kinetics exhibit a fast transition between T1 and T2 (10--100 ms) and a slow transition between T2 and T3 (100--1000 ms). The rates of both transitions depend likewise on Mg2+ concentration and temperature. The equilibrium and kinetic data clearly show the presence of strong and weak interactions between Mg2+ and tRNA. A cooperative model accounting for this behavior is developed. The ethidium probe behaves identically when located in different regions of the tRNA regarding both its distribution of states and its transition kinetics. This suggests that the different spectroscopic states report different conformations of the tRNA structure. The dependence of the three states on Mg2+ and spermine indicates that conformation T3 is closely related to or identical with the crystal structure. The rotational diffusion constants indicate that of all three states T3 is most extended while T2 is most compact. The thermodynamic analysis reveals that the strongly bound Mg2+ ions reduce both the activation entropy and enthalpy of all transitions. The weakly bound Mg2+ ions increase both the activation enthalpy and entropy of the slow transition between T2 and T3. It is suggested that the breaking of several intramolecular bonds, e.g., hydrogen bonds, is involved in this transition.     

Kinetic studies of calcium binding to parvalbumins from bullfrog skeletal muscle

In addition to steady-state properties of calcium binding to parvalbumins, kinetic studies are required for adequate evaluation of the physiological roles of parvalbumins. By using a dual-wavelength spectrophotometer equipped with a stopped-flow accessory, the transient kinetics of calcium binding to parvalbumins (PA-1 and 2) from bullfrog skeletal muscle was examined at 20 degrees C in medium containing 20 mM MOPS-KOH, pH 6.80, 0.13 mM tetramethylmurexide, 25 microM CaCl2, metal-deprived PA-1 or PA-2, various concentrations of Mg2+, and KCl to adjust the ionic strength of the medium to 0.106. The results can be explained in terms of the following rate constants under the conditions mentioned above when a second-order kinetic scheme is assumed. For PA-1, the association and apparent dissociation rate constants for Ca2+ are 1.5 X 10(7) M-1 X s-1 and 1.5 s-1, respectively, or more. The rate constants for Mg2+ are 7,500 M-1 X s-1 and 5-6 s-1, respectively. For PA-2, the rate constants for Ca2+ are 7 X 10(6) M-1 X s-1 and 1.16 s-1, respectively, and those for Mg2+ are 3,500 M-1 X s-1 and 3.5-4 s-1, respectively. Increased affinities for Ca2+ and Mg2+ at 10 degrees C are largely due to decreased apparent dissociation rate constants for these divalent cations, because no significant change in the association rate constants was found.     

Determination of the equilibrium association constant between Tet repressor and tetracycline at limiting Mg2+ concentrations: a generally applicable method for effector-dependent high-affinity complexes.

An analytical method for determining very high binding constants at equilibrium for reactions requiring an effector is proposed and applied to study the interaction of tetracycline with the repressor of the tetracycline resistance gene from Tn10. In this method complex formation is limited by low concentrations of the effector, which is Mg2+ for the interaction of tetracycline and Tet repressor. The binding of Mg2+ to tetracycline and subsequent formation of the ternary repressor-Mg(2+)-tetracycline complex are coupled reactions yielding a dependence of repressor-tetracycline-Mg2+ complex formation on the concentration of free Mg2+. The binding constants can be determined from the quantitative analysis of ternary complex formation with increasing Mg2+ concentrations. This method allows the determination of very high association constants at equilibrium in a large range of protein concentrations. In the case of repressor and tetracycline, the same affinity constant of 3 +/- 2 x 10(9) M-1 was found in the range of 0.1 to 5 microM of repressor. This result indicates that no association or dissociation of the repressor subunits occurs upon binding of tetracycline. Furthermore, the results show that a repressor dimer binds two effector molecules without significant cooperativity.     

Characterization of steady state, single-turnover, and binding kinetics of the TaqI restriction endonuclease.

The TaqI restriction endonuclease recognizes and cleaves the duplex DNA sequence T decreases CGA. Steady state kinetic analysis with a small oligodeoxyribonucleotide substrate showed that the enzyme obeyed Michaelis-Menten kinetics (Km = 53 nM, kcat = 1.3 min-1 at 50 degrees C and Km = 0.5 nM, kcat = 2.9 min-1 at 60 degrees C). At 0 degree C, the enzyme was completely inactive, while at 15 degrees C, turnover produced nicked substrate as the major product in excess of enzyme indicating dissociation between nicking events. Above 37 degrees C, both strands in the duplex were cleaved prior to dissociation. In contrast to the tight, temperature-dependent binding of substrate, binding of the Mg2+ cofactor was weak (Kd = 2.5 mM) and the same at either 50 degrees C or 60 degrees C. Single-turnover experiments using oligonucleotide substrate showed that hydrolysis of duplex DNA occurred via two independent nicking events, each with a first order rate constant (kst) of 5.8 min-1 at 60 degrees C and 3.5 min-1 at 50 degrees C. The pH dependence of Km (pKa = 9) and kst (pKa = 7) suggests Lys/Arg and His, respectively, as possible amino acids influencing these constants. Moreover, although kst increased significantly with pH, kcat did not, indicating that at least two steps can be rate-controlling in the reaction pathway. Binding of protein to canonical DNA in the presence of Mg2+ at 0 degree C or in the absence of Mg2+ at 50 degrees C was weak (Kd = 2.5 microM or 5,000-fold weaker than the optimal measured Km) and equal to the binding of noncanonical DNA as judged by retention on nitrocellulose. Similar results were seen in gel retardation assays. These results suggest that both Mg2+ and high temperature are required to attain the correct protein conformation to form the tight complex seen in the steady state analysis. In the accompanying paper (Zebala, J. A., Choi, J., Trainor, G. L., and Barany, F. (1992) J. Biol. Chem. 267, 8106-8116), we report how these kinetic constants are altered using substrate analogues and propose a model of functional groups involved in TaqI endonuclease recognition.     

Isocitrate lyase from Phycomyces blakesleeanus. The role of Mg2+ ions, kinetics and evidence for two classes of modifiable thiol groups.

Isocitrate lyase was purified from Phycomyces blakesleeanus N.R.R.L. 1555(-). The native enzyme has an Mr of 240,000. The enzyme appeared to be a tetramer with apparently identical subunits of Mr 62,000. The enzyme requires Mg2+ for activity, and the data suggest that the Mg2(+)-isocitrate complex is the true substrate and that Mg2+ ions act as a non-essential activator. The kinetic mechanism of the enzyme was investigated by using product and dead-end inhibitors of the cleavage and condensation reactions. The data indicated an ordered Uni Bi mechanism and the kinetic constants of the model were calculated. The spectrophotometric titration of thiol groups in Phycomyces isocitrate lyase with 5.5'-dithiobis-(2-nitrobenzoic acid) gave two free thiol groups per subunit of enzyme in the native state and three in the denatured state. The isocitrate lyase was completely inactivated by iodoacetate, with non-linear kinetics. The inactivation data suggest that the enzyme has two classes of modifiable thiol groups. The results are also in accord with the formation of a non-covalent enzyme-inhibitor complex before irreversible modification of the enzyme. Both the equilibrium constants for formation of the complex and the first-order rate constants for the irreversible modification step were determined. The partial protective effect of isocitrate and Mg2+ against iodoacetate inactivation was investigated in a preliminary form.     

Factors affecting the inhibition of yeast plasma membrane ATPase by vanadate

Inhibition of yeast plasma membrane ATPase by vanadate occurs only if either Mg2+ or MgATP2- is bound to the enzyme. The dissociation constant of the complex of vanadate and inhibitory sites is 0.14-0.20 microM in the presence of optimal concentrations of Mg2+ and of the order of 1 microM if the enzyme is saturated with MgATP2-. The dissociation constants of Mg2+ and MgATP2- for the sites involved are 0.4 and 0.62-0.73 mM, respectively, at pH 7. KCl does not increase the affinity of vanadate to the inhibitory sites as was found with (Na+ + K+)-ATPase. On the other hand, the effect of Mg2+ upon vanadate binding is similar to that upon (Na+ + K+)-ATPase, and the corresponding affinity constants of Mg2+ and vanadate for the two enzymes are of the same order of magnitude.     

Initial characterization of aldehyde dehydrogenase from rat testis cytosol

Aldehyde dehydrogenase was purified 187-fold from cytosol of rat testis by chromatographic methods and gel filtration with a yield of about 50%. The enzyme exhibits absolute requirement for exogenous sulfhydryl compounds and strong dependence on temperature. Addition of 0.4mM Ca2 or Mg2 ions results in 50% inhibition. Optimally active at pH 8.5 and 50 degrees C, aldehyde dehydrogenase displays broad substrate specificity; saturation curves with acetaldehyde and propionaldehyde are non-hyperbolic, with Hill coefficients comprised between 0.8 and 0.7. Strong substrate inhibition can be observed with both aromatic and long-chain alyphatic aldehydes. According to mathematical models, Km decreases from 246 microM for acetaldehyde to 4 microM for capronaldehyde and Ki decreases from about 4mM for butyraldehyde to 0.2 mM for capronaldehyde.     

The G-protein of retinal rod outer segments (transducin). Mechanism of interaction with rhodopsin and nucleotides

The mechanism of interaction of the G-protein of retinal rods with rhodopsin and with nucleotides has been investigated using two independent techniques, light-scattering and direct binding measurements with labeled nucleotides. Binding of photoexcited rhodopsin (R*) and nucleotides are shown to be antagonist, and three conformations of the G-protein are described, each of which is proposed to be related to a different level of light-scattering, as follows: (a) the "dark" state, stable in the absence of photoexcited rhodopsin, in which the nucleotide site is poorly accessible and has a high affinity (dissociation constants, 0.1 microM for GDP and 0.01 microM for GppNHp); (b) the R*-bound state in which the nucleotide site is rapidly accessible with a lower affinity (dissociation constants, about 20 microM for GDP and GTP; 20-100 microM for GppNHp). Binding of R* to the G-protein therefore enables rapid binding or exchange of the nucleotide; this in turn reduces the affinity of the G-protein for R* (dissociation constants, 0.2 microM for G-protein with GDP bound and 2-10 microM for G-protein with GppNHp bound, compared to 1 nM in absence of bound nucleotide); and (c) the third state, the activator of the phosphodiesterase. In the presence of GTP, an additional irreversible and fast step, which is proposed to be the dissociation of alpha-GTP from beta gamma, is shown to occur; a steady state equilibrium is obtained, and the dissociation constant measured between GTP and this third state of the G-protein in the presence of R* is an apparent constant which depends on the rate of transconformation between the first two states and on the rate of GTP hydrolysis. The minimum value of this apparent dissociation constant for GTP (0.05-0.1 (microM) is obtained at high levels of illumination. Finally, some results (number of nucleotide sites and saturation of the rate of the light-scattering signal) suggest an oligomeric association of the G-protein.     

Calcium and magnesium dependence of phospholipase A2-catalyzed hydrolysis of phosphatidylcholine small unilamellar vesicles.

The Ca2+ requirement for lipid hydrolysis catalyzed by phospholipase A2 from Agkistrodon piscivorus piscivorus (App-D49) and porcine pancreas has been examined using small, unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC SUV). Hydrolysis was affected by product inhibition even at early times, and the extent of this inhibition depended on the concentration of divalent cations. The Ca2+ requirement for half-maximal rates of hydrolysis reflected, in part, this non-catalytic role of divalent cations. The presence of 10 mM Mg2+, a cation which does not support catalysis, reduced the Ca2+ required for half-maximal rates of hydrolysis from millimolar concentrations to 40 microM for App-D49. Since the dissociation constant of the enzyme for Ca2+ in solution is 2 mM, these results indicate a change in the interaction of the enzyme with Ca2+ under catalytic conditions. The kinetic dissociation constant of Ca2+ for the pancreatic enzyme was 20 microM which is substantially lower than the dissociation constant in solution, 0.35 mM. The similarity of apparent kinetic dissociation constants for these enzymes suggests that structurally similar features determine the affinity for Ca2+ under catalytic conditions. Evidence is presented that the affinity of phospholipase A2 for Ca2+ changes subsequent to the initial interaction of the enzyme with the substrate interface. However, the apparent Michaelis constant, KMapp, for App-D49, 0.03-0.06 mM, is independent of [Ca2+] and is about the same as the equilibrium dissociation constant for DPPC SUV, 0.14 mM. We thus suggest that KMapp is a steady-state constant.     

Determining RuBisCO activation kinetics and other rate and equilibrium constants by simultaneous multiple non-linear regression of a kinetic model

The forward and reverse rate constants involved in carbamylation, activation, carboxylation, and inhibition of D-ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) have been estimated by a new technique of simultaneous non-linear regression of a differential equation kinetic model to multiple experimental data. Parameters predicted by the model fitted to data from purified spinach enzyme in vitro included binding affinity constants for non-substrate CO2 and Mg2+ of 200+/-80 microM and 700+/-200 microM, respectively, as well as a turnover number (k(cat)) of 3.3+/-0.5 s(-1), a Michaelis half-saturation constant for carboxylation (K(M,C)) of 10+/-4 microM and a Michaelis constant for RuBP binding (K(M,RuBP)) of 1.5+/-0.5 microM. These and other constants agree well with previously measured values where they exist. The model is then used to show that slow inactivation of RuBisCO (fallover) in oxygen-free conditions at low concentrations of CO2 and Mg2+ is due to decarbamylation and binding of RuBP to uncarbamylated enzyme. In spite of RuBP binding more tightly to uncarbamylated enzyme than to the activated form, RuBisCO is activated at high concentrations of CO2 and Mg2+. This apparent paradox is resolved by considering activation kinetics and the fact that while RuBP binds tightly but slowly to uncarbamylated enzyme, it binds fast and loosely to activated enzyme. This modelling technique is presented as a new method for determining multiple kinetic data simultaneously from a limited experimental data set. The method can be used to compare the properties of RuBisCO from different species quickly and easily.     

Kinetics of dissociation of parvalbumin complexes with calcium and magnesium ions

Dissociation kinetics of parvalbumin complexes with calcium and magnesium ions were studied by means of stopped-flow method employing intrinsic protein fluorescence registration. In the temperature range from 10 to 30 degrees C the kinetic curves of Ca2+ and Mg2+ dissociation are best fitted with a sum of two exponential terms, each term is ascribed to a dissociation process in one of two bindings sites of parvalbumin. Dissociation rate constants in this temperature range increase from 0.03 to 0.8 s-1 and from 0.18 to 5 s-1 for Ca2+, and from 0.9 to 4.5 s-1 and from 4 to 33 s-1 for Mg2+. Parvalbumin equilibrium binding constants of Ca2+ and Mg2+ were also measured in the same temperature range. It makes possible to estimate the rate constants of association of Ca2+ and Mg2+. In the case of Ca2+ the rate of association approaches the diffusion controlled limit.     

Magnesium, ADP, and actin binding linkage of myosin V: evidence for multiple myosin V-ADP and actomyosin V-ADP states

The [Mg(2+)] dependence of ADP binding to myosin V and actomyosin V was measured from the fluorescence of mantADP. Time courses of MgmantADP dissociation from myosin V and actomyosin V are biphasic with fast observed rate constants that depend on the [Mg(2+)] and slow observed rate constants that are [Mg(2+)]-independent. Two myosin V-MgADP states that are in reversible equilibrium, one that exchanges nucleotide and cation slowly (strong binding) and one that exchanges nucleotide and cation rapidly (weak binding), account for the data. The two myosin V-MgADP states are of comparable energies, as indicated by the relatively equimolar partitioning at saturating magnesium. Actin binding lowers the affinity for bound Mg(2+) 2-fold but shifts the isomerization equilibrium approximately 6-fold to the weak ADP binding state, lowering the affinity and accelerating the overall rate of MgADP release. Actin does not weaken the affinity or accelerate the release of cation-free ADP, indicating that actin and ADP binding linkage is magnesium-dependent. Myosin V and myosin V-ADP binding to actin was assayed from the quenching of pyrene actin fluorescence. Time courses of myosin V-ADP binding and release are biphasic, consistent with the existence of two (weak and strong) quenched pyrene actomyosin V-ADP conformations. We favor a sequential mechanism for actomyosin V dissociation with a transition from strong to weak actin-binding conformations preceding dissociation. The data provide evidence for multiple myosin-ADP and actomyosin-ADP states and establish a kinetic and thermodynamic framework for defining the magnesium-dependent coupling between the actin and nucleotide binding sites of myosin.     

The three conformations of the anticodon loop of yeast tRNA(Phe)

The complex conformational states of the anticodon loop of yeast tRNA(Phe) which we had previously studied with relaxation experiments by monitoring fluorescence of the naturally occurring Wye base, are analyzed using time and polarization resolved fluorescence measurements at varying counterion concentrations. Synchrotron radiation served as excitation for these experiments, which were analyzed using modulating functions and global methods. Three conformations of the anticodon loop are detected, all three occurring in a wide range of counterion concentrations with and without Mg2+, each being identified by its typical lifetime. The fluorescence changes brought about by varying the ion concentrations, previously monitored by steady state fluorimetry and relaxation methods, are changes in the population of these three conformational states, in the sense of an allosteric model, where the effectors are the three ions Mg2+, Na+ and H+. The population of the highly fluorescent M conformer (8ns), most affine to magnesium, is thus enhanced by that ligand, while the total fluorescence decreases as lower pH favors the H+-affine H conformer (0.6ns). Na+-binding of the N conformer (4ns) is responsible for complex fluorescence changes. By iterative simulation of this allosteric model the equilibrium and binding constants are determined. In turn, using these constants to simulate equilibrium fluorescence titrations reproduces the published results.     

Kinetic studies with myo-inositol monophosphatase from bovine brain

The kinetic properties of myo-inositol monophosphatase with different substrates were examined with respect to inhibition by fluoride, activation or inhibition by metal ions, pH profiles, and solvent isotope effects. F- is a competitive inhibitor versus 2'-AMP and glycerol 2-phosphate, but noncompetitive (Kis = Kii) versus DL-inositol 1-phosphate, all with Ki values of approximately 45 microM. Activation by Mg2+ follows sigmoid kinetics with Hill constants around 1.9, and random binding of substrate and metal ion. At high concentrations, Mg2+ acts as an uncompetitive inhibitor (Ki = 4.0 mM with DL-inositol 1-phosphate at pH 8.0 and 37 degrees C). Activation and inhibition constants, and consequently the optimal concentration of Mg2+, vary considerably with substrate structure and pH. Uncompetitive inhibition by Li+ and Mg2+ is mutually exclusive, suggesting a common binding site. Lithium binding decreases at low pH with a pK value of 6.4, and at high pH with a pK of 8.9, whereas magnesium inhibition depends on deprotonation with a pK of 8.3. The pH dependence of V suggests that two groups with pK values around 6.5 have to be deprotonated for catalysis. Solvent isotope effects on V and V/Km are greater than 2 and 1, respectively, regardless of the substrate, and proton inventories are linear. These results are consistent with a model where low concentrations of Mg2+ activate the enzyme by stabilizing the pentacoordinate phosphate intermediate. Li+ as well as Mg2+ at inhibiting concentrations bind to an additional site in the enzyme-substrate complex. Hydrolysis of the phosphate ester is rate limiting and facilitated by acid-base catalysis.     

Role of calcium as an inhibitor of rat liver carbamylphosphate synthetase I

The mechanism of Ca2+ inhibition of carbamylphosphate synthetase I has been investigated using purified enzyme obtained from livers of rats fed a high protein diet. Binding of Mn2+ to the enzyme was measured by EPR techniques at pH 7.8, and Scatchard plots of the data indicated one Mn2+-binding site with a K'd of 13 microM. From competition studies between Mn2+ and Ca2+ or Mg2+ binding, values of 180 microM were obtained for K'd (Mg) and 193 microM for K'd (Ca). A nonlinear least squares curve fitting program was used to calculate the K'm for MgATP2- at the metal-nucleotide binding sites using a simplified rate equation of the enzyme reaction mechanism. Values of 140 and 2420 microM were obtained for K'm (MgATP) at the first and second sites, respectively, at pH 7.8, with a free Mg2+ of 1 mM and other substrates and activators present at saturating concentrations. Variations of the bicarbonate, N-acetylglutamate, and ammonia concentrations in the absence and presence of different amounts of total calcium, from which free Ca2+, free Mg2+, MgATP2-, and CaATP2- concentrations were calculated, permitted values for K'i (CaATP) to be obtained by graphic procedures. Mean values of 375 and 120 microM were obtained for K'i (CaATP) at the first and second sites, respectively. Using the above kinetic constants, a computer model of the enzyme reaction was constructed and tested using two further sets of kinetic data obtained by varying the concentrations of Mg2+, Ca2+, MgATP2-, and CaATP2-. Poor fits were obtained unless the formation of a mixed complex involving CaATP2- competition with MgATP2- at the second metal-nucleotide-binding site was incorporated into the rate equation. Nonlinear least squares curve fitting of both sets of experimental data gave a well determined value of 124 microM for this final CaATP2- inhibitory constant. Sensitivity tests for variation of the primary kinetic constants with the computer model showed that the inhibitory effect of free Ca2+ was weak and that the observed calcium inhibition of carbamylphosphate synthetase can be accounted for primarily by competitive interaction of CaATP2- at the second MgATP2- binding site. With 1 mM free Mg2+ and 5 mM MgATP2-, half-maximal inhibition of enzyme activity was obtained with 0.2 mM CaATP2-.     

Kinetic model for the action of the inorganic pyrophosphatase from Streptococcus faecalis

Kinetic studies of the less active form of Streptococcus faecalis inorganic pyrophosphatase (EC 3.6.1.1), together with computational analysis, indicated that cooperativity in ligand binding contributes in a significant way to the behavior of this enzyme. The simplest model applicable to our data was a Monod-Wyman-Changeux-type, allosteric model, in which the enzyme is proposed to exist in two states, referred to as R and T states, respectively. In the absence of ligands, 94% of the enzyme was in the T state. MgPPi2- was the only substrate for the enzyme in the R form. This substrate was bound equally well by both enzyme forms, but it was hydrolyzed 5 times more efficiently by the R form than it was by the T form. Mg2PPi was bound exclusively to the T state of the enzyme, and it was hydrolyzed 25% as rapidly as MgPPi2- by the T form. Mg2PPi inhibited the hydrolysis of the more efficient substrate, MgPPi2-, by competing with MgPPi2- for the enzyme in the T form and by shifting the R----T equilibrium in favor of the T form. Mg2+ stabilized the R state, thus activating the hydrolysis of MgPPi2- and inhibiting that of Mg2PPi.     

Involvement of a divalent cation in the binding of fructose 6-phosphate to Trypanosoma cruzi phosphofructokinase: kinetic and magnetic resonance studies

When Mg2+ ions were replaced by Mn2+ in the assay of Trypanosoma (Schizotrypanum) cruzi phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) the Km for D-fructose 6-phosphate (F6P) was reduced threefold while the corresponding constant for ATP was essentially unaffected. A detailed kinetic investigation showed that the apparent Km for F6P decreased monotonically with increasing free Mn2+ concentrations, from a limiting value of 5.7 mM in its absence to a limiting value of 1.1 mM in the presence of saturating concentrations of the ion; the Vmax of the enzyme was, on the other hand, not affected by the concentration of Mn2+. Conversely, it was shown that the apparent Km for Mn2+ at fixed MnATP concentrations decreased with increasing F6P concentrations, from a limiting value of 30 microM in the absence of the sugar phosphate to 9 microM at saturating concentrations of the substrate, while the apparent Vmax increased monotonically from zero to its limiting value. Both electron paramagnetic resonance and water proton longitudinal relaxation studies showed binding of one Mn2+ ion per 18,000 Da catalytic subunit of enzyme in the absence of F6P, with a dissociation constant of 57 +/- 4 microM, comparable to the apparent Km for the ion in the absence of F6P. The presence of saturating level of F6P decreases the value of the dissociation constant of Mn2+ to a limiting value of 7.9 microM in agreement with the results of the kinetic analysis. The substrate F6P decreases the enhancement of the water proton longitudinal relaxation rate in a saturable fashion, suggesting displacement of water molecules coordinated to the enzyme-bound Mn2+ ion by the sugar phosphate. Computer fitting of the several dissociation constants and relaxation enhancements for binary and ternary complexes gives a value of 7.9 mM for the dissociation constant of the enzyme-F6P complex in the absence of Mn2+ and 1.1 mM in the presence of saturating concentrations of the ion, in excellent agreement with the respective Km values of F6P extrapolated to zero and saturating Mn2+, respectively. Studies of the frequency dependence of the water proton longitudinal relaxation rate enhancements in the presence of both binary (enzyme-Mn2+) and ternary (enzyme-Mn2(+)-F6P) complexes, are most simply explained by assuming two exchangeable water molecules in the coordination sphere of the enzyme-bound Mn2+ in the binary complex, while in the ternary complex the data are consistent with the displacement of one of the water molecule from the coordination sphere with no significant alteration of the correlation time. Overall, the kinetic and binding data are consistent with the formation of an enzyme-metal-F6P bridge complex at the active site of T. cruzi phosphofructokinase, a coordination scheme which is unique among the phosphofructokinases.