Purification and characterization of rat liver GTP cyclohydrolase I. Cooperative binding of GTP to the enzyme

GTP cyclohydrolase I, an enzyme that catalyzes the first step in the biosynthetic pathway of tetrahydrobiopterin, has been purified about 38,000-fold to apparent homogeneity from rat liver extract with a yield of 5%. The molecular weight of the enzyme was estimated to be 300,000 by gel filtration on Ultrogel AcA 34. The purified enzyme gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at a position corresponding to a molecular weight of 30,000. N-terminal amino acid sequence analysis gave a single amino acid at every step of the Edman degradation up to residue 10. These results suggest that the enzyme is probably a homopolymer. The enzyme showed positive cooperativity with a Hill coefficient of 2.4 at a substrate (GTP) concentration of 10-50 microM. The Vmax value of the enzyme was 45 nmol/min.mg protein. The GTP concentration producing half-maximal velocity was 30 microM at a KCl concentration of 0.1 M. This value increased as the KCl concentration rose, without any change in Vmax or Hill number. Biosynthesis of tetrahydrobiopterin may be controlled by the intracellular level of GTP.     

Human liver GTP cyclohydrolase I: purification and some properties

Human liver guanosine triphosphate (GTP) cyclohydrolase I has been purified more than 1,700-fold to what appears to be homogeneity. The active enzyme complex has an estimated molecular weight of 453,000 +/- 11,500 by gel filtration chromatography. It consists of a polypeptide of 149,000 +/- 4,000 mol wt by SDS-polyacrylamide gel electrophoresis. The activity of the enzyme is heat stable and is inhibited by di- and trivalent cations. The enzyme has an optimum pH of 7.7 in sodium phosphate buffer. It uses GTP as a sole substrate, with a Km of 116 microM.     

Purification of GTP cyclohydrolase I from human liver and production of specific monoclonal antibodies

GTP cyclohydrolase I, the first enzyme in the de novo biosynthesis of tetrahydrobiopterin, was enriched more than 13,000-fold from human liver by preparative isoelectric focusing using Sephadex G-200 SF gels. The pI of the active enzyme was determined as 5.6 by analytical isoelectric focusing in the same matrix. The native enzyme has an apparent molecular mass of 440 kDa and appears to be composed of eight 50-kDa subunits as estimated from SDS/PAGE. The enriched enzyme preparation was used to produce specific monoclonal antibodies. From 11 monoclonal antibodies obtained, one was extensively characterized for further applications. This monoclonal antibody belongs to the IgM class and shows immunoreactivity with GTP cyclohydrolase I both from man and from Escherichia coli. It is capable of highly sensitive detection of GTP cyclohydrolase I by ELISA and by Western blot analysis. The monoclonal antibody was used for the immunoenzymatic localisation of GTP cyclohydrolase I in human peripheral blood mononuclear cells. Furthermore, it was possible to demonstrate the absence of immunoreactivity in cells with GTP cyclohydrolase I deficiency. The antibody's use as a tool either for differential diagnosis of atypical phenylketonuria due to GTP cyclohydrolase I deficiency or prenatal diagnosis of this severe inherited metabolic disease is now under investigation.     

Partial purification of 6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropterin triphosphate synthetase from chicken liver.

An enzyme that catalyzes the formation of 6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropterin triphosphate (D-erythrodihydroneopterin triphosphate) and formic acid from GTP has been purified about 3700-fold from homogenates of chicken liver. The molecular weight of the enzyme, D-erythrodihydroneopterin triphosphate synthetase (GTP cyclohydrolase), has been estimated to be 125,000 by gel filtration on Ultrogel AcA-34. The enzyme functions optimally between pH 8.0 and 9.2 and is considerably heat-stable. No cofactors or metal ions have been demonstrated to be required for activity; however, the reaction is strongly inhibited by Cu2+ and Hg2+. GTP is the most efficient substrate, with GDP being 1/17 as active and guanosine, GMP, and ATP being inactive. The Km for GTP has been found to be 14 micrometer. Although the overall reaction catalyzed by D-erythrodihydroneopterin triphosphate synthetase from chicken liver is identical with that from Escherichia coli GTP cyclohydrolase, immunological studies show no apparent homology between the two enzymes.     

GTP cyclohydrolase I feedback regulatory protein-dependent and -independent inhibitors of GTP cyclohydrolase I

GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates the feedback inhibition of GTP cyclohydrolase I activity by (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) through protein complex formation. Since guanine and BH4 have a common pyrimidine ring structure, we examined the inhibitory effect of guanine and its analogs on the enzyme activity. Guanine, 8-hydroxyguanine, 8-methylguanine, and 8-bromoguanine inhibited the enzyme activity in a GFRP-dependent and pH-dependent manner and induced complex formation between GTP cyclohydrolase I and GFRP. The type of inhibition by this group is a mixed type. All these properties were shared with BH4. In striking contrast, inhibition by 8-azaguanine and 8-mercaptoguanine was GFRP-independent and pH-independent. The type of inhibition by 8-azaguanine and 8-mercaptoguanine was a competitive type. The two compounds did not induce complex formation between the enzyme and GFRP. These results demonstrate that guanine compounds of the first group bind to the BH4-binding site of the GTP cyclohydrolase I/GFRP complex, whereas 8-azaguanine and 8-mercaptoguanine bind to the active site of the enzyme. Finally, the possible implications in Lesch-Nyhan syndrome and Parkinson diseases of the inhibition of GTP cyclohydrolase I by guanine and 8-hydroxyguanine are discussed.     

Purification and properties of guanosine triphosphate cyclohydrolase II from Escherichia coli.

An enzyme that uses GTP as substrate for the formation in stoichiometric quantities of formate, inorganic pyrophosphate, and 2,5-diamino-6-hydroxy-4-(ribosylamino)pyrimidine-5'-phosphate has been purified 2200-fold from extracts of Escherichia coli B. This enzyme is named GTP cyclohydrolase II to distinguish it from a previously studied E. coli enzyme, named GTP cyclohydrolase (and called GTP cyclohydrolase I in this paper), that catalyzes the first of a series of enzymatic reactions leading to the biosynthesis of the pteridine portion of folic acid (Burg, A. W., and Brown, G. M. (1968) J. Biol. Chem. 243, 2349-2358). Some of the properties of GTP cyclohydrolase II are: (a) divalent cations are required for activity (Mg2+ is most effective); (b) its molecular weight, estimated by filtration on Sephadex G-200, is 44,000; (c) the K-m for GTP is 41 mum; (d) its pH optimum is 8.5; and (e) its activity is inhibited by inorganic pyrophosphate, one of the products of the reaction. Compounds not used as substrate are: GDP, GMP, guanosine, dGTP, ATP, ITP, and XTP. Properties a, b, c, and e (above), as well as the nature of the products, distinguish this enzyme from GTP cyclohydrolase I. Since GTP cyclohydrolase II apparently is not concerned with the biosynthesis of folic acid, the possible physiological role of this enzyme in the biosynthesis of riboflavin is considered in the light of the present investigations and the previously published work on riboflavin biosynthesis by other investigators.     

Localization of GTP cyclohydrolase I in human peripheral blood smears using a specific monoclonal antibody and an immune-alkaline phosphatase labeling technique

GTP cyclohydrolase I, the enzyme catalyzing the first step in the cofactor biosynthesis for the aromatic amino acid hydroxylases, has been localized in situ. By the use of a monoclonal antibody specific to human GTP cyclohydrolase I, the enzyme has been visualized immuno-enzymatically by alkaline phosphatase monoclonal anti-alkaline phosphatase labeling. In routine blood smears lymphocytes, monocytes/macrophages, and granulocytes show strong intraplasmatic staining. Premature erythrocytes show clear staining of the reticulated cytoplasmatic structure, while mature erythrocytes are completely negative. Neither is there any staining for GTP cyclohydrolase I in the blast cells of a case of T-cell acute lymphoblastic leukemia. These results closely confirm the prior finding that mature erythrocytes as well as most malignant mononuclear cells lack GTP cyclohydrolase I activity, and they indicate that in these cells the enzyme protein may be absent.     

Isolation and characterization of GTP cyclohydrolase I from mouse liver. Comparison of normal and the hph-1 mutant

GTP cyclohydrolase I, an enzyme that catalyzes the first reaction in the pathway for the biosynthesis of pterin compounds, was purified from of C3H mouse liver by 192-fold to apparent homogeneity, using Ultrogel AcA34, DEAE-Trisacryl, and GTP-agarose gels. Its native molecular weight was estimated at 362,000. When the enzyme was subjected to electrophoresis on a denaturing polyacrylamide gel, only one protein band was evident, and its molecular weight was estimated at 55,700. The NH2-terminal amino acid of this enzyme was serine. These results indicate the enzyme consists of six to eight subunits. No coenzyme or metal ion was required for activity. This enzyme activity was inhibited by most of divalent cations and was slightly activated by potassium ion. The Km value for GTP was determined to be 17.3 microM. The temperature and pH optima for the activity were 60 degrees C and pH 8.0-8.5, respectively. The expected products, a dihydroneopterin compound and formic acid, were found in a molar ratio of 1.01. A polyclonal antiserum generated against the purified enzyme was used to compare GTP cyclohydrolase I from the hph-1 mutant and normal mouse. The hph-1 mutant liver contained only 8% of normal specific activity, but a normal amount of GTP cyclohydrolase I antigen as compared with the C3H mouse. Subunit molecular weight and electrophoretic behavior of GTP cyclohydrolase I from hph-1 mutant were not different from those of the enzyme from C3H mouse. These results suggest that the hph-1 mutation may involve alteration of the catalytic site but does not detectably alter the whole enzyme structure.     

Control of drosopterin synthesis in Drosophila melanogaster: Mutants showing an altered pattern of GTP cyclohydrolase activity during development

The reaction catalyzed by GTP cyclohydrolase is the first unique step of pteridine biosynthesis in Drosophila melanogaster and is therefore likely to be an important control point. GTP cyclohydrolase activity varies during development, showing two distinct peaks of activity—one at pupariation and a much larger peak at emergence. Most of the early pupal enzyme is located in the body region, whereas in late pupal and early adult life most of the activity is found in the head. Mixing experiments indicate that developmental changes in activity are not due to changes in the level of a direct effector of GTP cyclohydrolase. The mutants raspberry and prune show an increased GTP cyclohydrolase activity at pupariation relative to wild type, but a decreased enzyme activity at emergence. The changes in GTP cyclohydrolase activity are reflected in changes in pteridine levels in these mutants. Several lines of evidence suggest that neither locus is the structural gene for GTP cyclohydrolase. The raspberry and prune gene products may play a specific role in regulating GTP cyclohydrolase activity during development.This work was supported by a grant from the Australian Research Grants Committee D2 75/15248.     

Correlation of guanosine triphosphate cyclohydrolase activity and the synthesis of pterins in Drosophila melanogaster

The enzyme guanosine triphosphate cyclohydrolase (GTP cyclohydrolase), which in bacteria is known to be the first enzyme in the biosynthetic pathway for the synthesis of pteridines, has been discovered in extracts of Drosophila melanogaster. Most of the enzyme (80%) is located in the head of the adult fly. An analysis of enzyme activity during development in Drosophila has revealed the presence of a relatively small peak of activity at pupariation and a much larger peak that appears at about the time of eclosion. Enzyme activity declines rapidly as the fly ages. Analyses for the production of the typical pteridine pigments of Drosophila have indicated that the small peak of GTP cyclohydrolase activity evident at pupariation coincides with the appearance of isoxanthopterin, sepiapterin, and pterin, and the larger peak at eclosion roughly corresponds to the accumulation of drosopterin as well as to the appearance in larger amounts of pterin and sepiapterin. These observations strongly suggest that in Drosophila, like bacteria, GTP cyclohydrolase is involved in the biosynthesis of pteridines. Analyses of a variety of zeste mutants of Drosophila melanogaster have shown that these mutants all contain GTP cyclohydrolase equal approximately to the amount found in the wild-type fly. These observations do not support the suggestions made by Rasmusson et al. (1973) that zeste is the structural locus for GTP cyclohydrolase.This work was supported by research grants from the National Institutes of Health (AM03442) and the National Science Foundation (GB33929).     

Presence of Escherichia coli of a deaminase and a reductase involved in biosynthesis of riboflavin.

Two enzymes have been partially purified from extracts of Escherchia coli B which together catalyze the conversion of the product of the action of GTP cyclohydrolase II, 2,5-diamino-6-oxy-4-(5'-phosphoribosylamine)pyrimidine, to 5-amino-2,6-dioxy-4-(5'-phosphoribitylamine)pyrimidine. These two compounds are currently thought to be intermediates in the biosynthesis of riboflavin. The enzymatic conversion occurs in two steps. The product of the action of GTP cyclohydrolase II first undergoes hydrolytic deamination at carbon 2 of the ring, followed by reduction of the ribosylamino group to a ribitylamino group. The enzyme which catalyzes the first step, herein called the "deaminase," has been purified 200-fold. The activity was assayed by detecting the conversion of the product of the reaction catalyzed by GTP cyclohydrolase II to a compound which reacts with butanedione to form 6,7-dimethyllumazine. The enzyme has a molecular weight of approximately 80,000 and a pH optimum of 9.1. The dephosphorylated form of the substrate is not deaminated in the presence of the enzyme. The assay for the enzyme which catalyzes the second step, referred to here as the "reductase," involves the detection of the conversion of the product of the deaminase-catalyzed reaction to a compound which, after treatment with alkaline phosphatase, reacts with butanedione to form 6,7-dimethyl-8-ribityllumazine. The reductase has a molecular weight of approximately 40,000 and a pH optimum of 7.5. Like the deaminase, the reductase does not act on the dephosphorylated form of its substrate. Reduced nicotinamide adenine dinucleotide phosphate is required as a cofactor; reduced nicotinamide adenine dinucleotide can be used about 30% as well as the phosphate form. The activity of neither enzyme is inhibited by riboflavin, FMN, or flavine adenine dinucleotide.     

Allosteric characteristics of GTP cyclohydrolase I from Escherichia coli.

The kinetic and regulatory properties of GTP cyclohydrolase I were investigated using an improved enzyme assay and direct determination of the product, dihydroneopterin triphosphate. The enzyme was purified from Escherichia coli to absolute homogeneity as demonstrated by N-terminal sequencing of up to 50 amino acid residues. A 30-residue internal fragment showed 42% similarity with rat liver GTP cyclohydrolase I. The enzyme did not obey Michaelis-Menten kinetics or show a sigmoid reaction curve. The substrate saturation kinetics were found to be slow with low response to minor changes in GTP concentrations. GTP cyclohydrolase I has a relatively high apparent Km. The values are slightly different for enzyme purified by GTP-agarose (100 microM) and UTP-agarose (110 microM). Low turnover numbers of 12/min and 19/min were calculated for the respective enzyme preparations. GTP-cyclohydrolase-I activity was modulated in Vmax by K+, divalent cations, UTP and tetrahydrobiopterin. Divalent cations, such as Mg2+, had an activating effect with an optimum at 8 mM Mg2+. A different catalytic function and formation of a new, unidentified product by GTP cyclohydrolase I was observed in the presence of Ca2+. In the presence of 1 mM EDTA and Mg2+, GTP-cyclohydrolase-I activity was strongly inhibited by chelate complexes. UTP proved not to be a competitive inhibitor, but a positive modulator. The inhibition by chelate complexes was totally abolished by UTP. Tetrahydrobiopterin showed an inhibitory effect, with 50% inhibition at 100 microM tetrahydrobiopterin. UTP was able to reduce the inhibition by tetrahydrobiopterin. Using monoclonal antibody 1F11 (related to the GTP-binding site), and monoclonal antibody NS7 (mimicking tetrahydrobiopterin), different binding sites were demonstrated for GTP and tetrahydrobiopterin on each enzyme subunit. Western-blot competition analysis revealed a UTP-binding site different from the binding sites of GTP and tetrahydrobiopterin. Based on the kinetic behaviour and the kind of modulations observed we defined GTP cyclohydrolase I as an M-class allosteric enzyme.     

Biochemical characterization of oligomerization of Escherichia coli GTP cyclohydrolase I

GTP cyclohydrolase I (E.C. 3.5.4.16) is a homodecameric protein that catalyzes the conversion of GTP to 7,8- dihydroneopterin triphosphate (H(2)NTP), the initial step in the biosynthesis of pteridines. It was proposed that the enzyme complex could be composed of a dimer of two pentamers, or a pentamer of tightly associated dimers; then the active site of the enzyme was located at the interface of three monomers (Nar et al. 1995a, b). Using mutant enzymes that were made by site-directed mutagenesis, we showed that a decamer of GTP cyclohydrolase I should be composed of a pentamer of five dimers, and that the active site is located between dimers, as analyzed by a series of size exclusion chromatography and the reconstitution experiment. We also show that the residues Lys 136, Arg139, and Glu152 are of particular importance for the oligomerization of the enzyme complex from five dimers to a decamer.     

Allosteric interactions in glycerol dehydratase. Purification of enzyme and effects of positive and negative cooperativity for glycerol

An improved method for the purification of the apoenzyme (AB complex) of glycerol dehydratase from Aerobacter aerogenes is presented. One hundredfold purification Was achieved. This purification was possible due to stabilization of the AB complex by glycerol. Using chromatography on Sephadex G-200, the highest degree of association of AB complex was found in glycine buffer in the presence of glycerol.Potassium ions, in contrast to glycerol, seem to weaken the forces which bind subunits A and B together. This may be the action of potassium necessary for the performance of glycerol dehydratase activity, since potassium is required for holoenzyme activity.From kinetic studies it appears that the enzyme exhibits homotrophic effects with regard to glycerol binding sites, which can he extended from positive (in the presence of glycine buffer) to negative (in the presence of ethanolamine buffer) cooperativity. The high cooperativity between glycerol binding sites on the enzyme, in glycine buffer, can be abolished by the addition of phosphate. By decreasing the value of the Hill coefficient and increasing the V of the enzymatic reaction, phosphate seems to act as an allosteric activator.     

Ligand binding to the inhibitory and stimulatory GTP cyclohydrolase I/GTP cyclohydrolase I feedback regulatory protein complexes

GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates feedback inhibition of GTP cyclohydrolase I activity by 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4), which is an essential cofactor for key enzymes producing catecholamines, serotonin, and nitric oxide as well as phenylalanine hydroxylase. GFRP also mediates feed-forward stimulation of GTP cyclohydrolase I activity by phenylalanine at subsaturating GTP levels. These ligands, BH4 and phenylalanine, induce complex formation between one molecule of GTP cyclohydrolase I and two molecules of GFRP. Here, we report the analysis of ligand binding using the gel filtration method of Hummel and Dreyer. BH4 binds to the GTP cyclohydrolase I/GFRP complex with a Kd of 4 microM, and phenylalanine binds to the protein complex with a Kd of 94 microM. The binding of BH4 is enhanced by dGTP. The binding stoichiometrics of BH4 and phenylalanine were estimated to be 10 molecules of each per protein complex, in other words, one molecule per subunit of protein, because GTP cyclohydrolase I is a decamer and GFRP is a pentamer. These findings were corroborated by data from equilibrium dialysis experiments. Regarding ligand binding to free proteins, BH4 binds weakly to GTP cyclohydrolase I but not to GFRP, and phenylalanine binds weakly to GFRP but not to GTP cyclohydrolase I. These results suggest that the overall structure of the protein complex contributes to binding of BH4 and phenylalanine but also that each binding site of BH4 and phenylalanine may be primarily composed of residues of GTP cyclohydrolase I and GFRP, respectively.     

Characterization of wild-type and mutants of recombinant human GTP cyclohydrolase I: relationship to etiology of dopa-responsive dystonia

To explore the molecular etiology of two disorders caused by a defect in GTP cyclohydrolase I--hereditary progressive dystonia with marked diurnal fluctuation (HPD), also known as dopa-responsive dystonia (DRD), and autosomal recessive GTP cyclohydrolase I deficiency--we purified and analyzed recombinant human wild-type and mutant GTP cyclohydrolase I proteins expressed in Escherichia coli. Mutant proteins showed very low enzyme activities, and some mutants were eluted at a delayed volume on gel filtration compared with the recombinant wild-type. Next, we examined the GTP cyclohydrolase I protein amount by western blot analysis in phytohemagglutinin-stimulated mononuclear blood cells from HPD/DRD patients. We found a great reduction in the amount of the enzyme protein not only in one patient who had a frameshift mutation, but also in an HPD/DRD patient who had a missense mutation. These results suggest that a dominant-negative effect of chimeric protein composed of wild-type and mutant subunits is unlikely as a cause of the reduced enzyme activity in HPD/DRD patients. We suggest that reduction of the amount of the enzyme protein, which is independent of the mutation type, could be a reason for the dominant inheritance in HPD/DRD.     

Effects of sepiapterin and 6-acetyldihydrohomopterin on the guanosine triphosphate cyclohydrolase I of mouse, rat and the fruit-fly Drosophila.

The regulation of GTP cyclohydrolase I would lead to the regulation of tetrahydrobiopterin, an important cofactor for synthesis of neurotransmitters. In an attempt to extend a previous finding [Bellahsene, Dhondt, & Farriaux (1984) Biochem. J. 217, 59-65] that GTP cyclohydrolase I of rat liver is inhibited by subnanomolar concentrations of reduced biopterin and sepiapterin, we found that this could not be verified with the enzyme from mouse liver, fruit-fly (Drosophila) heads or, indeed, from rat liver. It was shown, however, that 12 microM-sepiapterin inhibited mouse liver GTP cyclohydrolase I. Another compound, namely 6-acetyldihydrohomopterin, was also employed in the present study to explore its effect on enzymes that lead to its synthesis in Drosophila and for effects on mammalian systems; at 2-5 microM this compound was shown to stimulate one form of mouse liver GTP cyclohydrolase I and then to inhibit at higher concentrations (40 microM). Neither sepiapterin nor 6-acetyldihydrohomopterin caused any effect on the Drosophila head enzyme. On the other hand, the sigmoid GTP concentration curve for the Drosophila enzyme may indicate a regulatory characteristic of this enzyme. Another report, on the lower level of GTP cyclohydrolase I in mutant mouse liver [McDonald, Cotton, Jennings, Ledley, Woo & Bode (1988) J. Neurochem. 50, 655-657], was confirmed and extended. Instead of having 10% activity, we find that the hph-1 mouse mutant has less than 2% activity in the liver. These studies demonstrate that micromolar levels of reduced pterins may have regulatory effects on GTP cyclohydrolase I and that a mouse mutant is available that has low enough activity to be considered as a model for human atypical phenylketonuria.     

IMP dehydrogenase inhibitors reduce intracellular tetrahydrobiopterin levels through reduction of intracellular GTP levels. Indications of the regulation of GTP cyclohydrolase I activity by restriction of GTP availability in the cells.

GTP cyclohydrolase I exhibits a positive homotropic cooperative binding to GTP, which raises the possibility of a role for GTP in regulating the enzyme reaction (Hatakeyama, K., Harada, T., Suzuki, S., Watanabe, Y., and Kagamiyama, H. (1989) J. Biol. Chem. 264, 21660-21664). We examined whether or not the intracellular GTP level is within the range of affecting GTP cyclohydrolase I activity, using PC-12 rat pheochromocytoma and IMR-32 human neuroblastoma cells. Since GTP cyclohydrolase I was the rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin in these cell lines, the intracellular activities of this enzyme were reflected in the tetrahydrobiopterin contents. We found that the addition of guanine or guanosine increased GTP but not tetrahydrobiopterin in these cells. On the other hand, three IMP dehydrogenase inhibitors, tiazofurin, 2-amino-1,3,4-thiadiazole, and mycophenolic acid, decreased both GTP and tetrahydrobiopterin in a parallel and dose-dependent manner, and these effects were reversed by the simultaneous addition of guanine or guanosine. There was no evidence suggesting that these inhibitors inhibited other enzymes involved in the biosynthesis and regeneration of tetrahydrobiopterin. Comparing intracellular activities of GTP cyclohydrolase I in the inhibitor-treated cells with its substrate-velocity curve, we estimated that the intracellular concentration of free GTP is 150 microM at which point the activity of GTP cyclohydrolase I is elicited at its maximum velocity. Below this GTP concentration, GTP cyclohydrolase I activity is rapidly decreased. Therefore GTP can be a regulator for tetrahydrobiopterin biosynthesis.     

Effect of dodecylsulfate binding on the activity of creatine kinase

The number of lithium dodecylsulfate (LDS) molecules tightly bound to creatine kinase has been found by isotachophoresis to be 87 at 25°C upon saturation of the enzyme dimer with LDS. The binding shows positive cooperativity by both the Hill and Scatchard plots. The enzyme is completely inactivated when its high-affinity sites are fully occupied with LDS. However, at partial LDS saturation the activity remaining is definitely higher than can be accounted for by the amount of free enzyme left, showing the presence of species of active molecules with the tight LDS sites only partly saturated. The presence of ATP leads to a decrease in detergent bound at the high-affinity sites with partial restoration of activity.