Article

The active centre of triose phosphate isomerase

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Abstract

The molecular weight and amino acid composition of triose phosphate isomerase have been determined. The molecular weight (43000) is lower and the molecular activity (500000) higher than those of most other glycolytic enzymes. Reaction with iodoacetate (studied with radioactive reagent) takes place in two phases: in the first phase, at pH6.3, cysteine and methionine groups react and enzymic activity is unimpaired; in the second phase, histidine reacts and enzymic activity is lost. Photo-oxidation leads to inactivation, with loss of cysteine, of histidine and of tryptophan, but little loss of tyrosine. The mechanism postulated for the action of the enzyme demands the intervention of a group functioning as a base, and the results obtained are consistent with histidine's being the basic group in the active centre.

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... The heterogeneity of rabbit-muscle triosephosphate isomerase has been documented for a wide variety of animal [1,2] and human tissues [3,4]. Different research groups have reported the finding of one [5,8], three [6,7], four [8], five [9] or eight [2] bands of the rabbit-muscle enzyme in the polyacrylamide [5,7 -91 and starch-gel electrophoresis [I, 2,6,7], and by electrofocussing [8]. It has been suggested, that this multiformity is the result of an artefact of the isolation procedure [6], the presence of conformational isoenzymes [6a], or the existence of non-identical subunit,s [7,10]. ...
... The heterogeneity of rabbit-muscle triosephosphate isomerase has been documented for a wide variety of animal [1,2] and human tissues [3,4]. Different research groups have reported the finding of one [5,8], three [6,7], four [8], five [9] or eight [2] bands of the rabbit-muscle enzyme in the polyacrylamide [5,7 -91 and starch-gel electrophoresis [I, 2,6,7], and by electrofocussing [8]. It has been suggested, that this multiformity is the result of an artefact of the isolation procedure [6], the presence of conformational isoenzymes [6a], or the existence of non-identical subunit,s [7,10]. ...
... Different research groups have reported the finding of one [5,8], three [6,7], four [8], five [9] or eight [2] bands of the rabbit-muscle enzyme in the polyacrylamide [5,7 -91 and starch-gel electrophoresis [I, 2,6,7], and by electrofocussing [8]. It has been suggested, that this multiformity is the result of an artefact of the isolation procedure [6], the presence of conformational isoenzymes [6a], or the existence of non-identical subunit,s [7,10]. ...
Article
1Crystalline and specific-activity-constant rabbit-muscle triosephosphate isomerase was resolved into three major forms, α, β and γ (97%) and two minor components δ and ɛ (3%) through DEAE-cellulose chromatography.2Hybridization studies have shown the α and γ isoenzymes to be homodimers AA and BB, respectively, and β the heterodimer AB. Reassociation studies from 8 M urea have indicated that α, β and γ are not conformers. The evidence supports earlier data suggesting that the heterogeneity of triosephosphate isomerase is largely due to the random combination of two chemically non-identical subunits (A and B) to give three native dimeric forms.3Following hybridization, the pattern obtained with δ and ɛ is more complex. Reassociation of either form resulted in the formation of all five bands as well as two or four, respectively, additional more anodic bands.4Substrate and inhibition characterization have shown the isoenzymes to be “isokinetic”.5Titration with 5,5′-dithiobis(2-nitrobenzoate) has indicated two accessible sulfhydryl groups in the native molecule, one of which appears to be essential for activity.
... The production of phosphate was determined after incubation at 30 °C for 30 min. Triosephosphate isomerase [10], isocitrate lyase [11], malate synthase >* [12], acyl-CoA oxidase [13], fumarase [6] were assayed according to established methods. Galactosyl co transferase activity was determined analogous to the ^ procedure for glucosyl transferase [8]. ...
Article
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Diphosphatase (inorganic pyrophosphatase) activity was localized within compartments of cotyledons of germinating cucumber seeds during the stage of maximal conversion of fat into carbohydrates. At this stage, almost 2 mol pyrophosphate are produced during the formation of one mole sucrose from 0.28 mol triglyceride. When organelles of the 2000 x g pellet or 10,000 x g pellet were separated by density gradient centrifugation and gradient flotation, the diphosphatase activity paralleled the profiles of markers of the plastid stroma but was virtually absent from the glyoxysomes. Within the fraction of small vesicles and membranes, diphosphatase was attributed to the plasma membrane. The main portion of diphosphatase, contained in the plastids, was partially purified by chromatography on anion exchange resin and molecular sieving, leading to a 75-fold enrichment compared to the stroma fraction. Trace amounts of diphosphatase observed in the glyoxysomal fraction were analyzed in the same way. Comparison of the isoelectric points and the activity profile at different pH values and the inhibitory effect of the various cations indicated that the trace amounts of diphosphatase activity in the glyoxysome fraction represented contaminations originating from the plastids. The plasma membrane form of diphosphatase is an integral membrane protein which was solubilized with octylglucoside. It was shown to differ from the plastid form in pH optimum and sensitivity towards bivalent cations. All forms of diphosphatase were clearly distinguished from other phosphohydrolytic activities.
... Published estimates of the molecular weight of rabbit muscle triose phosphate isomerase (Meir & Cotton, 1966;Burton & Waley, 1966;Johnson & Waley, 1967;Krietsch, Pentchev, Klingenburg, Hofstatter & Bucher, 1970) vary somewhat, but are distributed around a value of 50 000. Hence the values in the second column of Table 2 have been calculated on this basis; our values agree reasonably well with those given by Krietsch et al. (1970), ...
Article
1. The nature of the subunits in rabbit muscle triose phosphate isomerase has been investigated. 2. Amino acid analyses show that there are five cysteine residues and two methionine residues/subunit. 3. The amino acid sequences around the cysteine residues have been determined; these account for about 75 residues. 4. Cleavage at the methionine residues with cyanogen bromide gave three fragments. 5. These results show that the subunits correspond to polypeptide chains, containing about 230 amino acid residues. The chains in triose phosphate isomerase seem to be shorter than those of other glycolytic enzymes.
... The activity of TPI was in large excess according to the Van Hoek assay, but the under the in vivo-like assay conditions it is of the same order of magnitude as the other enzyme activities. This was probably caused by the addition of phosphate, which is known to be an inhibitor of TPI [15]. Even in the in vivolike assay the activity of TPI was among the highest measured activities, in agreement with the fact that it is close to equilibrium in most cases [16]. ...
Article
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The aim of this study was to standardize the cultivation conditions of Saccharomyces cerevisiae and the in-vitro enzyme activity assays between different laboratories. Furthermore, the conditions under which the enzyme activity measurements were carried out were adapted such that they would be as close as possible to in vivo con-ditions, thus yielding results which are relevant for Systems Biology. This approach is different from the classical enzymologists' approach which is to optimize for the highest catalytic activity.
Article
The stochastic fluctuations in the initial phase of a one substrate one product enzyme system have been calculated for an actual enzyme reaction, using the model of Heyde & Heyde (1969). The experimental data are for the reaction catalysed by triose phosphate isomerase. It is shown that for this system the stochastic fluctuations in free enzyme, enzyme-substrate or enzyme-product complex, substrate and product are negligible. Using a more general stochastic model it is shown that stochastic fluctuations will in general be negligible for such systems except at the beginning of the transient phase.
Article
Rabbit triosephosphate isomerase, except for the occurence of substrate inhibition, obeys the simple Michaelis-Menton equation with glyceraldehyde-3-phosphate as substrate. But with dihydroxycetone phosphate as substrate, it exhibits sigmoidal kinetics. Its isomerase activity can be inhibited by various phosphates and metabolites.
Article
A procedure for the isolation of crystalline triosephosphate isomerase from human erythrocytes is described. The isolated enzyme, after a 4500-fold purification has a specific activity of approximately 8000 μmoles of d-glyceraldehyde 3-phosphate converted to dihydroxyacetone phosphate per minute per milligram. The enzyme is judged to be homogeneous by the criteria of analytical ultracentrifugation, zone electrophoresis, and rechromatography. Molecular weight determinations by sedimentation equilibrium ultracentrifugation and gel filtration yield a value of 56,000 daltons. The amino acid composition of the enzyme has also been established. The enzyme from human erythrocytes has been resolved into three catalytically active components by isoelectric focusing. The major peak containing 76% of the triosephosphate isomerase activity has an isoelectric pH of 6.0. The two minor peaks with isoelectric pH values of 5.5 and 6.4 contain 22 and 2% of the enzyme activity, respectively. Several possible explanations for this multiplicity are discussed.
Article
1. The equilibrium constant at 38 degrees and I 0.25 of the triose phosphate isomerase reaction was found to be 22.0 and that of the aldolase reaction, 0.99x10(-4)m. The [dihydroxyacetone phosphate]/[glyceraldehyde phosphate] ratio was found to be 9.3 in rat liver. The causes of the apparent deviation of the triose phosphate isomerase system from equilibrium in vivo have been investigated. 2. The equilibria of the triose phosphate isomerase and aldolase reactions were studied with relatively large concentrations of crystalline enzymes and small concentrations of substrates, approximating to those found in rat liver and muscle. There was significant binding of fructose diphosphate by aldolase under these conditions. There was no evidence that binding of glyceraldehyde phosphate by either enzyme affected the equilibria. 3. The deviation from equilibrium of the triose phosphate isomerase system in rat liver can be accounted for by the low activity of the enzyme, in relation to the flux, at low physiological concentrations of glyceraldehyde phosphate (about 3mum). It has been calculated that a flux of 1.8mumoles/min./g. wet weight of liver would be expected to cause the measured degree of disequilibrium found in vivo. 4. The conclusion that the triose phosphate isomerase is not at equilibrium is in accordance with the situation postulated by Rose, Kellermeyer, Stjernholm & Wood (1962) on the basis of isotope-distribution data. 5. The triose phosphate isomerase system is closer to equilibrium in resting muscle probably because of a very low flux and a high enzyme concentration. 6. The aldolase system deviated from equilibrium in rat liver by a factor of about 10 and by a much greater factor in resting muscle. 7. The measurement of total dihydroxyacetone phosphate and glyceraldehyde phosphate content indicates the concentrations of the free metabolites in the tissue. This may not hold for fructose diphosphate, a significant proportion of which may be bound to aldolase.
Article
The sequence of an active-site peptide from rabbit muscle triose phosphate isomerase, containing the essential glutamyl residue with which chloroacetol phosphate reacts, is Trp-Val-Leu-Ala-Tyr-Glu-Pro-Val-Ala-Trp-(Thr, Ile)-Gly-Gly-Lys.
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GLYCIDOL (2,3-epoxypropanol) phosphate inactivates and becomes covalently bound to triose phosphate isomerase (EC 5.3.1.1; TIM); the kinetics and stoichiometry of the reaction, and the structure of the reagent, indicate a specific reaction at the active centre of the enzyme1. The site that is labelled by this inhibitor, and by glycidol in the presence of inorganic phosphate, has now been identified, lodohydroxyacetone phosphate2 and bromohydroxyacetone phosphate3 are also specific inhibitors of TIM. The amino-acid sequence round the site in chicken muscle TIM labelled by the bromo compound has been established4; we were greatly helped by knowing the sequence of their peptide.
Article
Triose phosphate isomerase was isolated from brewer's yeast and rabbit liver and was obtained in crystalline form. Chemical, physical and kinetic properties were compared to rabbit muscle triose phosphate isomerase.1The molecular weight of all three enzymes is in the range of 56000 to 60000. In dodecyl sulfate or as modified maleylated protein, the enzymes dissociate into two polypeptide chains each having a molecular weight in the range of 24000 to 29000.2The rabbit muscle and liver enzymes appear to be indistinguishable in terms of their amino acid composition, electrophoretic mobility, kinetic properties, inhibition sensitivity, pH optimum, molecular weight and N-terminal amino acid (alanine).3The yeast enzyme, on the other hand, was found different in the following respects; it has a several fold lower content of sulfur amino acids, is highly resistant to inactivation through photooxidation as well as sulfhydryl and alkylating agents. Furthermore, it contains different N-terminal amino acids (valine and alanine) and has kinetic properties differing from those of the rabbit enzymes.4The crystalline liver and muscle enzymes could be resolved into three distinct electrophoretic forms in starch and polyacrylamide gels. The possible interpretation of the multiple forms in terms of hybrids or conformers is discussed.
Article
1. Details of an improved method for starch-gel electrophoresis of water-soluble muscle proteins are given. 2. Methods are described for detecting enzyme activities on the starch gel after electrophoresis, by using pieces of filter paper. 3. Compositions of incubation mixtures suitable for detecting any of the enzymes of glycolysis, and certain other enzymes, are given. 4. A comparison of the various enzymes in extracts of several muscles from one rabbit was made; most differences are quantitative only. 5. A detailed comparison of the mobilities of various enzymes in extracts of muscles from a wide variety of species was made. Each species was found to have a characteristic pattern of proteins on the starch gel, and the mobilities of individual enzymes varied considerably. 6. Potential uses and extensions of the methods are discussed.
Article
Rabbit muscle triosephosphate isomerase (EC 5.3.1.1) is inactivated by maleimides, Na2S4O6, organic mercurials, 5,5′-dithiobis (2-nitrobenzoic acid), Ag+, and Hg2+. Ag2+ and Hg2+ cause a decrease in the maximum velocity, and under specified conditions the other reagents induce an increase in the Michaelis constant.N-ethylmaleimide reacts with three sulfhydryl residues per mole of enzyme, and the maximum change in Km is about threefold. Mercurials cause a greater change in Km and react with more than three sulfhydryl groups, but subsequent precipitation prevents quantitative analysis after six residues have reacted (with p-hydroxymercuribenzoate).Experiments with several competitive inhibitors and the active-site affinity label, 3-chloroacetolphosphate, showed that the magnitude of the change in Michaelis constant was the same as the magnitude of the changes in the inhibition constants.The rabbit muscle and liver enzyme appear to have similar properties, but the chicken muscle enzyme is much less reactive, and the yeast enzyme does not become inactivated.Evidence is presented to show that the effects cannot be explained by assuming the hydrated substrates are bound to the enzyme as a result of sulfhydryl modification.
Article
The glycolytic enzyme, triose phosphate isomerase, has been purified and crystallized from bovine lens. The lens enzyme resembles the enzyme from rabbit muscle in its catalytic activity, and in its crystalline form. The electrophoretic mobility of the enzyme from bovine lens differs from that of the enzyme from rabbit muscle. This difference may be ascribed to species variation, rather than organ variation, since the enzymes from bovine lens and from bovine muscle have the same mobilities. These results, together with the other work discussed, suggest that the enzymes in the lens (unlike the bulk proteins) may be expected to be identical with the enzymes in other organs.
Article
1. Malate dehydrogenase of the mitochondrial type was prepared from an acetone-prepared powder of thoroughly washed minces of whole bovine heart by previously reported methods that were modified to give higher yields of the purified enzyme. 2. Determinations of the sedimentation and diffusion coefficients showed the molecular weight of the enzyme to be approx. 63000. The amino acid composition of the enzyme was also determined. Discrepancies between these data and similar data previously reported by Davies & Kun (1957) and Siegel & Englard (1962) were resolved. 3. ;Fingerprints' were made from tryptic digests of heat-denatured and of reduced and alkylated enzyme. These indicated that the enzyme is composed of a number of identical or similar sub-units.
Article
I have synthesized 1-hydroxy-3-iodo-2-propanone phosphate, a compound structurally similar to dihydroxyacetone phosphate, as a potential active-site specific reagent for triose phosphate isomerase. Under mild conditions, the reagent irreversibly inactivates the enzyme. The kinetics of the inactivation in the absence or presence of the competitive inhibitor α-glycerophosphate, the stoichimetric incorporation of the reagent, and the similarities of the pH dependencies of enzymic activity and of the inactivation rate are indicative of a specific modification at the active site of triose phosphate isomerase.
Article
Rabbit muscle phosphoglucose isomerase was subjected to methylene blue and rose bengal sensitized photooxidation. This treatment caused the enzyme to lose activity quite rapidly with half-lives of 2 to 10 minutes under the following conditions: pH 7.1; ionie strength, 0.11; temperature, 10°; enzyme concentration, 2 mg per ml. The time progress curve for inactivation was of apparent first order only until approximately 50% of the initial enzyme activity were lost. Quantitative rate studies were therefore restricted to a comparison of rates obtained during this initial period of the inactivation process. The thus obtained pseudo-first rate constants for inactivation were dependent on reaction volume, enzyme concentration, pH, and ionic strenth. The loss in enzyme activity was accompanied by an initial decrease in the histidine, cysteine, and methionine content followed, after more prologned oxidation, by a decrease also in tryptophan and tyrosine. Other amino acid residues were not affected, as determined by monitoring the change in total amino acid composition simultaneously with the loss in enzyme activiy as a function of the photooxidation time. A direct correlation between the rate of activity loss and the oxidation rate of a specific amino acid residue could not be established because of the multiplicity of the photocatalytic modifications. Kinetic studies of the photoinactivation process, however, implicate histidine as the residue whose modification is primarily involved in the loss of catalytic activity. A direct participation of methionine is considered unlikely on the basis of the ionic strength dependency of photoinactivation, while cysteine has been excluded on the basis of other studies with organic mercurials.
Article
The present work describes procedures in which seven major muscle enzymes and serum albumin can be simultaneously isolated from chicken skeletal muscles. The seven enzymes isolated were: phosphorylase, enolase, creatine-P kinase, aldolase, glyceraldehyde-3-P dehydrogenase, phosphoglycerate mutase, and triose-P isomerase. The proteins isolated by these methods were judged to be greater than 97% pure on the basis of electrophoretic analysis in sodium dodecyl sulfate polyacrylamide gels. The procedure is applicable for isolation of the enzymes from large (greater than 100 g) or small (less than 0.5 g) amounts of muscle tissue and the entire procedure can be completed within two days. Particularly useful features of the procedures are: (1) preferential solubilization of the enzymes from myofibrils by extraction of muscle specimens in solutions of different ionic strength; (2) specific precipitation of phosphorylase, creatine-P kinase, and glyceraldehyde 3-Phosphate dehydrogenase from solutions of specified pH and degrees of ammonium sulfate saturation; and (3) an alternate method for isolation of glyceraldehyde-3-P dehydrogenase by specific elution of the enzyme from phosphocellulose columns with ATP. Because of the ease, rapidity, and reproducibility of the procedures, these methods may be useful for the routine isolation of the muscle enzymes in studies on biochemical regulation, as well as for obtaining large quantitites of the enzymes for structural analysis.
Article
This chapter describes rapid and reproducible procedures for the routine isolation of several proteins from chicken breast muscle. The chicken enzymes isolated are phosphorylase, enolase, creatine kinase, fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglyceromutase, and triosephosphate isomerase. Another protein, serum albumin—shown to be identical to muscle β-actinin—is isolated from muscle specimens during the purification procedures. The chapter presents continuous spectrophotometric assays for these enzymes. The procedure for isolation of enzyme involves fractionation by ammonium sulfate precipitation, isolation of phosphorylase by ion exchange on diethylaminoethyl (DEAE)-cellulose, purification of serum albumin (β-actinin), phosphoglyceromutase, creatine kinase, enolase, aldolase, and glyceraldehyde-3-phosphate dehydrogenase by chromatography on phosphocellulose, isolation of triosephosphate isomerase, and purity of isolated enzymes. The versatility and reproducibility of the present procedures allow for the routine isolation of muscle enzymes in regulation studies and in obtaining large amounts of these proteins for structural analysis.
Article
Ferrate ion, a phosphate analog and a potent oxidizing agent, is known to inactivate a number of enzymes which interact with phosphoryl compounds. In contrast, enzymes which do not interact with phosphoryl compounds are not affected by comparable concentrations of ferrate. To further explore the specificity of ferrate as a reagent which is specific for phosphoryl binding sites, a study of its effect on human hemoglobin A was undertaken. In the deoxy form, this protein is known to interact with 2,3-bisphosphoglycerate, its natural allosteric inhibitor of cooperative binding of oxygen, while as oxyhemoglobin it does not interact with the inhibitor. Treatment with ferrate ion caused the loss of approximately three amino acid residues per beta chain of human deoxyhemoglobin, His-2, His-143, and Tyr-145, and one residue, presumably Tyr-42, per alpha chain. Oxyhemoglobin was not affected by the reagent. 2,3-Bisphosphoglycerate was found to protect deoxyhemoglobin from the action of ferrate. His-2 and His-143 are among the residues reported to be implicated in the binding of 2,3-bisphosphoglycerate by deoxyhemoglobin [A. Arnone (1972) Nature (London) 237, 146-148].
Article
Full-text available
Ferrate anion, an analog of orthophosphate anion, very rapidly inactivates triose phosphate isomerase from chicken muscle. The inactivation can be prevented by the presence of competitive inhibitors. Of the 247 amino acids known to be present in each of the identical monomers of this dimeric enzyme, Trp-168, located in the active site pocket, as well as Trp-191 and His-248 are destroyed. The partial loss of Tyr-164 also occurs. Trp-168 is known from published crystallographic studies to be located in the active site cavity harboring Glu-165. The gamma-carboxylate group of Glu-165 is believed to serve as the nucleophile which catalyzes the isomerization. Tyr-164, Glu-165, and Trp-168 are known to be conserved in all of the triose phosphate isomerases which have been sequenced, including those obtained from mammals, chicken, fish, yeast, and bacteria. It is suggested that the chemical modification of Trp-168 alters its shape and hydrophobic character in a manner that adversely affects the conformation of the active site cavity. When the chicken enzyme is treated with ferrate in the presence of the competitive inhibitor phosphoglycolate, only His-248 is destroyed. Thus, His-248, which is the COOH-terminal amino acid, cannot be essential for activity. This observation is consistent with the knowledge that it is not invariant in the enzyme from various species.
Article
1. Glycidol (2,3-epoxypropanol) phosphate is a specific irreversible inhibitor of rabbit muscle triose phosphate isomerase (EC 5.3.1.1); the site of attachment has now been studied. 2. The labelled enzyme was digested with pepsin and a modified peptide isolated. The sequence of the peptide is: Ala-Tyr-Glu-Pro-Val-Trp. 3. It is the glutamic acid residue in this peptide that is labelled: the peptide is thus a gamma-glutamyl ester derived from glycerol phosphoric acid. The same site is labelled by a mixture of glycidol and inorganic phosphate. 4. Kinetic and stereochemical features of these reactions are discussed.
Article
Full-text available
Crystalline triosephosphate isomerase from human erythrocytes can be resolved into three catalytically active forms by isoelectric focusing. Each variant (I, 1 to 5%, pI = 6.7; II, 70 to 75%, pI = 6.5; and III, 20 to 25%, pI = 6.1) refocuses as a single component and is a dimer (mol wt 56,000). Identical electrophoretic patterns are obtained from fresh and aged red cell lysates and are unchanged in the presence of reducing agents or proteolytic inhibitors. The enzyme can be dissociated in guanidinium chloride and reassociated into catalytically active enzyme. Dissociation and reassociation of Components I and III result only in the respective parent species, whereas dissociation and reassociation of Component II gives rise to all three forms of the enzyme. The amino acid compositions and tryptic peptide fingerprints of the components indicate several structural differences in the two types of subunits. The catalytic properties of the three forms are similar but consistent with the designation of Components I and III as homodimers AA and BB and Component II as the heterodimer AB.
Article
Triosephosphate isomerase exhibits acidic electrophoretic subforms in many tissues and these isozymes appear to increase during aging of erythrocytes and the eye lens. Incubation of the pure enzyme under mild alkaline conditions results in the generation of acidic forms which are identical to those found in vivo. Structural analysis of these isozymes from both in vivo and in vitro studies showed that they are the result of deamidation of two specific asparagines (Asn-15 and Asn-71). These labile asparagines are located in the subunit-subunit contact sites, and the deamidations introduce a total of four new negative charges in the contact site. The positions of the new aspartic acid residues are juxtaposed, thus creating charge-charge interactions which cause the dimeric enzyme to dissociate more readily. These studies (1) explain the molecular basis for the acidic isozymes observed in many tissues, (2) show that the deamination process is spontaneous and requires no intrinsic cell factors, (3) show that the deamination occurs in a sequential fashion with the deamidation of Asn-71 preceding the deamidation of Asn-15, and (4) suggest that proteolytic degradation processes may become altered during aging resulting in the accumulation of the deamidated intermediates of the normal catabolic process.
Chapter
Publisher Summary This chapter describes the molecular and catalytic properties of aldose–ketose isomerases. Aldose–ketose isomerases catalyze the interconversion of isomeric aldo- and keto sugars by causing the migration of carbon-bound hydrogen between carbons 1 and 2. These enzymes may be classified in two groups according to their action on free or on phosphorylated monosaccharide substrates. Those acting on free sugars appear to be confined mainly to microorganisms, whereas some of those acting on phosphorylated substrates are common to all living organisms. Prominent among the latter are glucose-6-phosphate isomerase, triosephosphate isomerase, and ribose- 5-phosphate isomerase. The basic function of glucose-6-phosphate isomerase is catalyzing an obligatory step in glycolysis. Its potential for exerting a regulatory influence on carbohydrate metabolism, however, must be considered uncertain. Despite its strategic location near the branching point of several pathways utilizing glucose 6-phosphate, three properties would appear to make this enzyme difficult to control: (1) its equilibrium constant being close to unity, (2) its ubiquitous presence in relatively high concentration, and (3) its high catalytic efficiency.
Article
The triosephosphate isomerase systems from two algal species, Ankistrodesmus Braunii and Scenedesmus acuminatus, have been isolated and partially purified by a series of solvent and salt fractionations. The enzymes from A. Braunii and S. acuminatus were electrophoretically homogeneous with uncorrected mobilities of −1·2 × 10−5 cm2 V−1 sec−1 and −1·01 × 10−5 cm2 V−1 sec−1 respectively. For both systems the pH optimum was sharp at pH 7·7 in 2·5 × 10−5 M Tris-HCl buffer. Kinetic analysis of the isomerase reactions gave Michaelis constants (Km) of 4·34 × 10−4 M and 9·7 × 10−4 M GAP respectively for the enzymes from A. Braunii and S. acuminatus. The algal isomerase systems were inhibited by sulfate, phosphate, and to a lesser degree, chloride ions.
Article
Triose phosphate isomerase was purified ca 250-fold from pea seed extracts. The km for D-glyceraldehyde-3-P was 0.44 mM and for dihydroxyacetone-P, 0.88 mM. P-enolpyruvate, 2-P-glycerate, 3-P-glycerate and 2-P-glycolate were strongly inhibitory. Pi and arsenate also inhibited pea seed triose phosphate isomerase.
Article
: Detergent extraction of brain slices and mouse fibroblast 3T3 cells was performed to determine rates and relative amounts of extraction of inositol versus the glycolytic enzymes. The two detergents, Triton X-100 and Brij 58, led to similar results for extraction of myo-inositol. The extraction of enzymes from brain slices or cells varied with the detergent. In brain slices, a buffered solution containing 0.2% of the detergent Brij 58 led to the extraction of 85% of the inositol before 3% of the aldolase or before 37% of either lactate dehydrogenase or triose phosphate isomerase was extracted. In contrast, with 0.1% Triton X-100 in isotonic phosphate-buffered saline, when 70% of the inositol was extracted, 33% of the aldolase and 48% of the triose phosphate isomerase were extracted. Lesser amounts of aldolase and glyceraldehyde phosphate dehydrogenase were extracted than most of the other glycolytic enzymes under all conditions, implying that these enzymes may be interacting with nonextractable subcellular components. In 3T3 cells, both detergents were of similar effectiveness for inositol extraction. Triton X-100 caused 89% of the inositol to be released and Brij 58 caused 84% to be released. With the enzymes, Brij 58 caused between 15 and 38% extraction and Triton X-100 caused between 61 and 85% extraction of the different glycolytic enzymes. Thus Brij 58 was as effective as Triton X-100 in inositol extraction but not nearly as effective in glycolytic enzyme extraction. The results demonstrate that inositol leakage from tissues or cells is a better indicator of detergent-mediated alterations in membrane porosity than glycolytic enzyme leakage. In addition, it may be suggested that Brij 58 caused plasma membrane perforations prior to destruction of the cytomatrix, whereas Triton X-100 appeared to affect both the membrane integrity and the cytomatrix as indicated by dramatic losses of both inositol and glycolytic enzymes. This distinction between detergents should be considered and used to advantage in the design of histochemical, immunocytochemical, or further biochemical studies.
Article
The direct analysis of both the position and abundance of 18O in d-glucose was accomplished by mass spectrometry of the pentaacetyl derivative. Synthetically prepared [1—18O]-, [2—18O]-, [3—18O]- and [6—18O]pentaacetylhexoses were prepared and used as standards to aid in the elucidation of fragmentation patterns. Ions were identified which contained specific oxygen atoms of glucose, permitting the measurement of 18O incorporated into these positions. This technique was used in the analysis of [4—18O] glucose prepared enzymatically.
Article
This chapter presents a technique, dye-sensitized photooxidation of enzymes for identification of critical residues at the catalytic site. The best illustrations of the method indicate that for some enzymes and under some circumstances, the method can indeed be a useful tool for tile determination of mechanisms or functional groups. The technique is no more complex than the irradiation of a solution containing protein, photosensitizing dye, and oxygen, long enough to measure a change in some property of the protein (usually catalytic activity). An apparatus is designed to ensure a high degree of reproducibility and given a method for calibrating this system. The active species in dyesensitive photooxidation include the activated dye itself, a dye-oxygen complex and oxygen in the triplet or one of two singlet energy states. The choice of dye and the pH of the reaction seem to be the most generally important determinants of specificity.
Article
Das Vorkommen von Histidin im aktiven Zentrum eines Enzyms lßt sich z. B. durch kinetische Messungen, chemische Modifizierung, NMR-Spektroskopie oder Röntgen-Strukturanalyse beweisen. Histidin ist die einzige natürliche Aminosure, die einen Imidazolrest als Seitenkette enthlt. Die Rolle des Histidins bei der Enzymkatalyse beruht u. a. auf den Besonderheiten des Imidazolrestes: So neigt er zur Bildung von Wasserstoffbrücken, vereinigt Donor- und Acceptoreigenschaften und kann in einer nucleophilen Katalyse oder in einer Basekatalyse wirksam werden. — Bei einigen dieser Enzyme ist die Lage jedes Atoms bekannt, und doch sind die Vorstellungen über den Ablauf der Katalyse auf molekularer Ebene kontrovers.
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If the pyridine nucleotide-linked dehydrogenases are related by evolution from one or a few common ancestral genes, it might be possible to suggest special evolutionary relationships among particular dehydrogenases by a comparison of structural and functional properties, even before extensive comparative primary sequence information is available.The dehydrogenases are compared using their amino acid compositions, isoelectric points, subunit molecular weights, and some functional properties. Amino acid compositions of dehydrogenases are tabulated per thousand total residues. The comparison of compositions is facilitated by using the difference index method developed by Metzger. The methods of comparison are evaluated using cytochromes c, proteases, and glyceraldehyde-3-phosphate dehydrogenases.Glutamate and glucose-6-phosphate dehydrogenases appear to be similar in many respects and mutually distinct from the other dehydrogenases. Similarities also appear to exist among the low molecular weight dehydrogenases: liver alcohol, mitochondrial malate, glycerol-3-phosphate, and NADP-linked isocitrate dehydrogenases. The Metzger difference index suggests that the primary sequence homology between H- and M-type lactic dehydrogenase subunits may be very extensive, perhaps being 75% identical.
Article
1.1.Triosephosphate isomerase has been purified from thoracic muscle of the mosquito, Aedes aegypti, and also from thoracic muscle of the housefly, Musca domestica.2.2.The estimated molecular weight for triosephosphate isomerase from the mosquito and housefly is 60,000.3.3.With dihydroxyacetone phosphate as substrate, triosephate isomerase from both sources shows sigmoidal kinetics. For the mosquito triosephosphate isomerase, the apparent dihydroxyacetone phosphate.4.4.With glyceraldehyde-3-phosphate as substrate, the housefly triosephosphate isomerase activity can be described by the simple Michaelis-Menton equation, except for the occurrence of substrate inhibition. The for the housefly.5.5.The mosquito triosephosphate isomerase can be inhibited by various phosphates, dicarboxylic acids, and arsenate.6.6.Insect triosephosphate isomerase is possibly a regulatory enzyme.
Article
Glycolytic enzymes were found to bind to isolated coated vesicles. From a preparation of rabbit muscle myogen mixed with clathrin coated vesicles greater than 75% of four enzymes, aldolase. glyceraldehydephosphate dehydrogenase, pyruvate kinase, and lactate dehydrogenase were found to pellet with isolated coated vesicles upon centrifugation at 60.000 g for 1 h. The binding of purified aldolase, glyceraldehydephosphate dehydrogenase, pyruvate kinase, the muscle form and the heart form of lactate dehydrogenase was characterized further. Substrates were found to elute three of the enzymes and binding was determined to be a function of ionic strength.
Article
The presence of histidine in the active center of an enzyme can be demonstrated by kinetic measurements, chemical modification, NMR spectroscopy or X-ray structure analysis. Histidine is the only naturally occurring amino acid to contain an imidazole residue as a side chain. The role of histidine in enzyme catalysis depends, inter alia, upon the special features of the imidazole residue: it thus tends to form hydrogen bonds, combines donor and acceptor properties and can take part in either nucleophilic or base catalysis. In some of these enzymes the position of each atom is known; however, the theories as to how the catalysis proceeds at a molecular level are controversial.
Article
Several glycolytic enzymes were observed to have between 40-90% of their activities associated with the particulate fractions of lysed nerve endings. The enzymes showing high particulate activity in lysed nerve endings were hexokinase (EC 2.7.1.1), aldolase (EC 4.1.2.13), glucosephosphate isomerase (EC 5.3.1.9), phosphofructokinase (EC 2.7.1.11), glyceraldehyde-phosphate dehydrogenase (EC 1.2.1.12), pyruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.27). With the exception of phosphofructokinase, 80% or more of the particle associated activity of each enzyme was solubilized by salt treatment indicating the association with particles was ionic. Sub-fractionation of lysed nerve endings showed hexokinase and fumarase (EC 4.2.1.2) had the highest specific activity in the same fractions which is consistent with observations indicating that hexokinase is associated with mitochondria. The other glycolytic zymes having high particulate activity, aldolase, glucosephosphate isomerase, phosphofructokinase, glyceraldehyde-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase, showed enrichment in fractions containing synaptosomal membranes, i.e. the fractions having highest specific activity of acetylcholinesterase (EC 3.1.1.7) and (Na+ + K+)-ATPase (EC 3.6.1.3).
The specific activities of glucosephosphate isomerase, aldolase, triosephosphate isomerase, glyceraldehydephosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, pyruvate kinase and lactate dehydrogenase were all higher in the synaptoplasmic fraction from rat brain than in 100,000 g supernatant fraction of rat brain homogenates when the supernatants were prepared in high ionic strength solutions. Four enzymes in synaptosomes and two enzymes in homogenates were associated with particulate fractions as indicated by the large increase in specific activity of the enzymes when samples were treated with 0.3 M KCl before centrifugation. Glucosephosphate isomerase, aldolase, pyruvate kinase and lactate dehydrogenase were the enzymes that showed a large increase in specific activity following salt treatment of isolated, synaptosomal membrane while aldolase and pyruvate kinase were the two enzymes which showed a large increase in specific activity in the high speed supernatant fractions. Because the specific activities of many enzymes are found to be elevated not only in synaptosomes but in synaptosomal membrane fractions it is suggested that these enzymes may provide the potential for significantly enhanced glycolysis at these locations.
Article
Detergent extraction of brain slices and mouse fibroblast 3T3 cells was performed to determine rates and relative amounts of extraction of inositol versus the glycolytic enzymes. The two detergents, Triton X-100 and Brij 58, led to similar results for extraction of myo-inositol. The extraction of enzymes from brain slices or cells varied with the detergent. In brain slices, a buffered solution containing 0.2% of the detergent Brij 58 led to the extraction of 85% of the inositol before 3% of the aldolase or before 37% of either lactate dehydrogenase or triose phosphate isomerase was extracted. In contrast, with 0.1% Triton X-100 in isotonic phosphate-buffered saline, when 70% of the inositol was extracted, 33% of the aldolase and 48% of the triose phosphate isomerase were extracted. Lesser amounts of aldolase and glyceraldehyde phosphate dehydrogenase were extracted than most of the other glycolytic enzymes under all conditions, implying that these enzymes may be interacting with non-extractable subcellular components. In 3T3 cells, both detergents were of similar effectiveness for inositol extraction. Triton X-100 caused 89% of the inositol to be released and Brij 58 caused 84% to be released. With the enzymes, Brij 58 caused between 15 and 38% extraction and Triton X-100 caused between 61 and 85% extraction of the different glycolytic enzymes. Thus Brij 58 was as effective as Triton X-100 in inositol extraction but not nearly as effective in glycolytic enzyme extraction. The results demonstrate that inositol leakage from tissues or cells is a better indicator of detergent-mediated alterations in membrane porosity than glycolytic enzyme leakage.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
Suppressor mutations in Bacillus subtilis cause the synthesis of a new protein with the enzymatic activity of l-leucine dehydrogenase and two groups of new proteins with the activity of triosephosphate isomerase. The new isoenzymes of triosephosphate isomerase are separable by zone electrophoresis and differ among themselves in elution behavior upon gel permeation chromatography. One group has an apparent average molecular weight of 120,000 to 135,000, which is more than twice that of the wild-type enzyme. Another group appears to be even higher in molecular weight. These data are consistent with the working hypothesis that the new isoenzymes are produced by extension of growing polypeptide chains through one or more chain-terminating triplets, although other mechanisms resulting in alteration of shapes, charges, or associations of the enzymes are not excluded.
Article
A study was made of the effects of 6,6'-dithiodinicotinic acid (DTDN) and 2,2'-dithiodipyridine (DTDP) on mouse Ehrlich ascites tumor cells. Both drugs inhibited glucose utilization and lactate production under aerobic conditions, DTDP being effective m lower concentrations than DTDN. Both drugs decreased the proportion of utilized glucose that is converted to lactate. This may indicate increased diversion of glucose into the phosphogluconate pathway. DTDN in concentrations inhibiting lactate production to a minimal degree greatly decreased the levels of dihydroxyacetone phosphate and increased those of glyceraldehyde-3-phosphate occurring in the cells after addition of glucose. This effect was not seen with DTDP. The effect of DTDN on the glycolytic intermediates may be due to inhibition of triosephosphate isomerase. Lack of the effect with DTDP may be due to more potent inhibition of an enzyme at a step prior to formation of fructose-l,6-diphosphate. Both drugs inhibit rabbit muscle triosephosphate isomerase in vitro. The drugs decreased the fall of extracellular pH due to lactic acid formation but did not lead to any unusual relationship between extracellular and intracellular pH. Both drugs caused loss of cellular K+ but only in concentrations higher than are required for complete suppression of glycolysis.
Article
1. Human skeletal muscle triosephosphate isomeras (D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1) was isolated and resolved by DEAE-cellulose chromatography into three major forms, A, B, and C, which comprise 97% of the total activity. The relative distribution was 25, 46 and 29% respectively. 2. The A and C forms are homodimers, alpha alpha and beta beta, and form B is the heterodimer, alpha beta. Reassociation studies from guanidinium chloride have indicated that A, B, and C are not conformers. Although these studies revealed the existence of two different chains, the amino acid analysis showed no significant variance. Since no differences were obsrved in Ouchterlony and Mancini tests or in immunotitration, the three fors are assumed to be immunologically identical. 3. The three forms have the same specific activity, Michaelis constants, pH optimum, activation energy, inhibition by metabolites and heat stability. Only with increasing ionic strength did the V and Km values differ. 4. The two poypeptide chains (alpha and beta) appear to be identical (amino acid composition, molecular weight and antigenity), and since the electrophoretic banding pattern changed with cell aging, it is concluded that the multiple forms of trisephosphate isomerase are the consequence of minor post-synthetic alteration(s) of form A.
Article
THE recently developed starch gel electrophoresis has proved to have a resolving power superior to any other type of zone electrophoresis1–4. I have applied this technique to the study of highly purified toxins3, but further work on the separated and eluted fractions of the toxin of Corynebacterium diphtheriae in tissue cultures and in animals indicated that the resolution of these proteins was not completely satisfactory in the buffer (0.03 M boric acid and 0.012 M sodium hydroxide) normally used in this type of zone electrophoresis. A more suitable system was found with a mixture of tris-(hydroxymethyl)-aminomethane-citrate (tris-citrate) used in conjunction with a boric acid buffer. Preliminary results obtained with toxins, enzymes and human sera are briefly reported here.
Article
Reimpresiones en 1961, 1962.
Article
1. Correlation between elution volume, V(e), and molecular weight was investigated for gel filtration of proteins of molecular weights ranging from 3500 (glucagon) to 820000 (alpha-crystallin) on Sephadex G-200 columns at pH7.5. 2. Allowing for uncertainties in the molecular weights, the results for most of the carbohydrate-free globular proteins fitted a smooth V(e)-log(mol.wt.) curve. In the lower part of the molecular-weight range the results were similar to those obtained with Sephadex G-75 and G-100 gels. 3. V(e)-log(mol.wt.) curves based on results with the three gels are taken to represent the behaviour of ;typical' globular proteins, and are proposed as standard data for the uniform interpretation of gel-filtration experiments. 4. Some glycoproteins, including gamma-globulins and fibrinogen, do not conform to the standard relationship. The effect of shape and carbohydrate content on the gel-filtration behaviour of proteins is discussed. 5. As predicted by the theoretical studies of other authors, correlation exists between the gel-filtration behaviour and diffusion coefficients of proteins. 6. The lower molecular-weight limit for complete exclusion of typical globular proteins from Sephadex G-200 varies with the swelling of the gel, but is usually >10(6). 7. The concentration-dependent dissociation of glutamate dehydrogenase was observed in experiments with Sephadex G-200, and the sub-unit molecular weight estimated as 250000. The free sub-units readily lose enzymic activity. 8. Recognition of the atypical gel-filtration behaviour of gamma-globulins necessitates an alteration to several molecular weights previously estimated with Sephadex G-100 (Andrews, 1964). New values are: yeast glucose 6-phosphate dehydrogenase, 128000; bovine intestinal alkaline phosphatase, 130000; Aerobacter aerogenes glycerol dehydrogenase, 140000; milk alkaline phosphatase, 180000.
Article
A technique for the analysis of certain amino acids in protein hydrolyzates is proposed which consists in one-dimensional paper chromatography followed by elution of the proper areas and colorimetric analysis by selective methods. Results are reported for the histidine, tyrosine, methionine, and arginine contents of a few crystalline protein.This method has been applied to the differentiation of some known and unknown strains of TMV. Application to DNP-derivatives of chymotrypsin and DFP-inhibited chymotrypsin has not yielded any evidence concerning the mechanism of the inhibition by the alkyl phosphates.
Article
A water-soluble (at pH 8) aromatic disulfide [5,5′-dithiobis(2-nitrobenzoic acid)] has been synthesized and shown to be useful for determination of sulfhydryl groups.Several applications have been made to show its usefulness for biological materials.A study of the reaction of this disulfide with blood has produced some evidence for the splitting of disulfide bonds by reduced heme.
Article
Detailed kinetic studies of mammary gland phosphoglucose isomerase have been made in both reaction directions. The influence of hydrogen ion concentration on Michaelis constants and maximum initial velocities was studied in 0.1 ionic strength Tris-acetate buffer at 30°. Kinetic data are interpreted in terms of a simple mechanism allowing for the ionization of the enzyme and the enzyme-substrate complexes. Values for the ionization constants of the enzyme and enzyme-substrate complexes have been calculated using a digital computer. The values of the "pH-independent" kinetic constants have also been calculated. The relationship between the kinetic parameters and the over-all equilibrium constant has been shown to be obeyed from pH 5.7 to 9.5.
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