Km Values For GTP Research Articles

Inhibition of Pig Phosphoenolpyruvate Carboxykinase Isoenzymes by 3-Mercaptopicolinic Acid and Novel Inhibitors.

There exist two isoforms of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) in pig populations that differ in a single amino acid (Met139Leu). The isoenzymes have different kinetic properties, affecting more strongly the Km and Vmax of nucleotides. They are associated to different phenotypes modifying traits of considerable economic interest. In this work we use inhibitors of phosphoenolpyruvate carboxykinase activity to search for further differences between these isoenzymes. On the one hand we have used the well-known inhibitor 3-mercaptopicolinic acid. Its inhibition patterns were the same for both isoenzymes: a three-fold decrease of the Ki values for GTP in 139Met and 139Leu (273 and 873 μM, respectively). On the other hand, through screening of a chemical library we have found two novel compounds with inhibitory effects of a similar magnitude to that of 3-mercaptopicolinic acid but with less solubility and specificity. One of these novel compounds, (N'1-({5-[1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl]-2-thienyl}methylidene)-2,4-dichlorobenzene-1-carbohydrazide), exhibited significantly different inhibitory effects on either isoenzyme: it enhanced threefold the apparent Km value for GTP in 139Met, whereas in 139Leu, it reduced it from 99 to 69 μM. The finding of those significant differences in the binding of GTP reinforces the hypothesis that the Met139Leu substitution affects strongly the nucleotide binding site of PEPCK-C.

Molecular cloning of a cDNA coding for GTP cyclohydrolase I from Dictyostelium discoideum

The GTP cyclohydrolase I (GTP-CH) gene of the cellular slime mould Dictyostelium discoideum has been cloned and sequenced. The 855 bp cDNA of this gene contains the open reading frame (ORF) encoding 232 amino acids with a predicted molecular mass of approx. 26 kDa. Southern blot analysis indicated the presence of a single gene for GTP-CH in Dictyostelium. PCR amplification of the ORF from chromosomal DNA and sequencing showed the existence of a 101 bp intron in the GTP-CH gene of Dictyostelium discoideum. The amino acid sequence has 47% and 49% positional identity to those of the human and yeast enzymes respectively. Most of the sequence variation between species is located in the N-terminal part of the protein. The overall identity with the E. coli protein is markedly lower. The enzyme was expressed in E. coli and purified as a 68 kDa fusion protein with the maltose-binding protein of E. coli. GTP-CH of Dictyostelium is heat-stable and showed maximal activity at 60 degrees C. The Km value for GTP is 50 microM.

Expression, purification, and characterization of a novel G protein, SGP, from Streptococcus mutans.

The sgp gene of Streptococcus mutans was recently detected immediately downstream from the dgk gene within the same operon. In this study, the sgp gene was subcloned into the pMAL-c2 vector and SGP (S. mutans G protein) was overexpressed in Escherichia coli as a fusion protein with the maltose-binding protein at a level of 40% of total cellular protein. One-step amylose affinity chromatography purification of this fusion protein yielded a product of approximately 95% purity. SGP was purified from this fusion protein following cleavage with protease factor Xa and DEAE-Sephacel chromatography. In nucleotide binding assays, recombinant SGP showed specific binding for GTP and GDP, but not ATP, CTP, and UTP, and also catalyzed efficient hydrolysis of GTP to GDP. Kinetic studies revealed that the SGP Km value for GTP in this reaction was approximately 5.9 microM. Mg2+ also served as a cofactor of SGP in this reaction. In vivo subcellular localization by immunogold labelling demonstrated that SGP was associated with both membrane and cytoplasmic fractions. SGP not only had structural similarities with other G proteins but also proved to have high-level intrinsic GTPase activity. Therefore, SGP appears to be a new member of the G protein superfamily and may participate in transmembrane signaling in the responses of S. mutans cells to environmental stimuli.

Identification of Arginine Residues in the Putative L-Aspartate Binding Site of Escherichiacoli Adenylosuccinate Synthetase

Three arginine residues in the putative aspartate binding site of Escherichia coli adenylosuccinate synthetase were changed to leucines by site-directed mutagenesis. The mutant enzymes R303L, R304L, and R305L were purified to homogeneity on the basis of sodium dodecyl sulfate polyacrylamide gel electrophoresis and characterized by CD spectrometry and initial rate kinetics. CD spectral analysis indicated no differences in secondary structure between the mutants and the wild-type enzyme in the absence of substrates. The Km values for GTP and IMP for the mutants and the wild-type enzyme were comparable. However, the mutant enzymes exhibited 50-200-fold increases in their values of Km for the substrate aspartate relative to the wild-type enzyme. Although the kcat values for the mutant enzymes decreased, the changes were not as dramatic as those observed for the Km of aspartate. The modeling of aspartate in the crystal structure of the complex of adenylosuccinate synthetase with IMP and MgGDP-1 is consistent with the results of mutagenesis, placing the alpha- and beta-carboxylates of aspartate near the side chains of Arg-131, -303, and -305.

Replacement of Asp333 with Asn by site-directed mutagenesis changes the substrate specificity of Escherichia coli adenylosuccinate synthetase from guanosine 5‘-triphosphate to xanthosine 5‘-triphosphate.

The aspartate residue of the (N/T)KXD concensus sequence for GTP-binding proteins is present in the eight available sequences of adenylosuccinate synthetase. Reported here is a comprehensive analysis of the substrate specificity of mutant enzymes, where the conserved Asp333 of the synthetase from Escherichia coli is changed to asparagine, glutamate, and glutamine by site-directed mutagenesis. The mutants D333N, D333E, and D333Q generally show decreased kcat values and increased Km values for GTP. The decreased values of kcat exhibited by the mutants indicate that the interactions between Asp333 and the guanine are relayed by some mechanism to the catalytic residues around the gamma-phosphate of GTP, and that the energy provided by the interaction between Asp333 and the guanine moiety of GTP is utilized for rearrangement of the catalytic residues. The three mutants each have higher affinity for xanthosine 5'-triphosphate (XTP) and ITP than does the wild-type enzyme. In fact, the D333N mutant uses XTP more effectively than the wild-type enzyme employs GTP as a substrate. The side-chain of Asp333 forms hydrogen bonds with the N-1 and the exocyclic amino group of the guanine base of GTP. In the D333N mutant, this interaction is probably replaced by hydrogen bonds between the amide side chain of Asn333 and N-1 and the 2-oxo group of XTP. The D333Q mutant can use UTP as a substrate more effectively than the wild-type enzyme. The longer side chain of glutamine at residue 333 favors pyrimidine nucleotides over the purine nucleotides, GTP, XTP, and ITP. These results demonstrate that Asp333 in the (N/T)KXD consensus sequence of adenylosuccinate synthetase from E. coli is a determinant for GTP-specificity.

Characterization of the phosphotransferase domain of casein kinase II by site-directed mutagenesis and expression in Escherichia coli.

The catalytic alpha subunit of casein kinase II contains the 11 conserved domains characteristic of all protein kinases. Domain II and VII are involved in nucleotide binding and phosphotransfer. Two residues of the alpha subunit, Val-66 (in domain II) and Trp-176 (in domain VII), were changed to Ala-66 and Phe-176, the residues present in more than 95% of the identified protein kinase sequences. These changes altered the selectivity of the alpha subunit for ATP and GTP. The Ala-66 mutant showed an increase in the Km value for GTP from 45 to 71 microM, while the Km value for ATP decreased from 13 to 9 microM. The Km value for ATP with the Phe-176 mutant showed a decrease from 13 to 7 microM. A double mutant of Ala-66/Phe-176 showed the combined effects, with a Km of 6 microM for ATP and 70 microM for GTP. Alteration of Trp-176 to Lys-176, an amino acid which is not present in the corresponding position of any known protein kinase, resulted in a lack of phosphotransferase activity. The mutations, Val-66 to Ala-66 and Trp-176 to Phe-176, also altered the interaction of the alpha subunit with the regulatory beta subunit. In contrast to the wild-type alpha subunit, which was stimulated 4-fold by addition of the beta subunit, the Ala-66 and Ala-66/Phe-176 mutants were not stimulated by the beta subunit, while the Phe-176 mutant was stimulated only 2.5-fold. All of the reconstituted holoenzymes were similar in molecular weight to the native holoenzyme. The stimulation of the phosphotransferase activity toward beta-casein B by spermine and polylysine, which is mediated by the beta subunit, was similar for holoenzymes reconstituted with either wild-type or mutant alpha subunits. Therefore, binding of the beta subunit appears to alter the active site of the alpha subunit directly or indirectly by inducing a conformational change. Ala-66 and Phe-176 mutations appear to change the structure of the alpha subunit sufficiently so that interaction of the subunits is altered and the stimulatory effect of the beta subunit is reduced or eliminated.

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.

Isolation, amino acid analyses and refolding of subunits of pig heart succinyl-CoA synthetase

For the first time, pig heart succinyl-CoA synthetase has been refolded from its isolated subunits after denaturation. Amino acid analyses of pig heart succinyl-CoA synthetase and its subunits were performed. Subunits were isolated by gel filtration in neutral 6 M-urea. The amino acid composition of the native enzyme bears a strong resemblance to that of the Escherichia coli enzyme. Application of the various methods for comparing amino acid compositions [Cornish-Bowden (1983) Methods Enzymol. 91, 60-75] shows that the degree of relatedness between the alpha-subunits of the pig heart and E. coli enzymes and between the beta-subunits of the two synthetases is intermediate between 'strong' and 'weak'. As for the E. coli synthetase, it is unlikely that the alpha-subunit arises from the larger beta-subunit by post-translational modification. The pig heart enzyme contains a single tryptophan residue, which is located in the beta-subunit. Excitation of the enzyme at 295 nm resulted in a typical tryptophan emission spectrum. Refolding of enzyme denatured in 6 M-guanidine hydrochloride or of alpha- and beta-subunits isolated in this solvent required the presence of either ethylene glycol or glycerol, optimally at 20-25% (v/v). GTP-Mg2+ did not stimulate reactivation of the enzyme, in contrast with the result obtained with ATP-Mg2+ in the reconstitution of the enzyme from E. coli. Yields of 60% and 40% were obtained in the refolding of denatured enzyme and isolated subunits respectively. The fluorescence spectrum of the refolded protein was essentially the same as that of native enzyme. Unrecovered activity could not be accounted for in the form of protein aggregates. The specific activity of refolded enzyme that had been separated from inactive protein on a Bio-Sil TSK 250 column was the same as that of native enzyme. Km values for GTP of 27 microM and 14 microM were determined for native and refolded enzyme respectively.

Autophosphorylation of v-Ha-ras p21 is modulated by amino acid residue 12.

The 21,000-dalton protein (p21) encoded by the ras oncogene of Harvey murine sarcoma virus (v-Ha-ras) becomes phosphorylated (pp21) in vivo and in vitro on threonine residue 59. p21 molecules encoded by cellular ras genes (c-Ha-ras-1) contain an alanine at position 59, and thus these p21 molecules are not phosphorylated. In this investigation, recombinant ras genes have been constructed between the 5' p21 coding region of normal (EC) or oncogenically activated (EJ) human c-Ha-ras-1 and the 3' p21 coding region of v-Ha-ras to generate p21 molecules containing a threonine phosphoacceptor site at position 59 and a glycine (EC/v-Ha) or valine (EJ/v-Ha) at residue 12. In transformed NIH 3T3 mouse fibroblast cells labeled with [35S]methionine, the ratio of pp21 to p21 was strikingly modulated by the amino acid at residue 12. v-Ha-ras p21 has an arginine at position 12, and 24% of total p21 was in the phosphorylated form. A glycine at residue 12 decreased the amount of pp21 to 14% of total p21, and a valine at residue 12 dramatically increased this value to 50%. In vitro, the valine form of p21 had 2.4- and 2.7-fold greater autophosphorylating activity than the glycine and arginine forms of p21, respectively, using [gamma-32P]GTP as phosphate donor, but the three p21 species had similar Km values for GTP (0.20-0.27 microM). These results indicate that a biochemical activity of p21 distinguishes between previously observed biological differences of normal and activated human ras genes.

Phosphorylation of glycogen synthase by cyclic AMP-independent casein kinase-2 from rabbit skeletal muscle.

Cyclic AMP-independent casein kinase-2 from rabbit skeletal muscle has been extensively purified by procedures including phosphocellulose, Bio-Gel A-1.5m, casein-Sepharose, and DEAE-cellulose column chromatographies. The casein kinase and glycogen synthase kinase activities are co-purified throughout the purification. The casein kinase-2 has Mr approximately equal to 135,000 as determined by glycerol density gradient centrifugation and by gel filtration. Analysis of the purified kinase by gel electrophoresis in the presence of sodium dodecyl sulfate reveals the presence of two major protein bands having Mr = 42,000 and 27,000 in a ratio of 0.85:1. The kinase phosphorylates glycogen synthase, casein, and phosvitin either in the presence of ATP or GTP; however, the Km values for GTP are slightly higher than those of ATP. The activity of this kinase is inhibited by heparin but is not affected by the addition of cyclic AMP, heat-stable inhibitor of cyclic AMP-dependent protein kinase, Ca2+, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, or the combination of Ca2+ and calmodulin. The phosphorylation of the synthase by the kinase results in the incorporation of approximately 0.8 mol of PO4/subunit and a reduction in the synthase activity ratio from 0.82 to 0.6. The extent of phosphorylation of the synthase catalyzed by casein kinase-2 is similar to that by phosphorylase kinase; however, the sites phosphorylated by these two kinases are different. The relationships of casein kinase-2 to the cyclic AMP-independent synthase kinases that have been described by other laboratories and to the casein kinases isolated from other tissues are discussed.

The coupling with polypeptide synthesis of the GTPase activity dependent on elongation factor G.

The coupling with polypeptide synthesis of the ribosome-elongation factor G (EF-G)-dependent GTPase activity was studied in a highly purified system with well characterized NH4Cl-washed ribosomes which were from 55 to 67% active in poly(U)-directed polyphenylalanine synthesis. The lowest stoichiometries of total GTP hydrolysis to polyphenylalanine incorporation (2.4 to 2.8) were observed at concentrations of MgCl2 (4 to 6.5 mM) slightly lower than the Mg2+ optimum for polyphenylalanine synthesis (7 to 8 mM), in a system containing 80 mM NH4Cl or KCl. For minimal stoichiometry, the concentration of EF-G should be rate-limiting, whereas that of EF-T (EF-Tu.EF-Ts) and aminoacyl-tRNA should be in excess, since the coupling of the EF-G GTPase activity depends on ribosomes in pretranslocative state. Under this condition, the apparent Km values for GTP of GTPase activity and polyphenylalanine synthesis are identical, and they are about an order of magnitude lower than the Km of the ribosome-EF-G-dependent GTPase activity uncoupled from polypeptide synthesis. The stoichiometry was calculated without the usual correction fro GTP hydrolysis obtained in the same system lacking elongation factor T or aminoacyl-tRNA. Such a correction causes overestimation of the uncoupled EF-G GTPase activity still present in the complete system, leading to artificially low stoichiometric values.

Endogenous activating factor for guanylate cyclase in synaptosomal-soluble fraction of rat brain.

When the crude mitochondrial fraction of rat brain was homogenized with distilled water and centrifuged, most of guanylate cyclase activity was detected in the soluble fraction. The total guanylate cyclase activity recovered in the soluble fraction was 5- to 8-fold higher than that of the crude mitochondrial fraction. The greater recovery of guanylate cyclase activity was found to be due to a release of an endogenous activating factor for guanylate cyclase. The activating factor was partially purified by acid extraction followed by a gel filtration and ion exchange resin columns. The factor was a dialyzable small molecule. The molecular weight was estimated to be between 300 and 600 by a Sephadex G-15 column and Diaflo ultrafilter membranes. It was stable in dilute acids, but labile in alkaline solution. It was readily soluble in water, but insoluble in organic solvents. Treatment with various enzymes, so far as tested, failed to abolish the activity. The activating factor stimulated the initial velocity of the reaction. It altered neither the Km value for GTP nor the dependency of the enzyme on divalent metals. The activation by the factor was due to an increase in the Vmax of the reaction. The activation was prevented by lysolecithin, Lubrol PX, hydroxylamine, methylhydroxylamine, or hemoglobin.

Purification of guanylate cyclase from human platelets and effect of arachidonic acid peroxide

Guanylate cyclase of human platelets was separated from cyclic nucleotide and GTP hydrolytic activities with a 104-fold purification over the homogenate. The purified guanylate cyclase preparation requires neither the GTP regenerating system nor cyclic GMP but is stimulated by about 2-fold by 2.5 mM cyclic GMP. The molecular weight of the enzyme was estimated as 180,000 and the Km value for GTP was 95 μM. Arachidonic acid peroxide stimulated the purified enzyme by increasing maximum velocity without changing Km value.