Negative Kinetic Cooperativity Research Articles

Pre-steady-state kinetics and solvent isotope effects support the "billiard-type" transport mechanism in Na+ -translocating pyrophosphatase.

Membrane‐bound pyrophosphatase (mPPase) found in microbes and plants is a membrane H+ pump that transports the H+ ion generated in coupled pyrophosphate hydrolysis out of the cytoplasm. Certain bacterial and archaeal mPPases can in parallel transport Na+ via a hypothetical “billiard‐type” mechanism, also involving the hydrolysis‐generated proton. Here, we present the functional evidence supporting this coupling mechanism. Rapid‐quench and pulse‐chase measurements with [32P]pyrophosphate indicated that the chemical step (pyrophosphate hydrolysis) is rate‐limiting in mPPase catalysis and is preceded by a fast isomerization of the enzyme‐substrate complex. Na+, whose binding is a prerequisite for the hydrolysis step, is not required for substrate binding. Replacement of H2O with D2O decreased the rates of pyrophosphate hydrolysis by both Na+‐ and H+‐transporting bacterial mPPases, the effect being more significant than with a non‐transporting soluble pyrophosphatase. We also show that the Na+‐pumping mPPase of Thermotoga maritima resembles other dimeric mPPases in demonstrating negative kinetic cooperativity and the requirement for general acid catalysis. The findings point to a crucial role for the hydrolysis‐generated proton both in H+‐pumping and Na+‐pumping by mPPases.

Constitutive inorganic pyrophosphatase as a reciprocal regulator of three inducible enzymes in Escherichia coli

BackgroundPrevious studies have demonstrated the formation of stable complexes between inorganic pyrophosphatase (PPase) and three other Escherichia coli enzymes – cupin-type phosphoglucose isomerase (cPGI), class I fructose-1,6-bisphosphate aldolase (FbaB) and l-glutamate decarboxylase (GadA). MethodsHere, we determined by activity measurements how complex formation between these enzymes affects their activities and oligomeric structure. ResultscPGI activity was modulated by all partner proteins, but none was reciprocally affected by cPGI. PPase activity was down-regulated upon complex formation, whereas all other enzymes were up-regulated. For cPGI, the activation was partially counteracted by a shift in dimer ⇆ hexamer equilibrium to inactive hexamer. Complex stoichiometry appeared to be 1:1 in most cases, but FbaB formed both 1:1 and 1:2 complexes with both GadA and PPase, FbaB activation was only observed in the 1:2 complexes. FbaB and GadA induced functional asymmetry (negative kinetic cooperativity) in hexameric PPase, presumably by favoring partial dissociation to trimers. ConclusionsThese four enzymes form all six possible binary complexes in vitro, resulting in modulated activity of at least one of the constituent enzymes. In five complexes, the effects on activity were unidirectional, and in one complex (FbaB⋅PPase), the effects were reciprocal. The effects of potential physiological significance include inhibition of PPase by FbaB and GadA and activation of FbaB and cPGI by PPase. Together, they provide a mechanism for feedback regulation of FbaB and GadA biosynthesis. General significanceThese findings indicate the complexity of functionally significant interactions between cellular enzymes, which classical enzymology treats as individual entities, and demonstrate their moonlighting activities as regulators.

Effects of Excluded Surface Area and Adsorbate Clustering on Surface Adsorption of Proteins. II. Kinetic Models

Models for equilibrium surface adsorption of proteins have been recently proposed (Minton, A. P., 2000. Biophys. Chem. 86:239–247) in which negative cooperativity due to area exclusion by adsorbate molecules is compensated to a variable extent by the formation of a heterogeneous population of monolayer surface clusters of adsorbed protein molecules. In the present work this concept is extended to treat the kinetics of protein adsorption. It is postulated that clusters may grow via two distinct kinetic pathways. The first pathway is the diffusion of adsorbed monomer to the edge of a preexisting cluster and subsequent accretion. The second pathway consists of direct deposition of a monomer in solution onto the upper (solution-facing) surface of a preexisting cluster (“piggyback” deposition) and subsequent incorporation into the cluster. Results of calculations of the time course of adsorption, carried out for two different limiting models of cluster structure and energetics, show that in the absence of piggyback deposition, enhancement of the tendency of adsorbate to cluster can reduce, but not eliminate, the negative kinetic cooperativity due to surface area exclusion by adsorbate. Apparently noncooperative (Langmuir-like) and positively cooperative adsorption progress curves, qualitatively similar to those reported in several published experimental studies, require a significant fraction of total adsorption flux through the piggyback deposition pathway. According to the model developed here and in the above-mentioned reference, the formation of surface clusters should be a common concomitant of non-site-specific surface adsorption of proteins, and may provide an important mechanism for assembly of organized “protein machines” in vivo.

Analysis of negative cooperativity for glutamate dehydrogenase

The empirical equation, which describes negative cooperativity in the enzyme kinetics, has been proposed. The equation is obtained from the Michaelis–Menten equation where the Michaelis constant is replaced by the effective Michaelis constant, which is a linear function of the v/ V max ratio ( v is the rate of the enzymatic reaction and V max is the limiting value of v at saturating concentrations of substrate). The equation allows the limiting values of the Michaelis constant at v/ V max → 0 and v/ V max → 1 to be estimated, K 0 and K lim, respectively. The K lim/ K 0 ratio is considered as a quantitative characteristic of negative cooperativity. The applicability of the equation has been demonstrated for the kinetic data obtained for glutamate dehydrogenases from various sources (negative kinetic cooperativity for coenzyme). The negative cooperativity for the functions of saturation of protein by ligand is also analyzed. The data on binding of spin-labeled NAD, NADH, and NADPH by beef liver glutamate dehydrogenase are used as examples.

Negative cooperativity in the steady-state kinetics of sugar oxidation by soluble quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus.

Steady-state-kinetics investigations were carried out for the oxidation of aldose sugars by soluble quinoprotein glucose dehydrogenase (GDH) from Acinetobacter calcoaceticus using N-methylphenazonium methyl sulfate (PMS) as artificial electron acceptor. As is not uncommon for a dye-linked dehydrogenase, the enzyme showed ping-pong behaviour and double-substrate inhibition. However, under conditions that avoided its masking by sugar-substrate inhibition as much as possible, negative kinetic cooperativity with respect to sugar substrate oxidation by this enzyme was demonstrated. Arguments are presented that exclude trivial factors as a cause for the phenomenon observed. Experimental data could be fitted with an equation accounting for biphasic cooperativity containing two sets of apparent kinetic parameters, V1 and K1, and V1 and K2, representing the enzyme's Michaelis-Menten behaviour at low and high substrate concentrations, respectively. Assuming that subunit interaction causes the cooperativity effect, the sets express the performance of soluble GDH's two subunits in two states of mutual interaction. From fitting the experimental data for several sugars with this equation, it appeared that their V1 values were similar, although their K1 values varied considerably. This showed that the cooperativity effect dramatically changes the performance of soluble GDH, as reflected by the V2 and K2 values for glucose (in phosphate buffer) being about 10-fold and 100-fold higher than the V1 and K1 values, respectively. Substituting the Ca2+ involved in activation of pyrroloquinoline quinone (PQQ) in soluble GDH by Sr2+ affected the cooperativity effect (an increase of the K1 value) but not the two turnover rates of the hybrid enzyme for glucose. The data suggest that the two catalytic cycles of soluble GDH have different rate-limiting steps compared with that of PQQ-containing methanol dehydrogenase.

Kinetics of the Slow pH-Mediated Transition of Polyphenol Oxidase

Catecholase activity of latent polyphenol oxidase from broad bean leaves showed a hysteresis phenomenon above pH 4, whereas a steady-state rate was reached immediately when pH values were lower, thus suggesting that slow pH-induced conformational changes in the protein occur during the assay. When the enzyme was activated by sodium dodecyl sulfate, the lag period completely disappeared. This transition was reversible, since a burst was observed when the enzyme was preincubated at acid pH, before being returned to its previous experimental conditions. The pKfor the isomerization process (pKH= 4.6) was estimated by preincubating the enzyme at different pH values and analyzing the product accumulation curves. Negative kinetic cooperativity was evident over a pH range in which the isomerization reaction was significant when the steady state was measured as a function of different substrate concentrations.

Spinach Chloroplast O‐Acetylserine (thiol)‐Lyase Exhibits two Catalytically Non‐Equivalent Pyridoxal‐5′‐Phosphate‐Containing Active Sites

A synthetic gene encoding the mature spinach- chloroplast O-acetylserine (thiol)-lyase was constructed and expressed in an Escherichia coli strain carrying the T7 RNA polymerase system. The pure recombinant protein was obtained at high yield (6 mg/l cell culture) using a new purification procedure that includes affinity chromatography on Green A agarose. Its specific activity was of the order of 1000 U/mg, and its physical properties were similar to those previously reported for the natural enzyme isolated from spinach chloroplasts. In particular the recombinant enzyme, as for the natural enzyme, behaved as a homodimer composed of two identical subunits each of Mr 35000. From steady-state kinetic studies using sulfide or 5-thio(2-nitrobenzoate) (Nbs) as alternative nucleophilic co-substrates, the enzyme exhibited positive kinetic co-operativity with respect to O-acetylserine [Ser(Ac)] in the presence of sulfide and a negative kinetic co-operativity in the presence of Nbs. Binding of Ser(Ac) to the enzyme was also investigated by absorbance and fluorescence measurements to obtain insight into the role of pyridoxal 5'-phosphate and of the single tryptophan residue (Trp176) present in the enzyme molecule. Addition of Ser(Ac) to the enzyme provoked the disappearance of the 409-nm absorbance band of the pyridoxal 5'-phosphate Schiff base and the appearance of two new absorbance bands, the one located between 320 nm and 360 nm and the other centered at 470 nm. Also, the fluorescence emission of the pyridoxal 5'-phosphate Schiff base was quenched upon addition of Ser(Ac) to the enzyme. These changes were most presumably due to the formation of a Schiff base intermediate between alpha-aminoacrylate and the pyridoxal 5'-phosphate cofactor. The fluorescence emission of Trp176 was also quenched upon Ser(Ac) binding to the enzyme. Quantitative analysis of the absorbance and fluorescence equilibrium data disclosed a co-operative behavior in Ser(Ac) binding, in agreement with the steady-state kinetic results. Fluorescence quenching experiments with the acrylamide and iodide revealed that the indole ring of Trp176 was largely exposed and located within the pyridoxal 5'-phosphate active site. These results are consistent with the finding that the native enzyme is composed of two identical subunits. Yet, presumably due to subunit-subunit interactions, the enzyme exhibits two non-equivalent pyridoxal-5'-phosphate-containing active sites.

Some regulatory properties of the 2-oxoglutarate dehydrogenase complex from European bison heart.

Basic regulatory properties of the 2-oxoglutarate dehydrogenase complex (OGDC) isolated and purified from the heart muscle of European bison (Bison bonasus) were studied. Kinetic studies have shown that in the absence of phosphate ions OGDC exhibits kinetic attributes of negative cooperativity with respect to 2-oxoglutarate. ADP and phosphate lower S0.5 value of OGDC for 2-oxoglutarate without changing the maximum reaction rate. NADH inhibits OGDC versus both 2-oxoglutarate and NAD+. Moreover, bison heart OGDC shows negative kinetic cooperativity for NAD+ and positive kinetic cooperativity for CoA at low CoA concentrations. The latter property has not been observed in earlier studies on OGDC from bovine and pig heart and other tissues of these animals.

Allosteric Properties, Substrate Specificity, and Subsite Affinities of Honeybee α-Glucosidase I

The substrate specificity of honeybee alpha-glucosidase I, a monomeric enzyme was kinetically investigated. Unusual kinetic features were observed in the cleavage reactions of sucrose, maltose, p-nitrophenyl alpha-glucoside, phenyl alpha-glucoside, turanose, and maltodextrin (DP = 13). At relatively high substrate concentrations, the velocities of liberation of fructose from sucrose, glucose from maltose, p-nitrophenol from p-nitrophenyl alpha-glucoside, and phenol from phenyl alpha-glucoside were accelerated, and so the Lineweaver-Burk plots were convex, indicating negative kinetic cooperativity: the Hill coefficients were calculated to be 0.50, 0.64, 0.50, and 0.67 for sucrose, maltose, p-nitrophenyl alpha-glucoside, and phenyl alpha-glucoside, respectively. For the degradation of turanose and maltodextrin, the enzyme showed a sigmoidal curve in v versus s plots and thus catalyzed the reaction with positive kinetic cooperativity. The Lineweaver-Burk plots were concave and the Hill coefficients were 1.2 and 1.5 for turanose and maltodextrin, respectively. These unique properties cannot be interpreted by the reaction mechanism that Huber and Thompson proposed: (1973) Biochemistry 12, 4011-4020. The rate parameters for the hydrolysis of sucrose, maltose, p-nitrophenyl alpha-glucoside and phenyl alpha-glucoside were estimated by extrapolating the linear part of the Lineweaver-Burk plots at low substrate concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Pig heart fumarase really does exhibit negative kinetic co-operativity at a constant ionic strength.

The kinetics of the action of fumarase on L-malate and fumarate were investigated at constant ionic strength. This was done to evaluate reports that fumarase follows simple Michaelis-Menten kinetics. However, when pH, buffer concentration and ionic strength are all maintained at constant values, the Lineweaver-Burk plots exhibit pronounced downward curvature, characteristic of negative kinetic co-operativity.

ATP hydrolysis kinetics by Na,K-ATPase in cataract

The steady-state kinetics of hydrolysis of Mg 2+ ATP by the epithelial Na,K-ATPase of individual human lenses were determined. Among the cataract lens population, four distinct kinetic types were observed: negative kinetic co-operativity, Michaelis-Menten kinetics, positive kinetic co-operativity, and substrate inhibition kinetics. Negative kinetic co-operativity and Michaelis-Menten kinetics were also observed in a group of presumably clear lenses from non-diabetic individuals ages 16–42 years. Substrate inhibition kinetics were found to be prevalent in individuals with mature onset diabetes. Substrate inhibition kinetics were also observed for Na,K-ATPase isolated from lenses which had been incubated in high glucose. It would appear that this modification leads to an inhibition of Na,K-ATPase-dependent K + influx into these cultured lenses.

Subunit coupling and kinetic co-operativity of polymeric enzymes. Amplification, attenuation and inversion effects

The principles of structural kinetics, as applied to dimeric enzymes, allow us to understand how the strength of subunit coupling controls both substrate-binding co-operativity, under equilibrium conditions, and kinetic co-operativity, under steady state conditions. When subunits are loosely coupled, positive substrate-binding co-operativity may result in either an inhibition by excess substrate or a positive kinetic co-operativity. Alternatively, negative substrate-binding co-operativity is of necessity accompanied by negative kinetic co-operativity. Whereas the extent of negative kinetic co-operativity is attenuated with respect to the corresponding substrate-binding co-operativity, the positive kinetic co-operativity is amplified with respect to that of the substrate-binding co-operativity. Strong kinetic co-operativity cannot be generated by a loose coupling of subunits. If subunit is propagated to the other, the dimeric enzyme may display apparently surprising co-operativity effects. If the strain of the active sites generated by subunit coupling is relieved in the non-liganded and fully-liganded states, both substrate-binding co-operativity and kinetic co-operativity cannot be negative. If the strain of the active sites however, is not relieved in these states, negative substrate-binding co-operativity is accompanied by either a positive or a negative co-operativity. The possible occurrence of a reversal of kinetic co-operativity, with respect to substrate-binding co-operativity, is the direct consequence of quaternary constraints in the dimeric enzyme. Moreover, tight coupling between subunits may generate a positive kinetic co-operativity which is not associated with any substrate-binding co-operativity. In other words a dimeric enzyme may well bind the substrate in a non co-operative fashion and display a positive kinetic co-operativity generated by the strain of the active sites.

Glucose-6-phosphate modification of bovine renal Na,K-ATPase: A model for changes occurring in the human renal medulla in diabetes

The kinetics of hydrolysis of ATP were determined for the renal Na,K-ATPase, in the K + conformation, modified with glucose-6-phosphate. There was a shift in the ATP hydrolysis kinetics from negative kinetic co-operativity for the control enzyme preparations to substrate inhibition kinetics for the modified enzyme preparations. The effect was reversible and stabilized after NaBH 4 reduction. Approximately 4 moles of glucose-6-phosphate were incorporated per mole of Na,K-ATPase (based on MW of 150,000 daltons). Similar substrate inhibition kinetics were observed for the renal Na,K-ATPase isolated from several human subjects with mature onset diabetes.

A new approach to understanding kinetic cooperativity when the Hill coefficients are less than 2

The simple enzyme kinetic equation, V ̄ v = 1 + K [ S] + D (A + [ S]) , allows one to obtain more information than does the Michaelis-Menten equation, from many nonlinear Lineweaver-Burk double-reciprocal plots in terms of four useful empirical parameters V ̄ , K, D, and A . Thus, in the absence of substrate inhibition, maximum or minimum Hill coefficients ( H) from 0 to 2 are given by the expression H 2 (1 − H) = 4K D . Only the parameter D can be negative and this is associated either with positive kinetic cooperativity (1 < H < 2) or substrate inhibition (( K + D) ≤, 0). By assuming that dissociation of the product is an exchange reaction coupled with either binding of the substrate or a surrogate ligand (competitive inhibitor), a minimal model was obtained that defines these four empirical parameters in terms of only five specific chemical reaction rate constants that can, in fact, be evaluated experimentally. In this model negative kinetic cooperativity (0 < H < 1) results when the substrate displaces the modifier more easily than the product and positive kinetic cooperativity (1 < H < 2) when the converse is true, whether this occurs by a direct displacement or through allosteric interactions. This simple minimal model successfully predicts how the parameters vàry with the concentration of surrogate ligand (competitive inhibitor) in several instances. In particular a new equation [ 1 (H − 1) = α + β[ modifier]] that relates the Hill coefficient ( H) to the modifier concentration was derived and shown to be empirically valid for both Eschericia coli phosphofructokinase and yeast pyruvate kinase even though they have Hill coefficients up to 4.

Self-organization and dynamics of an open futile cycle

The dynamic behaviour of an open futile cycle composed of two enzymes has been investigated in the vicinity of a steady-state. A necessary condition required for damped or sustained oscillations of the system is that enzyme E 2, which controls recycling of the substrate S 2, be inhibited by an excess of this substrate. In order for the system to be neutrally stable and therefore to exhibit sustained oscillations, it is not necessary for antagonist enzyme E 1 to be activated by its product S 2. If it is enzyme E 1 which is inhibited by an excess of its substrate S 1, the system has a saddle point. Other conditions for stability or instability of the system have been determined. If the enzyme E 1, which is not inhibited by the substrate, exhibits a slow conformational transition of the mnemonical type, this transition dramatically alters the stability behavior of the system. If the mnemonical enzyme E 1 were exhibiting a positive kinetic co-operativity, decreasing the rate of the conformational transition of the mnemonical enzyme will increase the stability of the whole system and will tend to damp the oscillations in the vicinity of the steady-state. If conversely the mnemonical enzyme E 1 were exhibiting a negative kinetic co-operativity, decreasing the rate of the enzyme conformational transition will decrease the stability of the system and will tend to create or amplify oscillations of the system taken as a whole. If these results may be extended to more complex metabolic cycles, involving more than two enzymes, it may be tentatively considered that positive co-operativity associated with slow transition has emerged in the course of evolution in order to limit temporal instabilities of metabolic cycles. Alternatively one may speculate that the “biological function” of negative co-operativity is to create or amplify these temporal instabilities.

Regulation of enzyme activity in adsorptive enzyme systems

The kinetic behaviour of adsorptive enzyme systems with free and adsorbed enzyme forms in rapid equilibrium has been analysed. It has been shown that the dependences of enzymic reaction rate on substrate or “adsorptive effector” concentrations reveal the deviations from simple kinetic laws of Michaelis-Menten type (positive or negative kinetic co-operativity). Such kinetic anomalies should be observed when adsorption of the enzyme results in the changing catalytic properties and when the state of the equilibrium between free and bound enzyme forms depends on the presence of low molecular substances (substrates, coenzymes and various cellular metabolites). The physiological significance of adsorption-desorption processes for the enzyme activity regulation has been emphasized.