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.     

UDPglucose 4-epimerase from Saccharomyces fragilis: desensitization with heat.

The allosteric kinetics exhibited by UDP glucose 4-epimerase from $\text{Saccharomyces fragilis}$ changes over to a normal hyperbolic kinetics when the enzyme is heated at 41° for 2 mins. The native enzyme is completely insensitive to inhibition by UMP in the allosteric region. The desensitized enzyme is however, strongly inhibited by UMP at this low concentrations. Apparently, desensitization by heat converts the enzyme to its ultimate catalytic form.     

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.     

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.     

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.     

The crystal complex of phosphofructokinase-2 of Escherichia coli with fructose-6-phosphate: kinetic and structural analysis of the allosteric ATP inhibition

Substrate inhibition by ATP is a regulatory feature of the phosphofructokinases isoenzymes from Escherichia coli (Pfk-1 and Pfk-2). Under gluconeogenic conditions, the loss of this regulation in Pfk-2 causes substrate cycling of fructose-6-phosphate (fructose-6-P) and futile consumption of ATP delaying growth. In the present work, we have broached the mechanism of ATP-induced inhibition of Pfk-2 from both structural and kinetic perspectives. The crystal structure of Pfk-2 in complex with fructose-6-P is reported to a resolution of 2 Å. The comparison of this structure with the previously reported inhibited form of the enzyme suggests a negative interplay between fructose-6-P binding and allosteric binding of MgATP. Initial velocity experiments show a linear increase of the apparent K_0.5 for fructose-6-P and a decrease in the apparent k_cat as a function of MgATP concentration. These effects occur simultaneously with the induction of a sigmoidal kinetic behavior (n_H of approximately 2). Differences and resemblances in the patterns of fructose-6-P binding and the mechanism of inhibition are discussed for Pfk-1 and Pfk-2, as an example of evolutionary convergence, because these enzymes do not share a common ancestor.     

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.     

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.     

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.     

Repair of deaminated cytosine residues of DNA: biological significance of the absence of uracil from DNA.

Uracil-DNA glycosylase of $\text{Bacillus}\text{subtilis}$ is involved in repair of deaminated cytosine residues of DNA. Survivals of SPO2 phage after treatment with bisulfite and weak alkali are considerably higher in wild type strains than in $\text{urg}$ mutants, which are deficient in the enzyme activity, whereas survivals of bisulfite/alkali-treated PBS1 phage in the two types of cells are essentially the same. The spontaneous mutation frequency of a $\text{urg}$ mutant is three fold higher than is that of a wild type strain.     

m-Octopamine as a substrate for monoamine oxidase.

$\text{m}$-Octopamine was characterized as substrate for monoamine oxidase (MAO) in rat brain and liver mitochondria. The $\text{K}$m and $\text{V}$max values of the brain enzyme were 735 μM and 32.5 nmoles/mg protein/30 min, and those of the liver enzyme 351 μM and 125 nmoles/mg protein/30 min, respectively. The inhibition experiments with clorgyline and deprenyl showed that $\text{m}$-octopamine was a common substrate for type A and type B MAO, though a major part of the activity was due to type A enzyme.     

D-Fructose-1,6-diphosphatase-AMP binding measurements using a nucleotide selective enzyme electrode.

A newly developed AMP selective enzyme electrode has been used to make direct binding measurements of the allosteric interaction between AMP and D-fructose-1,6-diphosphatase (E.C. No. 3.1.3.11). The proposed technique is based upon the ability of the enzyme electrode to distinguish between free and bound nucleotide. D-fructose-1,6-diphosphatase from rabbit muscle was found to have four binding sites for AMP with an average binding constant of 9 × 10⁴$\text{M}$^?1. The advantages of a direct electrode method for determining nucleotide binding constants are discussed.     

Molting fluid chitinase: a homotropic allosteric enzyme.

The molting fluid of the tobacco hornworm has chitinase activity which shows allosteric behavior with chitin. A parabolic curve is obtained on a double reciprocal plot of $\text{1}\text{v}$ vs. $\text{1}\text{[S]}$, and a sigmoid curve results when v (N-acetylglucosamine produced) is plotted against [S] (chitin concentration in terms of N-acetylglucosamine concentration). The Hill coefficient with insect chitin is 1.95 ± 0.08. Allostery of the chitinase enables the insect to exert additional control over the integrity of structure of its cuticle.     

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.     

Rapid inhibition by cycloheximide of rat hepatic nuclear free and engaged poly(A) polymerase activities

This paper reports the effect of cycloheximide on the activity of rat hepatic nuclear poly(A) polymerase. Three hours after cycloheximide treatment (3 mg/100 g body weight), total rat hepatic nuclear poly (A) polymerase activity was decreased to 50% of the normal level. This conclusion was reached when the enzyme activity was measured either in the whole nuclei $\text{in vitro}$ or with partly purified enzyme preparations. When examined at different times after a single injection of cycloheximide, it was observed that poly(A) polymerase activity decayed biphasically with an initial rapid decay phase reaching a minimum at one hour $\text{(}\text{t}\text{1}\text{2}\text{= 0.8}\text{hrs}\text{)}$, followed by a stable phase thereafter. These results have been interpreted to mean that poly(A) polymerase consists of either a mixture of two structurally distinct populations of enzymes with a different turnover rate, or of a single type of enzyme with a protein factor which is rapidly turning over and which is required for maximal enzyme activity.     

Interaction of wild-type and poky mitochondrial DNA in heterokaryons of Neurospora.

The mitochondrial phenotype of $\text{poky}$ and other extra-nuclear $\text{Neurospora}$ mutants is known to predominate over that of wild type in heteroplasms. This phenomenon was investigated in heterokaryons using normally occurring strain differences in restriction enzyme patterns to distinguish wild-type and $\text{poky}$ mitochondrial DNAs. Each of ten independent heterokaryons eventually showed the $\text{poky}$ phenotype as judged by slow growth rate and deficiency of 19 S RNA. Six heterokaryons contained mitochondrial DNAs with restriction enzyme patterns of the $\text{poky}$ parent whereas four contained DNAs which lacked restriction enzyme fragments characteristic of the $\text{poky}$ parent. The latter may be recombinants of wild-type and $\text{poky}$ mitochondrial DNA.     

Induction of penicillinase by bacitracin.

Bacitracin preparations are shown to induce penicillinase (β-lactamase I) formation in strain 569 of $\text{Bacillus cereus}$. At high bacitracin concentrations (2–4 mg/ml) the level of induced enzyme obtained reaches a maximum which is comparable to that induced by optimal concentrations (1–2 mcg/ml) of β-lactam antibiotics. Penicillinase formation induced by short exposure to bacitracin, continues at a normal rate after all free bacitracin has been removed. The inducing activity of bacitracin is highly, but not completely, resistant to β-lactamase and can be entirely eliminated by prolonged treatment with penicillinase of $\text{B. cereus}$. The site of induction by bacitracin is, however, different from that mediating induction by β-lactam antibiotics. The inducing component has been isolated by thin layer chromatography; it seems to be closely related to, but not identical with, bacitracin A,B or F.     

Chemical modification of opiate receptors with ethoxyformic anhydride and photo-oxidation: evidence for essential histidyl residues

In the presence of $\text{D}\text{=}$-carnitine significant decarboxylation of 2-oxoglutarate occurs with γ-butyrobetaine hydroxylase (EC 1.14.11.1) both from $\text{Pseudomonas}$ sp AK 1 and from human kidney. No product was formed from carnitine when $\text{D}\text{=}\text{L}\text{=}$-carnitine was incubated with either enzyme but succinate was formed in 1:1 stoichiometry to decarboxylation using $\text{D}\text{=}$-carnitine and the human enzyme. $\text{L}\text{=}$-Carnitine is also an uncoupler for the human enzyme. There is no significant decarboxylation of 2-oxoglutarate in the absence of a substrate, but during normal catalysis in the presence of γ-butyrobetaine the formation of CO₂ from 2-oxoglutarate exceeds carnitine formation with 20% for the human enzyme.     

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.