Km Values For ATP Research Articles

Identification and characterization of a novel type of ketohexokinase from the haloarchaeon Haloferax volcanii.

Ketohexokinase (KHK) catalyzes the ATP-dependent phosphorylation of fructose, forming fructose-1-phosphate and ADP. The enzyme is well studied in Eukarya, in particular in humans and other vertebrates, but homologs have not been identified in Bacteria and Archaea. Here we report the identification of a novel type of KHK from the haloarchaeon Haloferax volcanii (HvKHK). The encoding gene khk was identified as HVO_1812. The gene was expressed as a 90-kDa homodimeric protein, catalyzing the phosphorylation of fructose with a Vmax value of 59 U/mg and apparent KM values for ATP and fructose of 0.47 and 1.29 mM, respectively. Homologs of HvKHK were only identified in a few haloarchaea and halophilic Bacteria. The protein showed low sequence identity to characterized KHKs from Eukarya and phylogenetic analyses indicate that haloarchaeal KHKs are largely separated from eukaryal KHKs. This is the first report of the identification of KHKs in prokaryotes that form a novel cluster of sugar kinases within the ribokinase/pfkB superfamily.

Cloning and Characterization of Pyridoxal Kinase from Geobacillus sp. H6a

Pyridoxal kinase encoded by pdxK gene, is the important key enzyme in the salvage pathway of vitamin B6 biosynthesis. The enzyme catalyzes the phosphorylation of the 5′ alcohol groups of free form vitamin B6 into their 5′-phosphate forms that requires metal ion and ATP. Pyridoxal kinase have been reported in many organisms except in the thermophilic bacterium. Therefore, this study aimed to clone, express and characterize pyridoxal kinase of Geobacillus sp. H6a isolated from the hot spring in the North of Thailand. The GhpdxK gene (810 base pairs) was inserted into pET28a(+) plasmids at restriction site of NdeI and BamHI and transformed into E.coli BL21(DE3). The expressed pyridoxal kinase of this bacterium exhibits a homodimer, in which each subunit had a molecular mass of about 32 kDa when examined by SDS-PAGE and gel filtration. The enzyme showed maximal activity at 70°C and at pH 8.0. The expressed enzyme obtained in this study was found to be more active (>50%) in the broad pH range (6.0 – 9.0) than those previously reported. This enzyme prefers Mg2+ and also accepts other cations to the less extent. Under optimal conditions, the expressed enzyme has higher affinity toward PN (20 ± 1.35 µM), while it showed the same affinity to pyridoxal (100 ± 0.76 µM) and pyridoxamine (100 ± 1.21 µM). The Km value for ATP and 4-amino-5-hydroxymethyl-2-methylpyridine were 8.99 ± 1.76 µM and 19 ± 0.85 µM, respectively. With high activity at high temperature and active in the broad pH range, it could be considered as a potential candidate for future application particularly bioconversion of vitamin B6.

Kinetic and oligomeric study of Leishmania braziliensis nicotinate/nicotinamide mononucleotide adenylyltransferase

Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme involved in REDOX reactions and oxidative stress defense systems. Furthermore, NAD is used as substrate by proteins that regulate essential cellular functions as DNA repair, genetic, and signal transduction, among many others. NAD biosynthesis can be completed through the de novo and salvage pathways, which converge at the common step catalyzed by the nicotinate/nicotinamide mononucleotide adenylyltransferase (NMNAT EC: 2.7.7.1/18). Here, we report the kinetic characterization of the NMNAT of Leishmania braziliensis (LbNMNAT), one of the etiological agents of leishmaniasis, a relevant parasitic disease. The expression and homogeneous purification of the recombinant 6xHis-LbNMNAT protein was carried out and its kinetic study, which included analysis of Km, Vmax, Kcat and the equilibrium constant (KD) for both the forward and reverse reactions, was completed. The oligomeric state of the recombinant 6xHis-LbNMNAT protein was studied through size exclusion chromatography. Our results indicated the highest and lowest Km values for ATP and NAD, respectively. According to the calculated KD, the pyrophosphorolytic cleavage of NAD is favored in vitro. Moreover, the recombinant 6xHis-LbNMNAT protein showed a monomeric state, although it exhibits a structural element involved in potential subunits interaction. Altogether, our results denote notable differences of the LbNMNAT protein in relation to the human orthologs HsNMNAT1-3. These differences constitute initial findings that have to be continued to finally propose the NMNAT as a promissory pharmacological target in L. braziliensis.

Glu60 of α-Calcium/calmodulin dependent protein kinase II mediates crosstalk between the regulatory T-site and protein substrate binding region of the active site

Memory formation transpires to be by activation and persistent modification of synapses. A chain of biochemical events accompany synaptic activation and culminate in memory formation. These biochemical events are steered by interplay and modulation of various synaptic proteins, achieved by conformational changes and phosphorylation/dephosphorylation of these proteins. Calcium/calmodulin dependent protein kinase II (CaMKII) and N-methyl-d-aspartate receptors (NMDARs) are synaptic proteins whose interactions play a pivotal role in learning and memory process. Catalytic activity of CaMKII is modulated upon its interaction with the GluN2B subunit of NMDAR. The structural basis of this interaction is not clearly understood. We have investigated the role of Glu60 of α-CaMKII, a conserved residue present in the ATP binding region of kinases, in the regulation of catalysis of CaMKII by GluN2B. Mutation of Glu60 to Gly exerts different effects on the kinetic parameters of phosphorylation of GluN2B and GluN2A, of which only GluN2B binds to the T-site of CaMKII. GluN2B induced modulation of the kinetic parameters of peptide substrate was altered in the E60G mutant. The mutation almost abolished the modulation of the apparent Km value for protein substrate. However, although kinetic parameters for ATP were altered by mutating Glu60, modulation of the apparent Km value for ATP by GluN2B seen in WT was exhibited by the E60G mutant of α-CaMKII. Hence our results posit that the communication of the T-site of CaMKII with protein substrate binding region of active site is mediated through Glu60 while the communication of the T-site with the ATP binding region is not entirely dependent on Glu60.

Large-Scale Molecular Evolutionary Analysis Uncovers a Variety of Polynucleotide Kinase Clp1 Family Proteins in the Three Domains of Life

Clp1, a polyribonucleotide 5′-hydroxyl kinase in eukaryotes, is involved in pretRNA splicing and mRNA 3′-end formation. Enzymes similar in amino acid sequence to Clp1, Nol9, and Grc3, are present in some eukaryotes and are involved in prerRNA processing. However, our knowledge of how these Clp1 family proteins evolved and diversified is limited. We conducted a large-scale molecular evolutionary analysis of the Clp1 family proteins in all living organisms for which protein sequences are available in public databases. The phylogenetic distribution and frequencies of the Clp1 family proteins were investigated in complete genomes of Bacteria, Archaea and Eukarya. In total, 3,557 Clp1 family proteins were detected in the three domains of life, Bacteria, Archaea, and Eukarya. Many were from Archaea and Eukarya, but a few were found in restricted, phylogenetically diverse bacterial species. The domain structures of the Clp1 family proteins also differed among the three domains of life. Although the proteins were, on average, 555 amino acids long (range, 196–2,728), 122 large proteins with >1,000 amino acids were detected in eukaryotes. These novel proteins contain the conserved Clp1 polynucleotide kinase domain and various other functional domains. Of these proteins, >80% were from Fungi or Protostomia. The polyribonucleotide kinase activity of Thermus scotoductus Clp1 (Ts-Clp1) was characterized experimentally. Ts-Clp1 preferentially phosphorylates single-stranded RNA oligonucleotides (Km value for ATP, 2.5 µM), or single-stranded DNA at higher enzyme concentrations. We propose a comprehensive assessment of the diversification of the Clp1 family proteins and the molecular evolution of their functional domains.

Red-emitting chimeric firefly luciferase for in vivo imaging in low ATP cellular environments

Beetle luciferases have been adapted for live cell imaging where bioluminescence is dependent on the cellular availability of ATP, O2, and added luciferin. Previous Photinus pyralis red-emitting variants with high Km values for ATP have performed disappointingly in live cells despite having much higher relative specific activities than enzymes like Click Beetle Red (CBR). We engineered a luciferase variant PLR3 having a Km value for ATP similar to CBR and ∼2.6-fold higher specific activity. The red-emitting PLR3 was ∼2.5-fold brighter than CBR in living HEK293T and HeLa cells, an improvement consistent with the importance of the Km value in low ATP environments.

A Novel Streptavidin-luciferase Fusion Protein: Preparation, Properties and Application in Hybridization Analysis of DNA.

A streptavidin-luciferase fusion protein comprising the thermostable mutant form of firefly luciferase Luciola mingrelica and minimal core streptavidin was constructed. The streptavidin-luciferase fusion was mainly produced in a tetrameric form with high luciferase and biotin-binding activities. It was shown that fusion has the same Km values for ATP and luciferin and the bioluminescence spectra as initial luciferase. The linear dependence of the bioluminescence signal on the content of the fusion was observed within the range of 10-18 -10-13 mol per well. Successful application of obtained fusion in a biospecific bioluminescence assay based on biotin-streptavidin interactions was demonstrated by the example of a specific DNA hybridization analysis. A DNA hybridization analysis for Escherichia coli cells identification was developed using unique for these cells gadB fragment encoding glutamate decarboxylase. The amplified biotinylated GadB fragments were hybridized with the immobilized oligonucleotide probes; then, the biotin in the DNA duplexes was detected using the streptavidin-luciferase fusion protein. To reach the high sensitivity of the assay, we optimized the conditions of the assay. It was shown that the use of Pluronic for plate modification resulted in a significant reduction in the DNA detection limit which finally was 0.4 ng per well.

Identification and Partial Characterization of a Novel UDP-N-Acetylenolpyruvoylglucosamine Reductase/UDP-N-Acetylmuramate:l-Alanine Ligase Fusion Enzyme from Verrucomicrobium spinosum DSM 4136(T).

The enzymes involved in synthesizing the bacterial cell wall are attractive targets for the design of antibacterial compounds, since this pathway is essential for bacteria and is absent in animals, particularly humans. A survey of the genome of a bacterium that belongs to the phylum Verrucomicrobia, the closest free-living relative to bacteria from the Chlamydiales phylum, shows genetic evidence that Verrucomicrobium spinosum possesses a novel fusion open reading frame (ORF) annotated by the locus tag (VspiD_010100018130). The ORF, which is predicted to encode the enzymes UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) and UDP-N-acetylmuramate:l-alanine ligase (MurC) that are involved in the cytoplasmic steps of peptidoglycan biosynthesis, was cloned. In vivo analyses using functional complementation showed that the fusion gene was able to complement Escherichia coli murB and murC temperature sensitive mutants. The purified recombinant fusion enzyme (MurB/CVs) was shown to be endowed with UDP-N-acetylmuramate:l-alanine ligase activity. In vitro analyses demonstrated that the latter enzyme had a pH optimum of 9.0, a magnesium optimum of 10 mM and a temperature optimum of 44–46°C. Its apparent Km values for ATP, UDP-MurNAc, and l-alanine were 470, 90, and 25 μM, respectively. However, all attempts to demonstrate an in vitro UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) activity were unsuccessful. Lastly, Hidden Markov Model-based similarity search and phylogenetic analysis revealed that this fusion enzyme could only be identified in specific lineages within the Verrucomicrobia phylum.

The cold-adapted γ-glutamyl-cysteine ligase from the psychrophile Pseudoalteromonas haloplanktis.

A recombinant γ-glutamyl-cysteine ligase from the psychrophile Pseudoalteromonas haloplanktis (rPhGshA II) was produced and characterised. This enzyme catalyses the first step of glutathione biosynthesis by forming γ-glutamyl-cysteine from glutamate and cysteine in an ATP-dependent reaction. The other ATP-dependent enzyme, glutathione synthetase (rPhGshB), involved in the second step of the biosynthesis, was already characterised. rPhGshA II is a monomer of 58kDa and its activity was characterised through a direct radioisotopic method, measuring the rate of ATP hydrolysis. The enzyme was active even at cold temperatures in a moderately alkaline buffer containing a high concentration of Mg(++); 2-aminobutyrate could replace cysteine, although a lower activity was detected. The reaction rate of rPhGshA II at 15°C was higher than that reported for rPhGshB, thus suggesting that formation of γ-glutamyl-cysteine was not the rate limiting step of glutathione biosynthesis in P.haloplanktis. rPhGshA II had different affinities for its substrates, as evaluated on the basis of the KM values for ATP (0.093mM), glutamate (2.8mM) and cysteine (0.050mM). Reduced glutathione acted as an inhibitor of rPhGshA II, probably through the binding to an enzyme pocket different from the active site. Also the oxidised form of glutathione inhibited the enzyme with a more complex inhibition profile, due to the complete mono-glutathionylation of rPhGshA II on Cys 386, as proved by mass spectrometry data. When compared to rPhGshB, rPhGshA II possessed more typical features of a psychrophilic enzyme, as it was endowed with lower thermodependence and higher heat sensitivity. In conclusion, this work extends the knowledge on glutathione biosynthesis in the first cold-adapted source; however, another possible redundant γ-glutamyl-cysteine ligase (PhGshA I), not yet characterised, could participate in the biosynthesis of this cellular thiol in P.haloplanktis.

Novel inhibitors of acetate kinase from Francisella tularensis

Developed as a bioweapon in the 1940's, Francisella tularensis is the pathogenic bacteria responsible for tularemia, or rabbit fever. In order to develop new treatments for weaponized strains of F. tularensis, potential inhibitors F. tularensis acetate kinase were identified in silico and experimentally tested. Often kinase activity is probed using relatively expensive NADH‐linked assays. Here, we utilized an inexpensive kinetic assay that monitors the production of acetyl phosphate via reaction with hydroxylamine to rapidly characterize potential inhibitors of F. tularensis acetate kinase. The KM values for ATP and acetate were determined to be 1.4 and 15.4 mM, respectively, and the Vmax was determined to be 40.6 μmol min−1 mg−1. Computational screening using DOCK allowed the identification of potential inhibitors from the National Cancer Institute's Developmental Therapeutics Program. Ten compounds were obtained and tested, and two of these reduce the activity of F. tularensis acetate kinase significantly, though at relatively high concentrations. Using these structures as lead compounds, we are mining the database for similar compounds to test as inhibitors. This research was funded by the Wabash College Haines Biochemistry Fund.

Specific Domains in Yeast Translation Initiation Factor eIF4G Strongly Bias RNA Unwinding Activity of the eIF4F Complex toward Duplexes with 5′-Overhangs

During eukaryotic translation initiation, the 43 S ribosomal pre-initiation complex is recruited to the 5'-end of an mRNA through its interaction with the 7-methylguanosine cap, and it subsequently scans along the mRNA to locate the start codon. Both mRNA recruitment and scanning require the removal of secondary structure within the mRNA. Eukaryotic translation initiation factor 4A is an essential component of the translational machinery thought to participate in the clearing of secondary structural elements in the 5'-untranslated regions of mRNAs. eIF4A is part of the 5'-7-methylguanosine cap-binding complex, eIF4F, along with eIF4E, the cap-binding protein, and the scaffolding protein eIF4G. Here, we show that Saccharomyces cerevisiae eIF4F has a strong preference for unwinding an RNA duplex with a single-stranded 5'-overhang versus the same duplex with a 3'-overhang or without an overhang. In contrast, eIF4A on its own has little RNA substrate specificity. Using a series of deletion constructs of eIF4G, we demonstrate that its three previously elucidated RNA binding domains work together to provide eIF4F with its 5'-end specificity, both by promoting unwinding of substrates with 5'-overhangs and inhibiting unwinding of substrates with 3'-overhangs. Our data suggest that the RNA binding domains of eIF4G provide the S. cerevisiae eIF4F complex with a second mechanism, in addition to the eIF4E-cap interaction, for directing the binding of pre-initiation complexes to the 5'-ends of mRNAs and for biasing scanning in the 5' to 3' direction.

EMD57033 Acts as a Pharmacological Chaperone Stabilizing and Activating Myosin Motor Activity

Myosins play a crucial role for a wide range of physiological functions. The dysfunction or absence of myosin motors has been linked to diseases such as muscle wasting, heart failure, blood disorders, severe forms of senso-neuronal degeneration and cancer. Therefore, the development of selective and potent small-molecule myosin effectors holds the promise to open routes for the treatment of a wide range of disabling diseases. In this study, we show that the thiadiazinone derivative EMD57033 acts as a pharmacological chaperone for several members of the myosin family. Our results show that EMD57033 stimulates the actin-activated ATPase activity and motor activity of myosins from classes 2,5 and 6 up to 3-fold. The most potent interaction was observed with cardiac β-myosin. EMD57033 lowers the apparent second binding constant for F-actin binding in the presence of ATP from 31 μM (+/- 6 μM) to 4 μM (+/- 1 μM). The KM value for ATP remains unaffected by the drug. Furthermore, addition of 10 μM EMD57033 rescues the motor function of “aged” cardiac β-myosin preparations that display strongly reduced activity in the in vitro motility assay prior to addition of the drug. Structural studies indicate that EMD57033 binds in a pocket that is located between the N-terminal SH3-like subdomain and the motor domain-lever arm junction. The participation of the N-terminal SH3-like subdomain in EMD57033 binding is supported by the failure of class-1 myosins, which lack this domain, to productively interact with EMD57033.

Lobe-specific Functions of Ca2+·Calmodulin in αCa2+·Calmodulin-dependent Protein Kinase II Activation

N-Methyl-d-aspartic acid receptor-dependent long term potentiation (LTP), a model of memory formation, requires Ca2+·calmodulin-dependent protein kinase II (αCaMKII) activity and Thr286 autophosphorylation via both global and local Ca2+ signaling, but the mechanisms of signal transduction are not understood. We tested the hypothesis that the Ca2+-binding activator protein calmodulin (CaM) is the primary decoder of Ca2+ signals, thereby determining the output, e.g. LTP. Thus, we investigated the function of CaM mutants, deficient in Ca2+ binding at sites 1 and 2 of the N-terminal lobe or sites 3 and 4 of the C-terminal CaM lobe, in the activation of αCaMKII. Occupancy of CaM Ca2+ binding sites 1, 3, and 4 is necessary and sufficient for full activation. Moreover, the N- and C-terminal CaM lobes have distinct functions. Ca2+ binding to N lobe Ca2+ binding site 1 increases the turnover rate of the enzyme 5-fold, whereas the C lobe plays a dual role; it is required for full activity, but in addition, via Ca2+ binding site 3, it stabilizes ATP binding to αCaMKII 4-fold. Thr286 autophosphorylation is also dependent on Ca2+ binding sites on both the N and the C lobes of CaM. As the CaM C lobe sites are populated by low amplitude/low frequency (global) Ca2+ signals, but occupancy of N lobe site 1 and thus activation of αCaMKII requires high amplitude/high frequency (local) Ca2+ signals, lobe-specific sensing of Ca2+-signaling patterns by CaM is proposed to explain the requirement for both global and local Ca2+ signaling in the induction of LTP via αCaMKII.

Discovery of PDK1 Kinase Inhibitors with a Novel Mechanism of Action by Ultrahigh Throughput Screening

The phosphoinositide 3-kinase/AKT signaling pathway plays a key role in cancer cell growth, survival, and angiogenesis. Phosphoinositide-dependent protein kinase-1 (PDK1) acts at a focal point in this pathway immediately downstream of phosphoinositide 3-kinase and PTEN, where it phosphorylates numerous AGC kinases. The PDK1 kinase domain has at least three ligand-binding sites: the ATP-binding pocket, the peptide substrate-binding site, and a groove in the N-terminal lobe that binds the C-terminal hydrophobic motif of its kinase substrates. Based on the unique PDK1 substrate recognition system, ultrahigh throughput TR-FRET and Alphascreen screening assays were developed using a biotinylated version of the PDK1-tide substrate containing the activation loop of AKT fused to a pseudo-activated hydrophobic motif peptide. Using full-length PDK1, K(m) values were determined as 5.6 mum for ATP and 40 nm for the fusion peptide, revealing 50-fold higher affinity compared with the classical AKT(Thr-308)-tide. Kinetic and biophysical studies confirmed the PDK1 catalytic mechanism as a rapid equilibrium random bireactant reaction. Following an ultrahigh throughput screen of a large library, 2,000 compounds were selected from the reconfirmed hits by computational analysis with a focus on novel scaffolds. ATP-competitive hits were deconvoluted by dose-response studies at 1x and 10x K(m) concentrations of ATP, and specificity of binding was assessed in thermal shift assay. Inhibition studies using fusion PDK1-tide1 substrate versus AKT(Thr-308)-tide and kinase selectivity profiling revealed a novel selective alkaloid scaffold that evidently binds to the PDK1-interacting fragment pocket. Molecular modeling suggests a structural paradigm for the design of inhibitory versus activating allosteric ligands of PDK1.

Structure and Mechanism of the Lincosamide Antibiotic Adenylyltransferase LinB

Lincosamides make up an important class of antibiotics used against a wide range of pathogens, including methicillin-resistant Staphylococcus aureus. Predictably, lincosamide-resistant microorganisms have emerged with antibiotic modification as one of their major resistance strategies. Inactivating enzymes LinB/A catalyze adenylylation of the drug; however, little is known about their mechanistic and structural properties. We determined two X-ray structures of LinB: ternary substrate- and binary product-bound complexes. Structural and kinetic characterization of LinB, mutagenesis, solvent isotope effect, and product inhibition studies are consistent with a mechanism involving direct in-line nucleotidyl transfer. The characterization of LinB enabled its classification as a member of a nucleotidyltransferase superfamily, along with nucleotide polymerases and aminoglycoside nucleotidyltransferases, and this relationship offers further support for the LinB mechanism. The LinB structure provides an evolutionary link to ancient nucleotide polymerases and suggests that, like protein kinases and acetyltransferases, these are proto-resistance elements from which drug resistance can evolve.

Characterization of the ATP-dependent Sphingosine 1-Phosphate Transporter in Rat Erythrocytes

Sphingosine 1-phosphate (S1P) is a bioactive lipid signal transmitter present in blood. Blood plasma S1P is supplied from erythrocytes and plays an important role in lymphocyte egress from lymphoid organs. However, the S1P export mechanism from erythrocytes to blood plasma is not well defined. To elucidate the mechanism of S1P export from erythrocytes, we performed the enzymatic characterization of S1P transporter in rat erythrocytes. Rat erythrocytes constitutively released S1P without any stimulus. The S1P release was reduced by an ABCA1 transporter inhibitor, glyburide, but not by a multidrug resistance-associated protein inhibitor, MK571, or a multidrug resistance protein inhibitor, cyclosporine A. Furthermore, we measured S1P transport activity using rat erythrocyte inside-out membrane vesicles (IOVs). Although the effective S1P transport into IOVs was observed in the presence of ATP, this activity was also supported by dATP and adenosine 5'-(beta,gamma-imido)triphosphate. The rate of S1P transport increased depending on S1P concentration, with an apparent K(m) value of 21 microm. Two phosphorylated sphingolipids, dihydrosphingosine 1-phosphate and ceramide 1-phosphate, did not inhibit S1P transport. Similar to the intact erythrocytes, the uptake of S1P into IOVs was inhibited by glyburide and vanadate but not by the other ABC transporter inhibitors. These results suggest that S1P is exported from the erythrocytes by a novel ATP-dependent transporter.

A Real-Time Bioluminescent HTS Method for Measuring Protein Kinase Activity Influenced Neither by ATP Concentration Nor by Luciferase Inhibition

The firefly luciferin-luciferase reaction has been used to set up an assay for protein kinase based on measuring ATP consumption rate as the first-order rate constant for the kinase reaction. The assay obviates the problems encountered with previous bioluminescent protein kinase assays such as interference with the luciferase reaction from library compounds, nonlinear standard curves, and limited dynamic ranges. In the assay described in the present paper luciferase and luciferin are present during the entire kinase reaction, and the light emission can be measured continuously. In an HTS situation the light emission is measured only twice, i.e., initially and after a predetermined time. After a fivefold reduction of the ATP concentration a Z' value of 0.96 was obtained. Light emission data from samples with kinase are normalized with light emission data from blanks without kinase. First-order rate constants for the kinase reaction calculated from normalized light emission are not affected by a moderate degree of inactivation of luciferase and luciferin during the measuring time. The constants have the same value at all ATP concentrations much lower than the K(m) of the luciferase and the kinase. These factors make the assay very robust and influenced neither by ATP concentration nor by luciferase inhibition. The measuring time depends on the kinase activity and can be varied from minutes to more than 8 h provided the kinase is stable and the evaporation of water from the wells is acceptable. The assay is linear with respect to kinase activity over three orders of magnitude. The new reagents also allowed us to determine K(m) values for ATP and for Kemptide.

Site-Directed Mutagenesis and Structure/Function Studies of Casein Kinase II Correlate Stimulation of Activity by the β Subunit with Changes in Conformation and ATP/GTP Utilization

Casein kinase II exists in vivo as an active holoenzyme consisting of catalytic alpha and/or alpha' and regulatory beta subunits, which form a tetrameric structure of alpha 2 beta 2. Unlike most other protein kinases, casein kinase II uses both ATP and GTP effectively as phosphate donors. Two residues unique to the catalytic subunit of casein kinase II, Val66 and Trp176, were mutated to Ala66 and Phe176, respectively, the amino acids present in more than 95% of the identified protein kinase sequences. Using recombinant alpha subunit expressed in Escherichia coli, the mutations have been previously shown to reduce the utilization of GTP by changing Km values for ATP and GTP and to reduce the approximate fivefold stimulation observed upon addition of the regulatory subunit [Jakobi, R. & Traugh, J. A. (1992) J. Biol. Chem. 267, 23,894-23,902]. In this study, the mutations are shown to affect the catalytic activity of the reconstituted holoenzyme by changing both Km and Vmax values. The Vmax for ATP is reduced by the mutation of Trp176 to phenylalanine, but no change is observed with GTP. The Val66 to alanine and Val66/Trp176 to alanine/phenylalanine mutations reduce the Vmax values for ATP and GTP to levels comparable to those of the catalytic subunits alone, indicating that changes in the stimulation of activity by the beta subunit are due to changes in Vmax. Structural studies using ultraviolet CD spectroscopy show that changes in stimulation of Vmax by the beta subunit are correlated with changes in the secondary structure; the extent of these changes is reduced by both mutations. Correlation of changes in secondary structure and stimulation of activity by the beta subunit indicate that the formation of the wild-type holoenzyme causes conformational changes in the active site, leading to an increased rate of reaction. As shown by the mutations, Val66 and Trp176 are involved both in the conformational changes and in the selectivity of ATP and GTP.

Functional Evaluation of Conserved Basic Residues in Human Phosphomevalonate Kinase

Phosphomevalonate kinase (PMK) catalyzes the cation-dependent reaction of mevalonate 5-phosphate with ATP to form mevalonate 5-diphosphate and ADP, a key step in the mevalonate pathway for isoprenoid/sterol biosynthesis. Animal PMK proteins belong to the nucleoside monophosphate (NMP) kinase family. For many NMP kinases, multiple basic residues contribute to the neutralization of the negatively charged pentacoordinate phosphate reaction intermediate. Loss of basicity can result in catalytically impaired enzymes. On the basis of this precedent, conserved basic residues of human PMK have been mutated, and purified forms of the mutated proteins have been kinetically and biophysically characterized. K48M and R73M mutants exhibit diminished Vmax values in both reaction directions (>1000-fold) with only slight Km perturbations (<10-fold). In both forward and reverse reactions, R110M exhibits a large (>10,000-fold) specific activity diminution. R111M exhibits substantially inflated Km values for mevalonate 5-phosphate and mevalonate 5-diphosphate (60- and 30-fold, respectively) as well as decreases [50-fold (forward) and 85-fold (reverse)] in Vmax. R84M also exhibits inflated Km values (50- and 33-fold for mevalonate 5-phosphate and mevalonate 5-diphosphate, respectively). The Ki values for R111M and R84M product inhibition by mevalonate 5-diphosphate are inflated by 45- and 63-fold; effects are comparable to the 30- and 38-fold inflations in Km for mevalonate 5-diphosphate. R141M exhibits little perturbation in Vmax [14-fold (forward) and 10-fold (reverse)] but has inflated Km values for ATP and ADP (48- and 136-fold, respectively). The Kd of ATP for R141M, determined by changes in tryptophan fluorescence, is inflated 27-fold compared to wt PMK. These data suggest that R110 is important to PMK catalysis, which is also influenced by K48 and R73. R111 and R84 contribute to binding of mevalonate 5-phosphate and R141 to binding of ATP.

The Influence of Insertion of a Critical Residue (Arg356) in Structure and Bioluminescence Spectra of Firefly Luciferase

The firefly bioluminescence reaction, which uses luciferin, Mg-ATP, and molecular oxygen to yield an electronically excited oxyluciferin, is carried out by luciferase and visible light is emitted. The bioluminescence color of firefly luciferases is determined by the luciferase structure and assay conditions. Among different beetle luciferases, those from Phrixothrix railroad worm emit either yellow or red bioluminescence colors. Sequence alignment analysis shows that the red-emitter luciferase from Phrixothrix hirtus has an additional Arg residue at 353, which is absent in firefly luciferases. We report here the construction and purification of a mutant at residue Arg(356), which is not conserved in beetle luciferases. By insertion of an additional residue (Arg(356)) using site-specific insertion mutagenesis in a green-emitter luciferase (Lampyris turkestanicus) the color of emitted light was changed to red and the optimum temperature of activity was also increased. Insertion of this Arg in an important flexible loop showed changes of the bioluminescence color and the luciferase reaction took place with relatively retention of its basic kinetic properties such as Km and relative activity. Comparison of native and mutant luciferases using homology modeling reveals a significant conformational change of the flexible loop in the red mutant. Movement of flexible loop brought about a new ionic interaction concomitant with a change in polarity of the emitter site, thereby leading to red emission. It is worthwhile to note that the increased optimum temperature and emission of red light might make mutant luciferase a suitable reporter for the study of gene expression and bioluminescence imaging.