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Query: EC:3.4.16.2 (PCP)
 3,761 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The enthalpy changes that occur in the self-assembly of tubulin into microtubules were examined by adiabatic differential heat capacity microcalorimetry and by isothermal batch microcalorimetry. Tubulin solutions at concentrations between 7 and 17 mg/mL were heated from 0 to 40 degrees C at heating rates of 1 or 2 deg/min in pH 6.8 or 7.0 assembly buffers containing 20 mM MES, 100 mM glutamic acid, 5 mM MgCl2, 3.4 M glycerol, and either 0.5 mM GMP-PCP or 1 mM GTP. The assembly reaction in the presence of GTP was characterized by a complex heat-uptake pattern consisting of a broad endotherm with a sharper exotherm superimposed on it, similar to assembly in a GTP phosphate buffer [Hinz, H.-J., Gorbunoff, M.J., Price, B., & Timasheff, S.N. (1979) Biochemistry 18,3084]. Replacement of GTP by the nonhydrolyzable analogue resulted in a pattern typical for an endothermic reaction only. These results have permitted the assignment of the endothermic process to microtubule assembly and of the exothermic process to the resultant GTP hydrolysis. In these studies equilibration was found to be slow, several hours of cooling being required for the system to return to its original state. Turbidity scans also revealed hysteresis between consecutive scans and a displacement of the depolymerization transition midpoint to a lower temperature than that of assembly. The disassembly of microtubules was examined in batch calorimetry experiments in pH 7.0 phosphate, 1 mM GTP, 16 mM MgCl2, and 3.4 M glycerol, in which tubulin assembled into microtubules was diluted to below the critical concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Enthalpy changes in microtubule assembly from pure tubulin. 381 84

Elongation factor Ts (EF-Ts) catalyzes the reaction EF-Tu X GDP + nucleotide diphosphate (NDP) reversible EF-Tu X NDP + GDP where NDP is GDP, IDP, GTP, or GMP X PCP. The EF-Ts-catalyzed exchange rates were measured at a series of concentrations of EF-Tu X [3H] GDP and free nucleotide. Plotting the rate data according to the Hanes method produced a series of lines intersecting on the ordinate, a characteristic of substituted enzyme mechanisms. GDP is a competitive inhibitor of IDP exchange, a result predicted for the substituted enzyme mechanism but inconsistent with ternary complex mechanisms that involve an intermediate complex containing EF-Ts and both substrates. The exchange of both GTP and the GTP analog GMP X PCP also follow the substituted enzyme mechanism. The maximal rates of exchange of GDP and GTP are the same, which indicates that the rates of dissociation of EF-Ts from EF-Tu X GDP and EF-Tu X GTP are the same. The steady-state maximal exchange rate is slower by a factor of 20 than the previously reported rate of dissociation of GDP from EF-Ts X EF-Tu. This is interpreted to mean that the rate-determining step in the exchange reaction is the dissociation of EF-Ts from EF-Tu X GDP.
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PMID:A study of the kinetic mechanism of elongation factor Ts. 404 68

The requirement of initiation factors F(1) (highly purified) and F(2) (electrophoretically homogeneous) for ribosomal binding of N-formylmethionyl transfer RNA (fMet approximately tRNA) at low Mg(2+) concentration (3.5 mM), with the trinucleoside diphosphate ApUpG as messenger, was studied under various experimental conditions with 30S + 50S ribosomes and with 30S subunits alone. The results were qualitatively the same in both cases but the amount of binding was two to three times higher when both 30S and 50S subunits were present. Although there was a virtually absolute requirement for F(2) in all cases, considerable binding occurred at 0 degrees in the absence of added F(1). F(1) addition stimulated binding up to twofold under these conditions. However, at 25 degrees , the temperature at which the reaction is usually carried out, there was very little binding with F(2) alone and addition of F(1) stimulated the reaction five- to sixfold. Contrary to current belief, the GTP analog 5'-guanylyldiphosphonate (GMP-PCP) cannot replace GTP in the binding reaction. In particular, there was but little stimulation of binding (about 1.5-fold) by addition of F(1) to F(2)-containing samples when GMP-PCP was used. In marked contrast, binding was stimulated up to sevenfold by addition of F(1) when GTP was substituted for the analog. Under these conditions, there was an ApUpG and F(1)-dependent hydrolysis of GTP. This is observable with 30S subunits alone and can hardly be related to the occurrence of translocation. The results may be interpreted to mean that a complex relatively stable at 0 degrees , but less stable at 25 degrees , is formed upon addition of F(2) alone. Conversion of the less stable to the more stable form of complex is made possible by addition of F(1). This is accompanied or mediated by cleavage of GTP.
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PMID:Polypeptide chain initiation in E. coli: studies on the function of initiation factor F1. 489 78

The translocation of the mRNA in relation to the ribosome during peptide synthesis represents an example for a mechanochemical reaction in which the chemical bond energy of GTP is transformed into coordinated motion. We demonstrate here that translocation can be explained simply by binding equilibria between the tRNA, the mRNA, and their binding sites on the ribosome. The presence of two cognate tRNAs shifts the association constant for the 70 S ribosome . AUGU3 complex from 6.8 x 10(5) to 2.2 x 10(8) M-1. The elongation factor G and GTP or guanosine-5'-(beta,gamma-methylene)triphosphate GMP-PCP) displace the methionine tRNAs which can be formylated (tRNAfMet) from the quaternary complex 70 S . AUGU3 . tRNAfMet . tRNAPhe. Only the ternary complex Phe-tRNAPhe . elongation factor Tu . GMP-PCP shows an absolute preference for the aminoacyl-tRNA binding site (A site) (K a = 6.6 x 10(6) M-1). AcPhe-tRNAPhe, (N alpha-acetylphenylalanyl-tRNA) an analogue of a peptidyl-tRNA exhibits a 20-fold higher affinity to the peptidyl-tRNA binding site (P site) (K a = 3.5 x 10(6) M-1) as against the A site (K a = 1.8 x 10(6) M-1) at 8 mM Mg2+. Compared to aminoacyl-tRNA and tRNA, peptidyl-tRNA shows a 3- to 15-fold higher affinity toward complementary oligonucleotides both in the binary complex and in the presence of 70 S ribosomes (UUCA . AcPhe-tRNAPhe: K a = 1.9 x 10(5) M-1), UUCA . tRNAPhe:K a = 3.2 x 10(4) M-1). This indicates a stabilization of the peptidyl-tRNA . mRNA complex during translocation. Our data support a concept of mRNA translocation in which the removal of the deacylated tRNA from the P site requires GTP energy and a peptidyl-tRNA . mRNA complex diffuses from its low affinity site (A) to its high affinity binding site (P).
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PMID:Mechanism of translocation. Binding equilibria between the ribosome, mRNA analogues, and cognate tRNAs. 703 57

The double-stranded RNA bacteriophage phi 6 contains a virion-associated RNA-dependent RNA polymerase complex. Removal of the virus envelope and the nucleocapsid surface protein, P8, reveals a nucleocapsid core particle (proteins P1, P2, P4, P7) which is the viral polymerase complex, capable of synthesizing RNA strands of positive polarity. The in vitro plus strand synthesis (transcription) reaction of the particle obtained from the mature virion was optimized and its activation and inactivation were investigated. Purine nucleoside triphosphates (NTPs), binding to a low-affinity binding site in the polymerase complex, activated plus strand synthesis. GTP was the preferred NTP, but dGTP, ddGTP, and the noncleavable analog GMP-PCP could also switch on transcription. This NTP-binding site is probably different from that of the unspecific viral NTPase found in protein P4 and also from that of the rNTP-specific RNA polymerase active site. Binding of purine NTPs was sufficient for the switch-on; hydrolysis of the NTP was not required. Besides nucleotides, divalent cations had an effect on phi 6 in vitro plus strand synthesis. Magnesium ions are required for the activity but calcium ions inhibit the reaction. Manganese ions are shown to dissipate the effect of magnesium and calcium ions, leading to uncontrolled, exceptionally high level plus strand synthesis.
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PMID:In vitro transcription of the double-stranded RNA bacteriophage phi 6 is influenced by purine NTPs and calcium. 788 44

Six per cent of rat pheochromocytoma (PC12) cells extended neurites (processes greater than one cell diameter in length) in the presence of 300 microM extracellular GTP or 300 microM guanosine for 48 hr, compared to only 2.5% of cells in control cultures. In the presence of 40 ng/ml of 2.5S NGF, about 20-35% of PC12 cells had neurites after 48 hr, and the addition of 300 microM guanosine or GTP together with NGF synergistically increased the proportion of cells with neurites to 40-65%. GTP and guanosine also increased the average number of branches per neurite, from 0.6 in NGF-treated cultures to 1.2 (guanosine) or 1.5 (GTP). Neurites formed after exposure to NGF alone had axonal characteristics as determined by immunocytochemistry with antibody, SMI-31, against axonal-specific polyphosphorylated neurofilament epitopes. Neurites generated with the addition of both guanosine or GTP had the same characteristics. GTP probably did not exert its effects via the P2X or P2Y purinoceptors because the adenine nucleotides ATP, ATP gamma S, ADP beta S, and ADP, which are all agonists of these receptors, inhibited rather than enhanced, NGF-induced neurite outgrowth. UTP also enhanced the proportion of cells with neurites, although not to the same degree as did GTP. This may indicate activity through a P2U-like nucleotide receptor. However, the response profile obtained, GTP > UTP >> ATP, does not fit the profile of any known P2Y, P2X or P2U receptor. The poorly hydrolyzable GTP analogues, GTP gamma S and GDP beta s were also unable to enhance the proportion of cells with neurites. This implied that GTP may produce its effects through a GTP-specific ectoenzyme or kinase. This idea was supported by results showing that another poorly hydrolyzable analogue, GMP-PCP, competitively inhibited the effects of GTP on neurite outgrowth. GTP did not exert its effects after hydrolysis to guanosine since the metabolic intermediates GDP and GMP were also ineffective in enhancing the proportion of cells with neurites. Moreover, the effects of GTP and guanosine were mutually additive, implying that these two purines utilized different signal transduction mechanisms. The effects of guanosine were not affected by the nucleoside uptake inhibitors nitrobenzylthioinosine (NBTI) and dipyridamole, indicating that a transport mechanism was not involved. Guanosine also did not activate the purinergic P1 receptors, because the A2 receptor antagonists, 1,3-dipropyl-7-methylxanthine (DPMX) or CGS15943, and the A1 receptor antagonist, 1,3-dipropyl-8-(2-amino-4-chloro)xanthine (PACPX) did not inhibit its reaction. Therefore guanosine enhanced neurite outgrowth by a signal transduction mechanism that does not include the activation of the P1 purinoceptors. The enhancement of the neuritogenic effects of NGF by GTP and guanosine may have physiological implications in sprouting and functional recovery after neuronal injury in the CNS, due to the high levels of nucleosides and nucleotides released from dead or injured cells.
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PMID:GTP and guanosine synergistically enhance NGF-induced neurite outgrowth from PC12 cells. 877 5

The translation elongation factor (EF) Tu has chaperone-like capacity to promote renaturation of denatured rhodanese. This renaturation activity is greatly increased under conditions in which the factor can oscillate between the open and closed conformations that are induced by GDP and GTP, respectively. Oscillation occurs during GTP hydrolysis and subsequent replacement of GDP by EF-Ts which is then displaced by GTP. Renaturation of rhodanese and GTP hydrolysis by EF-Tu are greatly enhanced by the guanine nucleotide exchange factor EF-Ts. However, renaturation is reduced under conditions that stabilize EF-Tu in either the open or closed conformation. Both GDP and the nonhydrolyzable analog of GTP, GMP-PCP, inhibit renaturation. Kirromycin and pulvomycin, antibiotics that specifically bind to EF-Tu and inhibit its activity in peptide elongation, also strongly inhibit EF-Tu-mediated renaturation of denatured rhodanese to levels near those observed for spontaneous, unassisted refolding. Kirromycin locks EF-Tu in the open conformation in the presence of either GTP or GDP, whereas pulvomycin locks the factor in the closed conformation. The results lead to the conclusion that flexing of EF-Tu, especially as occurs between its open and closed conformations, is a major factor in its chaperone-like refolding activity.
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PMID:Renaturation of rhodanese by translational elongation factor (EF) Tu. Protein refolding by EF-Tu flexing. 940 22

GTP-binding (G) proteins regulate the flow of information in cellular signaling pathways by alternating between a GTP-bound "active" state and a GDP-bound "inactive" state. Cdc42, a member of the Rho family of Ras-related small G-proteins, plays key roles in the regulation of cell shape, motility, and growth. Here we describe the high resolution x-ray crystal structure for Cdc42 bound to the GTP analog guanylyl beta,gamma-methylene-diphosphonate (GMP-PCP) (i.e. the presumed signaling-active state) and show that it is virtually identical to the structures for the signaling-inactive, GDP-bound form of the protein, contrary to what has been reported for Ras and other G-proteins. Especially surprising was that the GMP-PCP- and GDP-bound forms of Cdc42 did not show detectable differences in their Switch I and Switch II loops. Fluorescence studies using a Cdc42 mutant in which a tryptophan residue was introduced at position 32 of Switch I also showed that there was little difference in the Switch I conformation between the GDP- and GMP-PCP-bound states (i.e. <10%), which again differed from Ras where much larger changes in Trp-32 fluorescence were observed when comparing these two nucleotide-bound states (>30%). However, the binding of an effector protein induced significant changes in the Trp-32 emission specifically from GMP-PCP-bound Cdc42, as well as in the phosphate resonances for GTP bound to this G-protein as indicated in NMR studies. An examination of the available structures for Cdc42 complexed to different effector proteins, versus the x-ray crystal structure for GMP-PCP-bound Cdc42, provides a possible explanation for how effectors can distinguish between the GTP- and GDP-bound forms of this G-protein and ensure that the necessary conformational changes for signal propagation occur.
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PMID:Effector proteins exert an important influence on the signaling-active state of the small GTPase Cdc42. 1834 80

Squid giant axons recover from acid loads by activating a Na(+)-driven Cl-HCO(3) exchanger. We internally dialyzed axons to an intracellular pH (pH( i )) of 6.7, halted dialysis and monitored the pH(i) recovery (increase) in the presence of ATP or other nucleotides, using cyanide to block oxidative phosphorylation. We computed the equivalent acid-extrusion rate (J(H)) from the rate of pH(i) increase and intracellular buffering power. In experimental series 1, we used dialysis to vary [ATP](i), finding that Michaelis-Menten kinetics describes J (H) vs. [ATP](i), with an apparent V(max) of 15.6 pmole cm(-2 )s(-1) and K (m) of 124 microM. In series 2, we examined ATP gamma S, AMP-PNP, AMP-PCP, AMP-CPP, GMP-PNP, ADP, ADP beta S and GDP beta S to determine if any, by themselves, could support transport. Only ATP gamma S (8 mM) supported acid extrusion; ATP gamma S also supported the HCO (3)(-) -dependent (36)Cl efflux expected of a Na(+)-driven Cl-HCO(3) exchanger. Finally, in series 3, we asked whether any nucleotide could alter J (H) in the presence of a background [ATP](i) of approximately 230 microM (control J (H) = 11.7 pmol cm(-2 )s(-1)). We found J (H) was decreased modestly by 8 mM AMP-PNP (J (H) = 8.0 pmol cm(-2 )s(-1)) but increased modestly by 1 mM ADP beta S (J (H) = 16.0 pmol cm(-2 )s(-1)). We suggest that ATP gamma S leads to stable phosphorylation of the transporter or an essential activator.
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PMID:ATP dependence of Na+-driven Cl-HCO3 exchange in squid axons. 1847 73

The archaeal cobY gene encodes the nonorthologous replacement of the bacterial NTP:AdoCbi kinase (EC 2.7.7.62)/GTP:AdoCbi-P guanylyltransferase (EC 3.1.3.73) and is required for de novo synthesis of AdoCbl (coenzyme B(12)). Here we show that ORF MJ1117 of the hyperthermophilic, methanogenic archaeon Methanocaldococcus jannaschii encodes a CobY protein (Mj CobY) that transfers the GMP moiety of GTP to AdoCbi-P to form AdoCbi-GDP. Results from isothermal titration calorimetry (ITC) experiments show that MjCobY binds GTP (K(d) = 5 muM), but it does not bind the GTP analogues GMP-PNP (guanosine 5'-(beta,gamma)-imidotriphosphate) or GMP-PCP (guanylyl 5'-(beta,gamma)-methylenediphosphonate) nor GDP. Results from ITC experiments indicate that MjCobY binds one GTP per dimer. Results from in vivo studies support the conclusion that the 5'-deoxyadenosyl upper ligand of AdoCbi-P is required for MjCobY function. Consistent with these findings, MjCobY displayed high affinity for AdoCbi-P (K(d) = 0.76 muM) but did not bind nonadenosylated Cbi-P. Kinetic parameters for theMj CobY reaction were determined. Results from circular dichroism studies indicate that, in isolation, MjCobY denatures at 80 degrees C with a concomitant loss of activity. We propose that ORF MJ1117 of M. jannaschii be annotated as cobY to reflect its involvement in AdoCbl biosynthesis.
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PMID:Biochemical characterization of the GTP:adenosylcobinamide-phosphate guanylyltransferase (CobY) enzyme of the hyperthermophilic archaeon Methanocaldococcus jannaschii. 1948 48


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