Phosphoryl Transfer Activity Research Articles

Trivalent rare earth metal cofactors confer rapid NP-DNA polymerase activity.

A DNA polymerase with a single mutation and a divalent calcium cofactor catalyzes the synthesis of unnatural N3'→P5' phosphoramidate (NP) bonds to form NP-DNA. However, this template-directed phosphoryl transfer activity remains orders of magnitude slower than native phosphodiester synthesis. Here, we used time-resolved x-ray crystallography to show that NP-DNA synthesis proceeds with a single detectable calcium ion in the active site. Using insights from isotopic and elemental effects, we propose that one-metal-ion electrophilic substrate activation is inferior to the native two-metal-ion mechanism. We found that this deficiency in divalent activation could be ameliorated by trivalent rare earth and post-transition metal cations, substantially enhancing NP-DNA synthesis. Scandium(III), in particular, confers highly specific NP activity with kinetics enhanced by more than 100-fold over calcium(II), yielding NP-DNA strands up to 100 nucleotides in length.

Inhibition of RNase L and RNA-dependent Protein Kinase (PKR) by Sunitinib Impairs Antiviral Innate Immunity

RNase L and RNA-dependent protein kinase (PKR) are effectors of the interferon antiviral response that share homology in their pseudokinase and protein kinase domains, respectively. Sunitinib is an orally available, ATP-competitive inhibitor of VEGF and PDGF receptors used clinically to suppress angiogenesis and tumor growth. Sunitinib also impacts IRE1, an endoplasmic reticulum protein involved in the unfolded protein response that is closely related to RNase L. Here, we report that sunitinib is a potent inhibitor of both RNase L and PKR with IC(50) values of 1.4 and 0.3 μM, respectively. In addition, flavonol activators of IRE1 inhibited RNase L. Sunitinib treatment of wild type (WT) mouse embryonic fibroblasts resulted in about a 12-fold increase in encephalomyocarditis virus titers. However, sunitinib had no effect on encephalomyocarditis virus growth in cells lacking both PKR and RNase L. Furthermore, oral delivery of sunitinib in WT mice resulted in 10-fold higher viral titers in heart tissues while suppressing by about 2-fold the IFN-β levels. In contrast, sunitinib had no effect on viral titers in mice deficient in both RNase L and PKR. Also, sunitinib reduced mean survival times from 12 to 6 days in virus-infected WT mice while having no effect on survival of mice lacking both RNase L and PKR. Results indicate that sunitinib treatments prevent antiviral innate immune responses mediated by RNase L and PKR.

Steady-State Kinetic Characterization of Kinase Activity and Requirements for Mg2+ of Interleukin-1 Receptor-Associated Kinase-4

Interleukin-1 receptor-associated kinase-4 (IRAK-4) is a Ser/Thr-specific protein kinase that plays a critical role in intracellular signaling cascades mediated by Toll-like and interleukin-1 (IL-1) receptors. Despite a growing body of information on the physiological functions of IRAK-4, its kinase activity remains poorly studied. The present study entails characterization of the steady-state kinetic properties and Mg(2+) requirements of full-length, recombinant human IRAK-4 preactivated by incubation with MgATP. In the presence of 20 mM Mg(2+), activated IRAK-4 herein is demonstrated to phosphorylate a peptide substrate (IRAK-1 peptide), derived from the activation loop of IRAK-1, with a k(cat) of 30 +/- 2.9 s(-1) and K(m) values of 668 +/- 120 and 852 +/- 273 microM for ATP and the peptide, respectively. Two-substrate, dead-end and product inhibition data, using analogues of ATP, are consistent with both a sequential ordered kinetic mechanism with ATP binding to the enzyme prior to the peptide and a sequential random mechanism. Investigation of the Mg(2+) requirements for phosphoryl transfer activity of IRAK-4 revealed that more than one Mg(2+) ion interacts with the enzyme and that the enzyme is maximally active in the presence of 5-10 mM free Mg(2+). While one divalent metal, as part of a chelate complex with ATP, is essential for catalysis, kinetic evidence is provided to show that uncomplexed Mg(2+) further enhances the catalytic activity of IRAK-4 by bringing about an approximately 3-fold increase in k(cat) and an approximately 6-fold reduction in the K(m) for ATP and by rendering the interaction between the nucleotide and peptide substrate binding sites less antagonistic.

Divergent Evolution of Function in the ROK Sugar Kinase Superfamily: Role of Enzyme Loops in Substrate Specificity

The d-allose and N-acetyl-d-mannosamine kinases of Escherichia coli K-12 are divergent members of the functionally diverse ROK (repressor, open reading frame, kinase) superfamily. Previous work in our laboratory has demonstrated that AlsK and NanK possess weak phosphoryl transfer activity toward the alternate substrate d-glucose. To gain insight into the evolutionary mechanisms that fuel the specialization of individual enzyme function, experimental laboratory evolution was conducted to improve the glucokinase activities of AlsK and NanK. Error-prone PCR was combined with in vivo functional selection in a glucokinase-deficient bacterium to identify four independent single nucleotide substitutions in the alsK and nanK genes that improve the glucokinase activity of each enzyme. The most advantageous substitutions, L84P in NanK and A73G in AlsK, enhance the kcat/Km values for phosphoryl transfer to glucose by 12-fold and 60-fold, respectively. Both substitutions co-localize to a variable loop region located between the fourth beta-sheet and the second alpha-helix of the ROK scaffold. A multiple sequence alignment of diverse ROK family members reveals that the A73G substitution in AlsK recapitulates a conserved glycine residue present in many ROK proteins, including some transcriptional repressors. Steady-state kinetic analyses of the selected AlsK and NanK variants demonstrate that their native activities toward d-allose and N-acetyl-d-mannosamine are largely unaffected by the glucokinase-enhancing substitutions. Substrate specificity profiling reveals that the A73G AlsK and L84P NanK variants display systematic improvements in the kcat/Km values for a variety of nonnative carbohydrates. This finding is consistent with an evolutionary process that includes the formation of intermediates possessing relaxed substrate specificities during the initial steps of enzyme functional divergence.

Elucidation of Human Choline Kinase Crystal Structures in Complex with the Products ADP or Phosphocholine

Choline kinase, responsible for the phosphorylation of choline to phosphocholine as the first step of the CDP-choline pathway for the biosynthesis of phosphatidylcholine, has been recognized as a new target for anticancer therapy. Crystal structures of human choline kinase in its apo, ADP and phosphocholine-bound complexes, respectively, reveal the molecular details of the substrate binding sites. ATP binds in a cavity where residues from both the N and C-terminal lobes contribute to form a cleft, while the choline-binding site constitutes a deep hydrophobic groove in the C-terminal domain with a rim composed of negatively charged residues. Upon binding of choline, the enzyme undergoes conformational changes independently affecting the N-terminal domain and the ATP-binding loop. From this structural analysis and comparison with other kinases, and from mutagenesis data on the homologous Caenorhabditis elegans choline kinase, a model of the ternary ADP·phosphocholine complex was built that reveals the molecular basis for the phosphoryl transfer activity of this enzyme.

Solution Structure of a Post-transition State Analog of the Phosphotransfer Reaction between the A and B Cytoplasmic Domains of the Mannitol Transporter IIMannitol of the Escherichia coli Phosphotransferase System

The solution structure of the post-transition state complex between the isolated cytoplasmic A (IIAMtl) and phosphorylated B (phospho-IIBMtl) domains of the mannitol transporter of the Escherichia coli phosphotransferase system has been solved by NMR. The active site His-554 of IIAMtl was mutated to glutamine to block phosphoryl transfer activity, and the active site Cys-384 of IIBMtl (residues of IIBMtl are denoted in italic type) was substituted by serine to permit the formation of a stable phosphorylated form of IIBMtl. The two complementary interaction surfaces are predominantly hydrophobic, and two methionines on IIBMtl, Met-388 and Met-393, serve as anchors by interacting with two deep pockets on the surface of IIAMtl. With the exception of a salt bridge between the conserved Arg-538 of IIAMtl and the phosphoryl group of phospho-IIBMtl, electrostatic interactions between the two proteins are limited to the outer edges of the interface, are few in number, and appear to be weak. This accounts for the low affinity of the complex (Kd approximately 3.7 mm), which is optimally tuned to the intact biological system in which the A and B domains are expressed as a single polypeptide connected by a flexible 21-residue linker. The phosphoryl transition state can readily be modeled with no change in protein-protein orientation and minimal perturbations in both the backbone immediately adjacent to His-554 and Cys-384 and the side chains in close proximity to the phosphoryl group. Comparison with the previously solved structure of the IIAMtl-HPr complex reveals how IIAMtl uses the same interaction surface to recognize two structurally unrelated proteins and explains the much higher affinity of IIAMtl for HPr than IIBMtl.

Synthesis and Stability of 1-Phenylethenyl Phosphate Derivatives and their Phosphoryl Transfer Activity

Synthesis and Stability of 1-Phenylethenyl Phosphate Derivatives and their Phosphoryl Transfer Activity

Multiple Phosphorylation Events Regulate the Activity of the Mannitol Transcriptional Regulator MtlR of the Bacillus stearothermophilus Phosphoenolpyruvate-dependent Mannitol Phosphotransferase System

D-mannitol is taken up by Bacillus stearothermophilus and phosphorylated via a phosphoenolpyruvate-dependent phosphotransferase system (PTS). Transcription of the genes involved in mannitol uptake in this bacterium is regulated by the transcriptional regulator MtlR, a DNA-binding protein whose affinity for DNA is controlled by phosphorylation by the PTS proteins HPr and IICB(mtl). The mutational and biochemical studies presented in this report reveal that two domains of MtlR, PTS regulation domain (PRD)-I and PRD-II, are phosphorylated by HPr, whereas a third IIA-like domain is phosphorylated by IICB(mtl). An involvement of PRD-I and the IIA-like domain in a decrease in affinity of MtlR for DNA and of PRD-II in an increase in affinity is demonstrated by DNA footprint experiments using MtlR mutants. Since both PRD-I and PRD-II are phosphorylated by HPr, PRD-I needs to be dephosphorylated by IICB(mtl) and mannitol to obtain maximal affinity for DNA. This implies that a phosphoryl group can be transferred from HPr to IICB(mtl) via MtlR. Indeed, this transfer could be demonstrated by the phosphoenolpyruvate-dependent formation of [(3)H]mannitol phosphate in the absence of IIA(mtl). Phosphoryl transfer experiments using MtlR mutants revealed that PRD-I and PRD-II are dephosphorylated via the IIA-like domain. Complementation experiments using two mutants with no or low phosphoryl transfer activity showed that phosphoryl transfer between MtlR molecules is possible, indicating that MtlR-MtlR interactions take place. Phosphorylation of the same site by HPr and dephosphorylation by IICB(mtl) have not been described before; they could also play a role in other PRD-containing proteins.

Human Nucleoside Diphosphate Kinase B (Nm23-H2) from Melanoma Cells Shows Altered Phosphoryl Transfer Activity Due to the S122P Mutation

The Ser122 --> Pro mutation in human nucleoside diphosphate kinase (NDK)-B/Nm23-H2 was recently found in melanoma cells. In comparison to the wild-type enzyme, steady state activity of NDKS122P with ATP and TDP as substrates was slowed down 5-fold. We have utilized transient kinetic techniques to analyze phosphoryl transfer between the mutant enzyme and various pairs of nucleoside triphosphates and nucleoside diphosphates. The two half-reactions of phosphorylation and dephosphorylation of the active site histidine residue (His118) were studied separately by making use of the intrinsic fluorescence changes which occur during these reactions. All apparent second order rate constants are drastically reduced, falling 5-fold for phosphorylation and 40-200-fold for dephosphorylation. Also, the reactivity of the mutant with pyrimidine nucleotides and deoxy nucleotides is more than 100-fold reduced compared with the wild-type. Thus, the rate-limiting step of the NDK-BS122P-catalyzed reaction is phosphoryl transfer from the phospho-enzyme intermediate to the nucleoside diphosphate and not phosphoryl transfer from the nucleoside triphosphate to the enzyme as was found for the wild-type protein. This results in a pronounced shift of the equilibrium between unphosphorylated and phosphorylated enzyme. Moreover, like the Killer-of-prune mutation in Drosophila NDK and the neuroblastoma Ser120 --> Gly mutation in human NDK-A/Nm23-H1, the Ser122 --> Pro substitution in NDK-B affects the stability of the protein toward heat and urea. These significantly altered properties may be relevant to the role of the mutant enzyme in various intracellular processes.

Phosphorylation and functional properties of the IIA domain of the lactose transport protein of Streptococcus thermophilus.

The lactose-H+ symport protein (LacS) of Streptococcus thermophilus has a carboxyl-terminal regulatory domain (IIALacS) that is homologous to a family of proteins and protein domains of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in various organisms, of which IIAGlc of Escherichia coli is the best-characterized member. On the basis of these similarities, it was anticipated that IIALacS would be able to perform one or more functions associated with IIAGlc, i.e., carry out phosphoryl transfer and/or affect other catabolic functions. The gene fragment encoding IIALacS was overexpressed in Escherichia coli, and the protein was purified in two steps by metal affinity and anion-exchange chromatography. IIALacS was unable to restore glucose uptake in a IIAGlc-deficient strain, which is consistent with a very low rate of phosphorylation of IIALacS by phosphorylated HPr (HPr approximately P) from E. coli. With HPr approximately P from S. thermophilus, the rate was more than 10-fold higher, but the rate constants for the phosphorylation of IIALacS (k1 = 4.3 x 10(2) M-1 s-1) and dephosphorylation of IIALacS approximately P by HPr (k-1 = 1.1 x 10(3) M-1 s-1) are still at least 4 orders of magnitude lower than for the phosphoryltransfer between IIAGlc and HPr from E. coli. This finding suggests that IIALacS has evolved into a protein domain whose main function is not to transfer phosphoryl groups rapidly. On the basis of sequence alignment of IIA proteins with and without putative phosphoryl transfer functions and the known structure of IIAGlc, we constructed a double mutant [IIALacS(I548E/G556D)] that was predicted to have increased phosphoryl transfer activity. Indeed, the phosphorylation rate of IIALacS(I548E/G556D) by HPr approximately P increased (k1 = 4.0 x 10(3) M-1 s-1) and became nearly independent of the source of HPr approximately P (S. thermophilus, Bacillus subtilis, or E. coli). The increased phosphoryl transfer rate of IIALacS(I548E/G556D) was insufficient to complement IIAGlc in PTS-mediated glucose transport in E. coli. Both IIALacS and IIALacS(I548E/G556D) could replace IIAGlc, but in another function: they inhibited glycerol kinase (inducer exclusion) when present in the unphosphorylated form.

Phosphoryl transfer between phosphorylated histidine-containing protein and histidine-containing protein is not autocatalytic

Histidine-containing protein, HPr, is a phosphocarrier protein that is part of the bacterial phosphoenolpyruvate:sugar phosphotransferase system. HPr is phosphorylated by enzyme I, and P-HPr transfers the phosphoryl group to the IIA domain of a number of sugar-specific enzyme II complexes. Autocatalytic phosphoryl transfer between P-HPr and HPr has recently been reported [van Dijk, A. A., Eisermann, R., Hengstenberg, W., & Robillard, G. T. (1991) Biochemistry 30, 2876-2882]. Our results show that this phosphoryl transfer is due to an unidentified contaminant of HPr preparations. The phosphoryl transfer activity is not present in all HPr preparations. When present, the phosphoryl transfer activity can be removed by further purification or destroyed over time by resuspension of HPr preparations in water. There is no autocatalytic phosphoryl transfer between P-HPr and HPr.

Evidence of acid phosphatase in the cytoplasm as a distinct entity

A study of subcellular acid phosphatase distribution in mammalian tissues shows that isozymes with specific functions are compartmentalized in the cells. The enzyme may be generalized into two types: type A and type B. They are shown by several means to be distinct entities. Type A is confined to the cytoplasm and is inhibited by Cu 2+, HCHO, and the coupling agents (for enzyme staining) fast blue RR salt and fast Garnet GBC salt (newly discovered inhibitors), but is insensitive to fluoride and L-(+)-tartrate. Type B is localized in the organelles, presumably lysosomes, in both soluble form and membrane-bound form, with inhibitor sensitivity exactly opposite to that of type A enzyme. Types A and B consist of different sets of isozymes, with sensitivities to inhibitors resembling those observed with the crude extracts of subcellular fractions. Acid phosphatase that exhibits a phosphoryl transfer property was identified as type A enzyme. Type A enzyme has a slightly higher optimal pH and is inhibited by alloxan, whereas for type B, the addition of alloxan broadens the optimal pH to a higher range and elevates the activity at pH 7.4 from negligible to about 30–40% of that obtained under optimal conditions. The alloxan-mediated elevation of type B enzyme activity to this level at the physiological pH may be of considerable significance. Type B enzyme has a high affinity for metabolic intermediates and nucleotides, while type A has an extremely low affinity for these substrates. Cytoplasmic acid phosphatase (type A) is a significant enzyme population and its activity is not related to the lysosome density in the cells. Type A enzyme in the cytoplasm is thus shown to be an entity distinctly different from type B enzyme in the lysosomes. These findings suggest that the physiological functions of type A acid phosphatase, such as metabolic regulatory processes, merit further studies because of the phosphoryl transfer activity and cytoplasmic localization of the enzyme.

Partial characterization of a phosphoryl group transferring enzyme in the membrane of catecholamine storage vesicles

Phosphoryl group transfer from ATP to ADP occurred in the isolated membrane of catecholamine storage vesicles. The reaction was accelerated by extraction of the membranes with 50% (v/v) acetone and by treatment with 1% (v/v) Triton X-100. The phosphoryl group transfer reaction was activated by Mg2+ and by Mn2+. The activation profile differed from that obtained for the ATPase activity. The Michaelis-Menten kinetics of the phosphoryl transfer reaction were not entirely linear. From the linear parts of the double reciprocal plots KmATP approximately equal to 1 mM and KmADP approximately equal to 0.4 mM was obtained. All lines of the double reciprocal plots intersected indicating a sequential reaction mechanism. The reaction exhibited a narrow specificity for nucleoside diphospate and a broader one for nucleoside triphosphate indicating that ADP was the true substrate. The transfer reaction was slightly inhibited by AMP, orthophosphate and P1, P5-di(adenosine-5')pentaphosphate. The thiol reagents, N-ethylmaleimide and para-chloromercuribenzoate (PCMB), affected the ATPase activity and the phosphoryl transfer activity differently: with the blockade of 2.4 essential thiol equivalents by N-ethylmaleimide the ATPase was inhibited 50% and net uptake of catecholamine ceased, while the phosphoryl transfer remained unimpaired. PCMB affected both, the ATPase activity and phosphoryl transfer reaction. Treatment of the membranes with dithioerythritol prevented the PCMB-induced inhibition of the phosphoryl transfer, but was ineffective in protecting the ATPase activity, indicating that different thiol groups must be involved in the both enzymatic activities.