The theoretical analysis of kinetic behaviour of "hysteretic" allosteric enzymes. IV. Kinetics of dissociation-association processes of allosteric enzymes.

The kinetics of equilibration of dissociating enzyme system

$\text{2}\text{p}\text{?}\text{k ? 0}\text{k + 0}\text{P}$

(P is enzyme oligomer which is able to dissociate reversibly forming two identical halves p) is analysed after changes in storage conditions or after addition of allosteric ligands. The expressions for changes in the proportions of p and P forms with time and the expressions for dependence of initial rate of dissociation-association processes and half-life time on enzyme concentration or allosteric ligand concentration are deduced. It is shown that the dependences of intial rate of dissociation-association processes on allosteric ligand concentration has co-operative character at definite values of kinetic parameters. The graphic methods of determination of the first-order rate constant for dissociation (k_?0) and the second-order rate constant for association (k₊₀) are developed. The experimental kinetic data of dissociation of L-threonine dehydratase, glycogen phosphorylase a and aspartic-β-semialdehyde dehydrogenase are used for illustration of applicability of deduced expression.     

ADPglucose pyrophosphorylase: Evidence for a lysine residue at the activator site of the Escherichia coli B enzyme

Pyridoxal-P can be covalently linked to $\text{E. coli}$ B ADPglucose pyrophosphorylase by reduction with sodium borohydride. The modified enzyme is almost fully active when less than 1 mole of pyridoxal-P is incorporated per mole of enzyme subunit and is no longer dependent on the presence of allosteric activators in reaction mixtures for high activity. The allosteric activators, fructose-P₂ or hexanediol 1,6 bisphosphate, decrease the incorporation of pyridoxal-P into enzyme suggesting that the pyridoxal-P is linked at or near the allosteric activator binding site. Acid hydrolysis of the modified enzyme yields pyridoxyllysine suggesting that the epsilon amino group of lysine is functional in the binding of the allosteric activators of the enzyme.     

UDP glucose-4 epimerase from Saccharomyces fragilis: interaction with sugar phosphates at an effector site

UDP glucose-4 epimerase from $\text{Saccharomyces fragilis}$ was found to be activated at low substrate concentrations by some metabolically related sugar phosphates. The stimulation of the enzyme activity showed a sigmoidal response to the increasing concentration of glucose-6 phosphate at a fixed substrate concentration. The activated enzyme was allosterically inhibited by UMP which otherwise acted as a strictly competitive inhibitor for the enzyme. The interaction with sugar phosphates was not accompanied by any change in the aggregation state of the molelcule.     

Kinetic properties of pyruvate kinase isolated from rat hepatic tumours.

The results demonstrate the existence of L and M forms of pyruvate kinase in rat hepatomas. Tumours were induced by feeding N-Nitrosodiethylamine. The kinetic properties of the L-type tumour enzyme was markedly different from the L-enzyme form found in normal liver. The L-form of tumour enzyme was purified by DEAE cellulose-Sephadex G200 chromatography (Sp. activity 41 units/mg). $\text{MgADP}{}^{\mathrm{?}}\text{ADP}{}^{\mathrm{2?}}$ of $\text{20}\text{1}$ gave optimum activity for both the intrinsic and F1,6di-P stimulated reactions. ATP did not inhibit the enzyme. Alanine (2.5 nM) caused 60% inhibition at low PEP concentrations (0.25 mM). The homotropic effector (PEP) exhibited a complex allosteric pattern and saturation kinetics were not observed for either the intrinsic or F1,6di-P stimulated reactions with PEP concentrations as high as 10 mM.     

Effects of 3′-deoxyadenosine triphosphate on in vitro RNA synthesis of plant RNA-dependent RNA polymerases

3′-deoxyadenosine triphosphate inhibited $\text{in}\text{vitro}$ [³H]UMP incorporation by RNA-dependent RNA polymerases from tobacco and cowpea plants. The inhibition of [³H]UMP incorporation could be reversed by simultaneous addition of higher ATP concentrations but not with increasing concentrations of UTP or when excess ATP was added 10 min after the inhibitor. These results suggest 3′-deoxyadenosine triphosphate competes specifically with ATP in reaction mixtures and results in premature termination of RNA synthesis $\text{in}\text{vitro}$ by RNA-dependent RNA polymerase.     

Glutamine-dependent carbamoyl phosphate synthetase: polyamines inhibit the activity and modify the activating effect of 5-phosphoribosyl 1-pyrophosphate.

Mammalian glutamine-dependent carbamoyl phosphate synthetase (EC 2.7.2.9), the first enzyme of $\text{de novo}$ pyrimidine nucleotide biosynthesis, was strongly inhibited by polyamines at concentrations of 10^?4 to 10^?3 M. Spermine was the most effective, followed in order by spermidine and putrescine. The inhibition was partially reversed by increasing the concentration of Mg²⁺ or MgATP^2?, or by adding low concentrations of 5-phosphoribosyl 1-pyrophosphate, an allosteric activator of the enzyme. Polyamines increased the apparent $\text{K}{}_{\mathrm{a}}$ value of the enzyme for phosphoribosyl pyrophosphate. A possible physiological role of polyamines in widening the range of the effective concentrations of phosphoribosyl pyrophosphate as the activator for the enzyme is suggested.     

Allosteric and isosteric modifiers of NADH binding to cytoplasmic malic dehydrogenase

The binding of NADH to cytoplasmic malic dehydrogenase is shown to be affected by a number of added ligands. One class of ligands appear to be analogs of a substrate for the enzyme, $\text{L}$-malate. These alter the binding constant for NADH without affecting the cooperativity of binding. In contrast, fructose-1,6-diphosphate behaves as an allosteric inhibitor at low enzyme concentrations, apparently by shifting the monomer-dimer equilibrium of the protein to the cooperatively binding dimer. The significance of these results are discussed in terms of a proposed regulatory function for the enzyme.     

Evidence for allosteric NADH regulation of Acinetobacter alpha-oxoglutarate dehydrogenase from multiple-inhibition studies.

Previous studies have suggested that the E1 component (α-oxoglutarate dehydrogenase) of the α-oxoglutarate dehydrogenase enzyme complex from $\text{Acinetobacter lwoffi}$ is inhibited by the end-product NADH. We have now carried out multiple-inhibition studies in the simultaneous presence of NADH and α-oxoadipate, both competitive inhibitors with respect to α-oxoglutarate. The results indicate that NADH acts at an allosteric site within the multi-enzyme complex.     

Studies on the mechanism of activation of mitotic histone H1 kinase

A chromatin-associated histone H1 kinase has been detected in synchronized Novikoff hepatoma cells. Enzyme specific activity increased 4 to 6-fold from late G-2 to mid-metaphase, then decayed exponentially ($\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, 28.5 min) to the interphase level. Extracts of the mitotic kinase retained the ability to decay $\text{in}\text{vitro}$ at 37°C but not at 0°C ($\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, 24 min), under conditions in which interphase activity was stable. Sedimentation rates in sucrose density gradients of interphase and mitotic enzymes (before and after decay) were identical. Purification did not alter the rate of enzyme decay. However, high ionic strength prevented decay of crude but not purified preparations of mitotic enzyme. The results are discussed in terms of an allosteric mechanism for reversible activation of enzyme activity.     

Membrane lipid fatty acids and regulation of membrane-bound enzymes. Allosteric behaviour of erythrocyte Mg2+-ATPase, (Na+ + K2+)-ATPase and acetylcholineasterase from rats fed different fat-supplemented diets

Studies were carried out to determine the Hill coefficients for the inhibition by F^? of the erythrocyte membrane-bound Mg²⁺-ATPase, $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ and acetylcholinesterase from rats fed with seven different diets. Five groups were fed with different natural fats or oil supplements, one with a hydrogenated fat supplement and the other with fat-free diet. The responses of the red cell fatty acids to dietary fats were recorded. The value of $\text{n}$ for the inhibition by F^? of the three enzymes revealed a particular and different behaviour in each group. Correlations between the fatty acid compositions of erythrocyte membranes and cooperativity of each enzyme were calculated. The results indicate that neither the essential fatty acid family nor the non-essential ones are particularly involved in the allosteric phenomena. The increase of the double bond index/saturation ratio of fatty acids, which is taken as indicative of membrane fluidity, was accompanied in an inverse manner by changes in allosteric transitions of the $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ and acetylcholinesterase, whereas the Mg²⁺-ATPase was not dependent on this ratio. Diminution of membrane fluidity, carried out by in vitro increase of its cholesterol content, yields confirmatory results of this regulatory mechanism since the value of $\text{n}$ for acetylcholinesterase shifted as predicted.These facts indicate that the membrane fluidity is a physiological regulator for the allosteric behaviour of the membrane-bound enzymes and that each enzyme exhibits a particular behaviour in this phenomenon.     

Dihydroorotase from Clostridium oroticum is an allosteric enzyme.

Dihydroorotase from $\text{Clostridium}$$\text{oroticum}$ exhibits allosteric behavior with respect to both of its substrates. L-dihydroorotate dependence reflects a positive homotropic interaction for which the Hill coefficient is 1.3–1.6, depending upon the preparation. Conversely, a negative homotropic response is observed when L-ureidosuccinate serves as substrate, as characterized by a Hill coefficient of 0.65–0.75. Interaction between L-dihydroorotate binding sites is a labile characteristic lost during enzyme purification. Negative cooperativity of ureidosuccinate binding appears to be more stable. The effects of purification and medium are also discussed.     

Relationship between ligand binding and conformational change of macromolecules in the two-state allosteric model

The relationship between the binding function $\text{Y}$ and the state function $\text{R}$ of an oligomeric protein has been analysed for the general two-state allosteric model. It is shown that this relation is determined by the numerical values of the inherent parameters of the model. The shape of the function $\text{Y}\text{= f (}\text{R}\text{)}$ can therefore be strictly concave, strictly convex or inverse sigmoidal according to the conditions. In the two-state allosteric model only a dimeric protein can display a linear relationship between $\text{Y}$ and $\text{R}$.In the paper general criteria for the estimation of the state function $\text{R}$ from experimentally obtained conformational parameters are discussed.     

The self-association of chorismate mutase/prephenate dehydratase from Escherichia coli K12

The state of association of chorismate mutase/prephenate dehydratase (EC 5.4.99.5/ 4.2.1.51) from E. coli K12 has been studied using ultracentrifugal techniques. The smallest species inferred is a dimer of molecular weight 73,000–84,000, with a $\text{s}{}_{\mathrm{<mtext>20,w</mtext>}}{}^0$ of 5.02 S at pH 8.2, I = 0.013 M. This species undergoes a concentration-dependent self-association which results in an equilibrium mixture of dimer, tetramer, and probably octamer, with a M_r of 164,000 at an enzyme concentration of 8.0 mg/ml under the same conditions. Addition of the feedback inhibitor phenylalanine (2 mm) or increase in ionic strength (I = 0.40 M), or a decrease in pH to 7.4 displaces this equilibrium toward the higher-molecular-weight forms of the enzyme, resulting in M_r values of 273,000, 254,000, and 257,000, respectively. This behavior partially explains the allosteric kinetics and inhibitor binding observed previously with this enzyme.     

Modification of ribulose bisphosphate carboxylase by 2,3-butadione

D-ribulose-1,5-bisphosphate carboxylases purified from barley or formate-grown $\text{Pseudomonas oxalaticus}$ were inactivated by 2,3-butadione. Pseudo first-order inactivation depended on the presence of borate and was reduced by product 3-phosphoglycerate. The half-times at 30°C and pH 8.3 in the presence of 2 mM 2,3-butadione are 10 and 60 minutes for the enzymes from $\text{P. oxalaticus}$ and barley, respectively. Saturation kinetics and arginine modification were demonstrated for the enzyme from $\text{P. oxalaticus}$.     

Ca 2+ -dependent allosteric regulation of nicotinamide nucleotide transhydrogenase from Pseudomonas aeruginosa

Nicotinamide nucleotide transhydrogenase from $\text{Pseudomonas}\text{aeruginosa}$ exhibits allosteric properties and has been shown to be regulated by the prevailing $\text{[NADPH]}\text{[NADP}{}^{\mathrm{+}}\text{]}$ ratio or by 2′-AMP. The present data obtained with membrane fragments from $\text{P.}\text{aerug}$. show that Ca²⁺ strongly influences the concentration of 2′-AMP or NADPH required for half-maximal stimulation. Saturating concentrations of Ca²⁺ cause full activation of the enzyme; Mn²⁺, Mg²⁺ and K⁺ are considerably less efficient and antagonistic to Ca²⁺. Some implications of these findings for the regulatory mechanism and possible physiological function of the enzyme are considered.     

Allosteric and non-allosteric E. coli phosphofructokinases: effects on growth.

In wild type $\text{Escherichia coli}$ K-12 ca. 90% of phosphofructokinase is known to be the allosteric enzyme Pfk-1, and the rest is Pfk-2, a non-allosteric enzyme. An isogenic strain series has now been constructed with varying combinations and amounts of Pfk-1 and Pfk-2 (e.g., no Pfk-1, high level of Pfk-2; normal level Pfk-1, high level Pfk-2, etc.). In minimal medium with glucose, glucose-6-P, and glycerol, aerobically and anaerobically, provided there is adequate total amount of enzyme, what allosteric type it is does not make much difference to growth rate or yield of this organism.     

Controlled reduction of cytochrome b in succinate-cytochrome c reductase complex by succinate in the presence of ascorbate and antimycin.

$\text{Bacillus subtilis}$ membranes can transfer either N-acetylmuramyl-pentapeptide phosphate or N-acetylglucosaminyl phosphate from UMP directly onto undecaprenyl phosphate. Tunicamycin blocks only the latter transfer and inhibits peptidoglycan synthesis by toluenized cells of $\text{Bacillus megaterium}$ utilizing added nucleotide sugar precursors or cell wall synthesis by intact cells of $\text{B. subtilis}$. Tunicamycin prevents formation of the cell wall disaccharide lipid intermediate by blocking transfer of N-acetylglucosamine onto undecaprenyl muramyl pentapeptidyl pyrophosphate.     

Allosteric regulation of platelet actomyosin

The kinetic properties of platelet actomyosin have been examined to understand the mode of hydrolysis of its substrate ATP. In the presence of divalent cations, ATP hydrolysis deviated from Michaelis-Menten kinetics in such a way as to indicate cooperative effects, with a sigmoidal velocity $\text{vs.}$ substrate curve and a Hill slope of 2.4. In the absence of added divalent cations, linear Michaelis-Menten kinetics were obtained and the Hill slope reduced to 1.0. These results indicate an allosteric regulatory site on platelet actomyosin.     

Further proof for the catalytic role of the larger subunit in the spinach leaf ribulose-1,5-diphosphate carboxylase

The catalytically active oligomeric form of the larger subunit, A_m, obtained from spinach leaf ribulose-1,5-diphosphate carboxylase by pretreatment with p-mercuribenzoate at pH 7.5 followed by incubation at pH 9.0, was free of the smaller subunit based on C-terminal amino acid analyses. Valine was the predominant C-terminus of the A_m preparations, the release of tyrosine being negligibly small [cf. Sugiyama and Akazawa, $\text{Biochemistry}$ 9 (1970) 4499]. The pH optimum of the ribulose-1,5-diphosphate carboxylase reaction by A_m was about 8.5, in comparison to the native enzyme which showed an alkaline pH optimum only in the absence of Mg²⁺. The substrate saturation curve of the catalytic subunit with respect to bicarbonate followed the Michaelis-Menten equation, as contrasted to the anomalous reaction kinetics of the native ribulose-1,5-diphosphate carboxylase molecule reported previously. These overall results indicate that the allosteric properties of spinach ribulose-1,5-diphosphate carboxylase are possibly conveyed by a unique structural conformation that requires the presence of the smaller subunit in association with the larger catalytic subunit component of the enzyme molecule.     

Stimulation of brain aromatic alkylamine N-methyltransferase activity by FAD and methylcobalamin

We studied the effects of methylcobalamin alone and with various reducing systems (FADH₂, FAD or its analogues in combination with NADH or β-mercaptoethanol) on the activity of partially purified aromatic alkylamine N-methyltransferase (AANMT) from rat brain. As expected, the specific activity of AANMT in the presence of methylcobalamin alone was enhanced (125% of control). Surprisingly, in the presence of FAD alone (but not FADH₂$\text{et cetera}$) it was 175% of control; and int the presence of methylcobalamin plus FAD plus β-mercaptoethanol it reached 307% of control. Preliminary evidence suggests that FAD induces allosteric kinetics for the activated enzyme with respect to the methyl donor 5-methyltetrahydrofolic acid (5-MTHF).