Freezing of dCMP aminohydrolase in the activated conformation by glutaraldehyde.

dCMP aminohydrolase, which is an allosteric enzyme, was reacted with glutaraldehyde in the presence of the allosteric activator deoxycytidine-5′-triphosphate and of the competitive inhibitor deoxyadenosine-5′-monophosphate. The isolated modified enzyme is no longer sensitive to the effect of the allosteric ligands and shows kinetics typical of the activated enzyme. Gel electrophoresis demonstrated that glutaraldehyde, under our experimental conditions, does not produce intermolecular cross-links but fixes 80% of the enzyme in a stable hexameric form by intramolecular cross-links.The kinetic and molecular data are explained assuming that glutaraldehyde freezes the enzyme in the activated conformation.     

Reversal of the effect of the allosteric ligands of dCMP-aminohydrolase and stabilization of the enzyme in the T form

The 5-mercury derivative of dCMP is a substrate of deoxycytidylate aminohydrolase in the presence of mercaptoethanol. With this substrate a reversal of the effect of the allosteric ligands of the enzyme is observed. dCTP, which is an allosteric activator for aminohydrolysis of dCMP, becomes an inhibitor for the mercury substrate, whilst dTTP, an allosteric inhibitor for dCMP, becomes an activator for the mercury substrate.This observation has been interpreted by assuming that dCMP-Hg-S-CH₂-CH₂-OH is a substrate of the T form of the enzyme. By reacting dCMP-aminohydrolase in the T form (in the presence of dTTP) with glutaraldehyde, an enzyme has been isolated that is no longer active with dCMP, while it is fully active with the mercurated analog. Gel electrophoresis demonstrated that glutaraldehyde does not produce intermolecular crosslinks, but fixes 95% of the enzyme in a stable hexameric form by intramolecular crosslinks. The data are explained by assuming that glutaraldehyde stabilizes the enzyme in the T conformation.     

Characterization of the aspartate kinase from Saccharomyces cerevisiae and of its interaction with threonine

Aspartate kinase (AK) from Saccharomyces cerevisiae has been characterized to elucidate its quaternary structure and the effect of the allosteric inhibitor threonine on the enzyme conformation. The homogeneously purified enzyme was inhibited by threonine (K(i) 1.4 mM) and was found to bind this compound (K(d) 0.97 mM) in a hyperbolic manner. Gel filtration and native gel electrophoresis indicated that yeast AK is a homohexamer of 346 kDa composed by 58 kDa subunits. Threonine caused a decrease in the apparent molecular mass of AK as evidenced by size-exclusion chromatography (from 345 to 280 kDa) and blue native gel electrophoresis (from 346 to 297 kDa); no other molecular species were detected. This shift in the hydrodynamic size was threonine-specific and was reversed by rechromatography in the absence of threonine. No change in the apparent molecular mass was induced by threonine in an AK mutant insensitive to inhibition by this amino acid, which was observed to be unable to bind threonine. These results indicate that the allosteric transition elicited by binding of threonine to yeast AK involves a large conformational change of the protein that isomerizes from a relaxed active conformation to a more compact inactive one of smaller molecular dimensions.     

Purification, kinetic behavior, and regulation of NAD(P)+ malic enzyme of tumor mitochondria

The purification and kinetic characterization of an NAD(P)+-malic enzyme from 22aH mouse hepatoma mitochondria are described. The enzyme was purified 328-fold with a final yield of 51% and specific activity of 38.1 units/mg of protein by employing DEAE-cellulose chromatography and an ATP affinity column. Sephadex G-200 chromatography yielded a native Mr = 240,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major subunit with Mr = 61,000, suggesting a tetrameric structure, and also showed that the preparation contained less than 10% polypeptide impurities. Use of the ATP affinity column required the presence of MnCl2 and fumarate (an allosteric activator) in the elution buffers. In the absence of fumarate, the Michaelis constants for malate, NAD+, and NADP+ were 3.6 mM, 55 microM, and 72 microM, respectively; in the presence of fumarate (2 mM), the constants were 0.34 mM, 9 microM, and 13 microM, respectively. ATP was shown to be an allosteric inhibitor, competitive with malate. However, the inhibition by ATP displayed hyperbolic competitive kinetics with a KI (ATP) of 80 microM (minus fumarate) and 0.5 mM (plus 2 mM fumarate). The allosteric properties of the enzyme are integrated into a rationale for its specific role in the pathways of malate and glutamate oxidation in tumor mitochondria.     

Purification and general properties of aspartate aminotransferase of ox heart

1. A five-step procedure for preparing highly purified aspartate aminotransferase from ox heart is described. 2. The homogeneity of the pure enzyme was established by criteria such as ultracentrifugation and electrophoresis in starch gel and in polyacrylamide gel. 3. The pure enzyme has an isoelectric point of about pH5, and E(1%) (1cm.) 14.40 at 278mmu. 4. The molecular weight of the pure enzyme was determined as 96000 by sedimentation equilibrium. 5. The pH optimum for the pure enzyme was about 8. It was determined by a new assay technique. 6. A difference in the electrophoretic migration rate between the enzyme from ox heart and brain and the enzyme from pig heart and brain suggests a species specificity rather than an organ specificity. 7. A new effect of deionization on the visible-absorption spectrum of the enzyme was observed.     

AMP-deaminase from normal and cirrhotic human liver: A comparative study

AMP-deaminase (EC 3.5.4.6) is an enzyme of nucleotide breakdown involved in regulation of adenine nucleotide pool in mammalian cells. Reaction catalysed by AMP-deaminase constitutes a rate-limiting step in adenine nucleotide catabolism in liver. In this study kinetic and regulatory properties of AMP-deaminase purified from normal and cirrhotic human liver were investigated. In comparison to AMP-deaminase extracted from the normal human liver, AMP-deaminase extracted from the cirrhotic liver was less sensitive towards substrate analogues, and only a very limited response towards pH and adenylate energy charge changes tested for enzyme isolated from this tissue source had been observed. At physiological pH 7.0, in the absence and in the presence of important allosteric effectors (ATP, ADP, GTP and orthophosphate), AMP-deaminases from the two sources studied manifested different regulatory profiles, with half-saturation constant (S0.5) values being distinctly higher for the enzyme extracted from the pathological organ. In contrast to AMP-deaminase isolated from the normal, healthy liver, where presence of relatively large (68 kDa) protein fragment was also detected, only smaller protein fragments were identified, while SDS-PAG electrophoresis of AMP-deaminase isolated from the cirrhotic liver was performed. The obtained results indicate clearly that advanced proteolytic processes occurring in the cirrhotic liver may affect structural integrity of AMP-deaminase studied, making enzyme less active and less sensitive to regulatory action of important allosteric effectors.     

Allosteric transition and substrate binding are entropy-driven in glucosamine-6-phosphate deaminase from Escherichia coli

Glucosamine-6P-deaminase (EC 3.5.99.6, formerly glucosamine-6-phosphate isomerase, EC 5.3.1.10) from Escherichia coli is an attractive experimental model for the study of allosteric transitions because it is both kinetically and structurally well-known, and follows rapid equilibrium random kinetics, so that the kinetic K(m) values are true thermodynamic equilibrium constants. The enzyme is a typical allosteric K-system activated by N-acetylglucosamine 6-P and displays an allosteric behavior that can be well described by the Monod-Wyman-Changeux model. This thermodynamic study based on the temperature dependence of allosteric parameters derived from this model shows that substrate binding and allosteric transition are both entropy-driven processes in E. coli GlcN6P deaminase. The analysis of this result in the light of the crystallographic structure of the enzyme implicates the active-site lid as the structural motif that could contribute significantly to this entropic component of the allosteric transition because of the remarkable change in its crystallographic B factors.     

AMP deaminase from baker's yeast. Kinetic and molecular properties.

The kinetic and molecular properties of AMP deaminase [AMP aminohydrolase, EC 3.5.4.6] purified from baker's yeast (saccharomyces cerevisiae) were investigated. The enzyme was activated by ATP and dATP, but inhibited by Pi and GTP in an allosteric manner. Alkali metal ions and alkaline earth metal ions activated the enzyme to various extent. Kinetic negative cooperativity was observed in the binding of nucleoside triphosphates. Kinetic analysis showed that the number of interaction sites for AMP (substrate) and Pi (inhibitor) is two each per enzyme molecule. The molecular weight of the native enzyme was estimated to be 360,000 by sedimentation equilibrium studies. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the enzyme gave a single polypeptide band with a molecular weight of 83,000, suggesting that the native enzyme has a tetrameric structure. Baker's yeast AMP deaminase was concluded to consist of two "promoter" units which each consist of two polypeptide chains with identical molecular weight.     

Purification and properties of rat liver mitochondrial glutathione peroxidase.

Glutathione peroxidase (glutathione:hydrogen peroxide oxidoreductase, EC 1.11.1.9) was purified from rat liver mitochondria. The enzyme was shown to be pure by polyacrylamide-gel electrophoresis and to contain multiple forms that differed in charge. Selenium was specifically associated with the enzyme. The enzyme was inhibited by iodoacetic acid and iodoacetamide in an unusual pattern of reduction by sulfhydryl compounds and pH dependency. The mitochondrial and cytoplasmic forms of the enzyme were compared, and an explanation of the inhibition patterns is offered.     

Interaction of rose bengal with mung bean aspartate transcarbamylase

The fluorescein dye, rose bengal in the dark: (i) inhibited the activity of mung bean aspartate transcarbamylase (EC 2.1.3.2) in a non-competitive manner, when aspartate was the varied substrate; (ii) induced a lag in the time course of reaction and this hysteresis was abolished upon preincubation with carbamyl phosphate; and (iii) converted the multiple bands observed on polyacrylamide gel electrophoresis of enzyme into a single band. The binding of the dye to the enzyme induced a red shift in the visible spectrum of dye suggesting that it was probably interacting at a hydrophobic region in the enzyme. The dye, in the presence of light, inactivated the enzyme and the inactivation was not dependent on pH. All the effects of the dye could be reversed by UMP, an allosteric inhibitor of the enzyme. The loss of enzyme activity on photoinactivation and the partial protection afforded by N-phosphonoacetyl-L-aspartate, a transition state analog and carbamyl phosphate plus succinate, a competitive inhibitor for aspartate, as well as the reversal of the dye difference spectrum by N-phosphonoacetyl-L-aspartate suggested that in the mung bean aspartate transcarbamylase, unlike in the case ofEscherichia coli enzyme, the active and allosteric sites may be located close to each other.     

N‐ and C‐terminal peptide sequences are essential for enzyme assembly, allosteric, and/or catalytic properties of ADP‐glucose pyrophosphorylase

ADP-glucose pyrophosphorylase is a key regulatory enzyme in starch synthesis in most plant tissues. Unlike the allosteric regulatory dependent properties of the leaf enzyme, the enzymes from non-photosynthetic tissues exhibit varying levels of sensitivity to allosteric regulation, a behavior which may be an inherent property of the enzyme or a product of post-translational modification. As partial proteolysis of the holoenzyme may account for the wide variation of allosteric regulatory behavior exhibited by enzymes from non-photosynthetic tissues, small N- and C-terminal peptide deletions were made on either the potato large and small subunit and co-expressed with the counterpart wild-type subunit in Escherichia coli. Removal of the putative carboxy-terminal allosteric binding region from either subunit type results in an abolishment of enzyme formation indicating that the carboxy terminus of each subunit type is essential for proper subunit folding and/or enzyme assembly as well as its suggested role in allosteric regulation. Removal of a small 10 amino acid peptide from the N-terminus of the small subunit increased its resistance to the allosteric inhibitor Pi as well as its sensitivity to heat treatment. Likewise, removal of the corresponding peptide (17 residues) at the N-terminus of the large subunit also increased its resistance towards Pi inhibition but, in addition, increased its sensitivity to 3-PGA activation. Deletion of an additional 11 residues reversed these changes in allosteric properties but at the expense of a reduced catalytic turnover rate. Combined, these results indicate that the N- and C-terminal regions are essential for the proper catalytic and allosteric regulatory properties of the potato ADP-glucose pyrophosphorylase. The possible significance of these results on the observed insensitivity to effector molecules by ADP-glucose pyrophosphorylases from other non-photosynthetic tissues is discussed.     

Alpha-L-fucosidase from bull seminal plasma: its purification and acrosome reaction promoting property

Alpha-L-fucosidase was purified from the bull seminal plasma by chromatography on DEAE-disk, octyl sepharose hydrophobic column and HPLC. The enzyme appeared to be pure as judged by the polyacrylamide gel electrophoresis both under the nondenaturing and denaturing conditions. The pure enzyme promoted the acrosome reaction of guinea pig spermatozoa in vitro. This is the first report showing that an acrosomal enzyme induces acrosome reaction which is an essential pre-requisite for the gamete interaction and fertilization.     

Reversible association of ox liver glutamate dehydrogenase with the inner mitochondrial membrane

A comparative study on the catalytic and allosteric properties of particulate and soluble forms of ox liver glutamate dehydrogenase has been carried out. The response of the bound enzyme to release by various effectors was investigated. The particulate enzyme was found to have catalytic activities similar to the free enzyme in contrast to its behaviour when bound to pure anionic phospholipids. Possible reasons for such outstanding differences are discussed.     

Pyruvate kinase “Göttingen1,2”: Congenital hemolytic anemia,evidence of double heterozygosity,and lack of enzyme cooperativity

Summary Double heterozygosity of pyruvate kinase (PK) deficiency associated with hereditary hemolytic anemia is emphasized by studies of a kindred harboring two distinct mutant forms of this enzyme. The hematologically unaffected parents exhibit slightly reduced PK activity, a normal Hill coefficient, and a normal thermodynamic dissociation constant for the overall reaction. The paternal enzyme is characterized by normal substrate affinities and decreased activities with the substrate analogues CDP and GDP, whereas the maternal enzyme shows normal affinity for PEP, but an increased affinity for ADP and low thermostability. It is assumed that the erythrocytes of the parents contain a mixture of normal PK and a functionally abnormal isoenzyme, the latter differing between the parents. The two children suffer from hereditary hemolytic anemia. Their PK must be a combination of the mutant paternal and maternal isoenzymes, and their activities are reduced to about 30%. These enzymes are characterized by an increased affinity for PEP and a decreased affinity for ADP, a Hill coefficient of about 1 (indicating lack of cooperativity due to a loss of its allosteric properties), a decreased overall catalytic activity, and a higher resistance to heat denaturation. Further differences are observed in the SDS-gel electrophoresis between the two patients' enzymes. From the enzymological point of view it is impossible to characterize true PK variants in such double heterozygous cases which contain a combination of two different isoenzymes. The cause of chronic hemolysis appears to depend mainly on the loss of the allosteric properties, i.e., the lack of enzyme cooperativity.     

Deoxycytidine kinase from human leukemic spleen: preparation and characteristics of homogeneous enzyme

Deoxycytidine kinase from human leukemic spleen has been purified 6000-fold to apparent homogeneity with an overall yield of 10%. The purification was achieved by using DEAE chromatography, hydroxylapatite chromatography, and affinity chromatography on dTTP-Sepharose. Only one form of deoxycytidine kinase activity was found during all the chromatographic procedures. The subunit molecular mass, as judged by sodium dodecyl sulfate--polyacrylamide gel electrophoresis, was 30 kilodaltons. The pure enzyme phosphorylates deoxycytidine, deoxyadenosine, and deoxyguanosine, demonstrating for the first time that the same enzyme molecule has the capacity to use these three nucleosides as substrates. The apparent molecular weight of the active enzyme, determined by gel filtration and glycerol gradient centrifugation, was 60,000. Thus, the active form of human deoxycytidine kinase is a dimer. The kinetic behavior of pure human deoxycytidine kinase was studied in detail with regard to four different phosphate acceptors and two different phosphate donors. The apparent Km values were 1, 20, 150, and 120 microM for deoxycytidine, arabinosylcytosine, deoxyguanosine, and deoxyadenosine, respectively. The Vmax values were 5-fold higher for the purine nucleosides as compared to the pyrimidine substrates. We observe competitive inhibition of the phosphorylation of one substrate by the presence of either of the three other substrates, but the apparent Ki values differed greatly from the corresponding Km values, suggesting the existence of allosteric effects. The double-reciprocal plots for ATP-MgCl2 as phosphate donor were convex, indicating negative cooperative effects. In contrast, plots with varying dTTP-MgCl2 concentration as phosphate donor were linear with an apparent Km of 2 microM. The enzyme activity was strongly inhibited by dCTP, in a noncompetitive way with deoxycytidine and in a competitive way with ATP-MgCl2.     

Kinetic and allosteric properties of L-threonine-L-serine dehydratase from human liver]

It was shown that low concentrations of ATP (1..10(-4)M) and 10-fold concentrations of AMP (1.10(-3)M) at three constant L-threonine concentrations activated the L-threonine dehydratase activity of L-threonine-L-serine dehydratase from human liver, but had no effect on the L-serine dehydratase activity of this enzyme. Higher concentrations of both nucleotides inhibited the enzyme. The effects of ATP and AMP were specific. The activating and inhibiting effects of various concentrations of ATP and AMP were revealed as changes in the shapes of the curves for the initial reaction rate of the L-threonine dehydratase reaction versus initial substrate concentration. For this reaction the curves were not hyperbolic and were characterized by intermediary plateaux. ATP and AMP also influenced the maximal rate of the enzymatic reaction. Using the desensitization method it was shown that the activating effects of ATP and AMP are of allosteric nature. Thus, human liver L-threonine-L-serine dehydratase is an allosteric enzyme, for which positive allosteric effectors are low concentrations of ATP and AMP and negative allosteric effectors are high concentrations of these nucleotides. A possible mechanism of allosteric regulation of the enzyme under catalysis of the L-threonine dehydratase reaction and the lack of regulation under catalysis of the L-serine dehydratase reaction as well as specificity of the allosteric sites of this enzyme to the two nucleotides and the physiological significance of this process are discussed.     

Regulation of mitochondrial malate dehydrogenase: kinetic modulation independent of subunit interaction

Porcine heart mitochondrial malate dehydrogenase (EC 1.1.1.37), a dimeric enzyme of Mr = 70,000, is both allosterically activated and inhibited by citrate. Using an affinity elution procedure based upon citrate binding to malate dehydrogenase, the isolation of pure heterodimer (a dimeric species with one active subunit and one iodoacetamide-inactivated subunit) has been achieved. Investigations utilizing this heterodimer in conjunction with resin-bound monomers of malate dehydrogenase have allowed the formulation of a definite conclusion concerning the role of subunit interactions in catalysis and regulation of this enzyme. The citrate kinetic effects, oxaloacetate inhibition, malate activation, and the effects of 2-thenoyl-trifluoroacetone (TTFA) are shown to be independent of interaction between catalytically active subunits. Previous kinetic data thought to support a reciprocating catalytic mechanism for this enzyme may be reinterpreted upon closer analysis in relation to an allosteric, conformationally specific binding model for malate dehydrogenase.     

Equilibrium analysis of allosteric interactions shows zero-order effects

The binding of effector to an allosteric protein exhibits a non-Michaelis-Menten behavior, resulting in either ultrasensitive or subsensitive response. In the present work, a modular approach has been developed to determine the response curve for allosteric systems at higher concentration of allosteric enzyme than that of effector (zero-order sensitivity, as observed in enzyme cascades) by equilibrium analysis. The analysis shows that, in an allosteric system, the zero-order effect can make the response ultrasensitive or subsensitive with respect to the enzyme concentration. The response is dependent on the number of binding sites, cooperativity, and the total effector concentration. The framework was further applied to a well studied allosteric protein, the Escherichia coli aspartate transcarbamoylase. The predictions are found to be consistent with the reported experimental data.     

Properties of highly purified lysyl oxidase from embryonic chick cartilage.

Lysyl oxidase the enzyme which oxidately deaminates lysine residues in collagen and elastin, was purified from embryonic chick cartialge by employing an affinity column of lathyritic rat skin collagen coupled to Sepharose, followed by separation on DEAE-cellulose. An enzyme preparation was obtained which was pure as shown by polyacrylamide gel electrophoresis. The specific activity was 1800-fold higher than that of the original extract. The pure enzyme utilized both collagen and elastin substrate. Furthermore, the ratios of enzyme activity with elastin substrate versus that with collagen substrate were the same at all stages of purity. Only one protein band was found after polyacrylamide gel electrophoresis of the pure lysyl oxidase in sodium dodecyl sulfate and mercaptoethanol. The molecular weight was estimated to be 28000. It was found that the enzyme contained a large number of cysteine and tyrosine residues. Evidence was obtained for molecular heterogeneity of lysyl oxidase. The enzyme eluted from DEAE-cellulsoe in at least four distinct regions. When the peaks were rechromatographed separately, they eluted at salt concentrations similar to those of the original chromatogram. However, the substrate specificity and the electrophoretic mobility on polyacrylamide gel were the same for all enzyme fractions.