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• W2026692906 abstract "Within the family of large GTP-binding proteins, human guanylate binding protein 1 (hGBP1) belongs to a subgroup of interferon-inducible proteins. GTP hydrolysis activity of these proteins is much higher compared with members of other GTPase families and underlies mechanisms that are not understood. The large GTP-binding proteins form self-assemblies that lead to stimulation of the catalytic activity. The unique result of GTP hydrolysis catalyzed by hGBP1 is GDP and GMP. We investigated this reaction mechanism by transient kinetic methods using radioactively labeled GTP as well as fluorescent probes. Substrate binding and formation of the hGBP1 homodimer are fast as no lag phase is observed in the time courses of GTP hydrolysis. Instead, multiple turnover experiments show a rapid burst of Pi formation prior to the steady state phase, indicating a rate-limiting step after GTP cleavage. Both molecules are catalytically active and cleave off a phosphate ion in the first step. Then bifurcation into catalytic inactivation, probably by irreversible dissociation of the dimer, and into GDP hydrolysis is observed. The second cleavage step is even faster than the first step, implying a rapid rearrangement of the nucleotide within the catalytic center of hGBP1. We could also show that the release of the products, including the phosphate ions, is fast and not limiting the steady state activity. We suggest that slow dissociation of the GMP-bound homodimer gives rise to the burst behavior and controls the steady state. The assembled forms of the GDP- and GMP-bound states of hGBP1 are accessible only through GTP binding and hydrolysis and achieve a lifetime of a few seconds. Within the family of large GTP-binding proteins, human guanylate binding protein 1 (hGBP1) belongs to a subgroup of interferon-inducible proteins. GTP hydrolysis activity of these proteins is much higher compared with members of other GTPase families and underlies mechanisms that are not understood. The large GTP-binding proteins form self-assemblies that lead to stimulation of the catalytic activity. The unique result of GTP hydrolysis catalyzed by hGBP1 is GDP and GMP. We investigated this reaction mechanism by transient kinetic methods using radioactively labeled GTP as well as fluorescent probes. Substrate binding and formation of the hGBP1 homodimer are fast as no lag phase is observed in the time courses of GTP hydrolysis. Instead, multiple turnover experiments show a rapid burst of Pi formation prior to the steady state phase, indicating a rate-limiting step after GTP cleavage. Both molecules are catalytically active and cleave off a phosphate ion in the first step. Then bifurcation into catalytic inactivation, probably by irreversible dissociation of the dimer, and into GDP hydrolysis is observed. The second cleavage step is even faster than the first step, implying a rapid rearrangement of the nucleotide within the catalytic center of hGBP1. We could also show that the release of the products, including the phosphate ions, is fast and not limiting the steady state activity. We suggest that slow dissociation of the GMP-bound homodimer gives rise to the burst behavior and controls the steady state. The assembled forms of the GDP- and GMP-bound states of hGBP1 are accessible only through GTP binding and hydrolysis and achieve a lifetime of a few seconds. GTP-binding proteins control a multitude of cellular functions ranging from protein translation, signal transduction, and regulation of the cytoskeleton to endocytosis and immunological response. Timed and specific action of these proteins is achieved by adopting different conformations when in complex with either GDP or GTP (1Bourne H.R. Sanders D.A. McCormick F. Nature. 1990; 348: 125-132Crossref PubMed Scopus (1832) Google Scholar). Only in one of the two states, in most cases the GTP-bound form, is the GTPase capable of fulfilling its function by specific interaction with other proteins. They can flip between these two states by GTP hydrolysis and by GDP dissociation followed by rebinding of abundant GTP. For many GTP-binding proteins, regulatory proteins have been identified and characterized, which can promote hydrolysis and dissociation, respectively, and thereby control the action of the GTPase. Therefore, exploring the mechanism of GTP hydrolysis is important for understanding the biological function on the molecular level. Large GTPases make up a protein superfamily with biological functions that can be grouped in two classes (2Praefcke G.J. McMahon H.T. Nat. Rev. Mol. Cell Biol. 2004; 5: 133-147Crossref PubMed Scopus (1104) Google Scholar). Dynamin and dynamin-like proteins play a key role in membrane scission and fusion, whereas interferon-induced proteins like Mx and hGBP1 (human guanylate binding protein 1) are involved in antiviral defense (3Haller O. Kochs G. Traffic. 2002; 3: 710-717Crossref PubMed Scopus (371) Google Scholar, 4Anderson S.L. Carton J.M. Lou J. Xing L. Rubin B.Y. Virology. 1999; 256: 8-14Crossref PubMed Scopus (217) Google Scholar). Being a member of this superfamily is not decided only by the molecular size of the proteins, ranging between 65 and 100 kDa, but also by common biochemical properties and structural features. In contrast to small GTPases like Ras, Rab, Rho, etc. and α-subunits of heterotrimeric G proteins, they bind the guanine nucleotides GDP and GTP not very tightly thus showing Kd values in the micromolar range (5Uthaiah R.C. Praefcke G.J. Howard J.C. Herrmann C. J. Biol. Chem. 2003; 278: 29336-29343Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). This low affinity is caused by fast dissociation rates as compared with small GTPases. Also GTP hydrolysis is faster and, more strikingly, is enhanced by self-association of the large GTPases. For dynamin and Mx, the formation of helical assemblies after binding of GTP is described (6Hinshaw J.E. Schmid S.L. Nature. 1995; 374: 190-192Crossref PubMed Scopus (656) Google Scholar, 7Zhang P. Hinshaw J.E. Nat. Cell Biol. 2001; 3: 922-926Crossref PubMed Scopus (208) Google Scholar, 8Accola M.A. Huang B. Al M.A. McNiven M.A. J. Biol. Chem. 2002; 277: 21829-21835Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 9Stowell M.H. Marks B. Wigge P. McMahon H.T. Nat. Cell Biol. 1999; 1: 27-32Crossref PubMed Scopus (312) Google Scholar), whereas hGBP1 forms homodimers when bound to GTP (10Ghosh A. Praefcke G.J. Renault L. Wittinghofer A. Herrmann C. Nature. 2006; 440: 101-104Crossref PubMed Scopus (153) Google Scholar, 11Prakash B. Praefcke G.J. Renault L. Wittinghofer A. Herrmann C. Nature. 2000; 403: 567-571Crossref PubMed Scopus (249) Google Scholar). Regarding the structure the most conspicuous difference to other GTPases is a large helical domain extending to the C terminus of the protein. Biochemical work on dynamin and Mx has shown that a C-terminal subdomain takes part in the activation of GTP hydrolysis and is therefore named GTPase effector domain. How exactly the extra domains work in respect to enhanced GTP hydrolysis and nucleotide dissociation and how far they might replace regulatory proteins that are observed for other GTPases have not been resolved (12Sever S. Muhlberg A.B. Schmid S.L. Nature. 1999; 398: 481-486Crossref PubMed Scopus (313) Google Scholar, 13Marks B. Stowell M.H. Vallis Y. Mills I.G. Gibson A. Hopkins C.R. McMahon H.T. Nature. 2001; 410: 231-235Crossref PubMed Scopus (373) Google Scholar, 14Flohr F. Schneider-Schaulies S. Haller O. Kochs G. FEBS Lett. 1999; 463: 24-28Crossref PubMed Scopus (117) Google Scholar, 15Janzen C. Kochs G. Haller O. J. Virol. 2000; 74: 8202-8206Crossref PubMed Scopus (68) Google Scholar, 16Sever S. Damke H. Schmid S.L. J. Cell Biol. 2000; 150: 1137-1148Crossref PubMed Scopus (194) Google Scholar). Mutations of residues critical for catalysis of GTP hydrolysis were identified in many GTPases resulting in malfunction and diseases. The most prominently known are point mutations in small GTP-binding proteins like Ras and Rho leading to cancer (17Barbacid M. Annu. Rev. Biochem. 1987; 56: 779-827Crossref PubMed Scopus (3768) Google Scholar), but also mutations in large GTPases like dynamin, atlastin, and OPA1 leading to impairment of GTP hydrolysis may result in disease (18Bitoun M. Maugenre S. Jeannet P.Y. Lacene E. Ferrer X. Laforet P. Martin J.J. Laporte J. Lochmuller H. Beggs A.H. Fardeau M. Eymard B. Romero N.B. Guicheney P. Nat. Genet. 2005; 37: 1207-1209Crossref PubMed Scopus (333) Google Scholar, 19Delettre C. Lenaers G. Griffoin J.M. Gigarel N. Lorenzo C. Belenguer P. Pelloquin L. Grosgeorge J. Turc-Carel C. Perret E. Starie-Dequeker C. Lasquellec L. Arnaud B. Ducommun B. Kaplan J. Hamel C.P. Nat. Genet. 2000; 26: 207-210Crossref PubMed Scopus (1144) Google Scholar, 20Alexander C. Votruba M. Pesch U.E. Thiselton D.L. Mayer S. Moore A. Rodriguez M. Kellner U. Leo-Kottler B. Auburger G. Bhattacharya S.S. Wissinger B. Nat. Genet. 2000; 26: 211-215Crossref PubMed Scopus (1052) Google Scholar, 21Sauter S.M. Engel W. Neumann L.M. Kunze J. Neesen J. Hum. Mutat. 2004; 23: 98Crossref PubMed Scopus (64) Google Scholar). Although all large GTPases are extraordinary in respect to the role and the mechanism of GTP hydrolysis being linked to self-assembly, hGBP1 is even more unique as the result of catalyzed GTP hydrolysis is phosphate and GMP to the major part and only a small fraction of GDP (22Schwemmle M. Staeheli P. J. Biol. Chem. 1994; 269: 11299-11305Abstract Full Text PDF PubMed Google Scholar). Earlier we have characterized affinity and binding kinetics of all three guanine nucleotides and have shown homodimer formation of hGBP1 only upon binding to GTP analogs (11Prakash B. Praefcke G.J. Renault L. Wittinghofer A. Herrmann C. Nature. 2000; 403: 567-571Crossref PubMed Scopus (249) Google Scholar, 23Praefcke G.J. Geyer M. Schwemmle M. Kalbitzer H.R. Herrmann C. J. Mol. Biol. 1999; 292: 321-332Crossref PubMed Scopus (99) Google Scholar, 24Praefcke G.J. Kloep S. Benscheid U. Lilie H. Prakash B. Herrmann C. J. Mol. Biol. 2004; 344: 257-269Crossref PubMed Scopus (90) Google Scholar). In contrast, the GDP as well as the GMP form of hGBP1 are monomers, and GDP hydrolysis is not detected. This study addresses the mechanism of GTP hydrolysis by a transient kinetic approach. Using rapid flow techniques allows us to resolve successive steps in the course of GTP hydrolysis. Protein Preparation—hGBP1 with an N-terminal His6 tag was synthesized from a pQE9 vector (Qiagen, Hilden, Germany) in Escherichia coli strain BL21(DE3) and purified as described previously (23Praefcke G.J. Geyer M. Schwemmle M. Kalbitzer H.R. Herrmann C. J. Mol. Biol. 1999; 292: 321-332Crossref PubMed Scopus (99) Google Scholar). The concentration of hGBP1 was calculated from the absorbance at 276 nm in 20 mm potassium phosphate, pH 6.5, using a molar absorption coefficient of 45,400 m–1 cm–1 (23Praefcke G.J. Geyer M. Schwemmle M. Kalbitzer H.R. Herrmann C. J. Mol. Biol. 1999; 292: 321-332Crossref PubMed Scopus (99) Google Scholar). Nucleotides—GTP was purchased from Sigma and was purified by ion exchange chromatography as described by Lenzen et al. (25Lenzen C. Cool R.H. Wittinghofer A. Methods Enzymol. 1995; 255: 95-109Crossref PubMed Scopus (121) Google Scholar). Mant 2The abbreviations used are: mant-, N-methyl-anthraniloyl-; MDCC, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide; PBP, phosphate-binding protein. -nucleotides were synthesized according to Ref. 25Lenzen C. Cool R.H. Wittinghofer A. Methods Enzymol. 1995; 255: 95-109Crossref PubMed Scopus (121) Google Scholar. The purity of the nucleotides was greater than 98% as verified by high pressure liquid chromatography (25Lenzen C. Cool R.H. Wittinghofer A. Methods Enzymol. 1995; 255: 95-109Crossref PubMed Scopus (121) Google Scholar). Nucleotide concentrations were determined from the absorbance at 253 nm using the absorption coefficients 13,700 and 22,600 m–1 cm–1 for nonlabeled and mant-nucleotides, respectively (26Hiratsuka T. Biochim. Biophys. Acta. 1983; 742: 496-508Crossref PubMed Scopus (395) Google Scholar). Rapid Quench Flow Kinetics—Transient enzyme kinetics were investigated with the help of the rapid quench flow technique using an RQF-3 instrument (KinTek Corp., Austin, TX). To obtain time courses of GTP hydrolysis, solutions of (nucleotide-free) hGBP1 and GTP containing 10 nCi/μl [α-32P]- or [γ-32P]GTP (Amersham Biosciences) were rapidly mixed (15 μl each) in a first mixing device. The reactions were stopped after different time intervals by using 1 m perchloric acid as the quenching solution in the second mixing step. The pH value of quenched reaction mixtures was raised to about 5 by addition of potassium acetate. The solutions were centrifuged (3 min at 15,000 × g) to remove precipitated potassium perchlorate and protein, and the supernatant was subjected to further analysis for substrate and product concentrations. Nucleotides and phosphate were separated by TLC on polyethyleneimine-cellulose (Merck) with 0.65 m potassium phosphate, pH 3.5, as developing solution. The relative amounts were quantified using a FLA-3000 PhosphorImager (Fuji, Kanasawa, Japan). In cold substrate chase experiments, the acid was replaced by highly concentrated GTP as quenching solution. After a second incubation time, the reaction was finally stopped by manual addition of acid, and the solution was processed and analyzed as described above. Stopped-flow Kinetics of Nucleotide Binding and Dissociation—Stopped-flow measurements were performed in an SFM400 apparatus with MOS-200 optics (Bio-Logic, Grenoble, France). In competitive binding experiments, hGBP1 was mixed with solutions of constant mant-GMP and increasing GTP concentrations. Nucleotides were in at least 5- or 10-fold excess over protein, providing pseudo-first order conditions in case the dissociation rates of both nucleotides can be neglected (27Nowak E. Goody R.S. Biochemistry. 1988; 27: 8613-8617Crossref PubMed Scopus (16) Google Scholar, 28John J. Sohmen R. Feuerstein J. Linke R. Wittinghofer A. Goody R.S. Biochemistry. 1990; 29: 6058-6065Crossref PubMed Scopus (344) Google Scholar). Dissociation rate constants of GDP and GMP were measured by displacing the (nonlabeled) nucleotides from the preformed complexes by a 20- or 40-fold excess of mant-GDP. Fluorescence was excited at 295 nm and detected after passing through a 400 nm cut-off filter. In both types of experiments fluorescence resonance energy transfer between tryptophan residues in hGBP1 and the mant-fluorophor on the nucleotide was used because direct excitation of the mant-group leads to high fluorescence background. Kinetics of Pi Release—Pi release was studied according to the method of Brune et al. (29Brune M. Hunter J.L. Corrie J.E. Webb M.R. Biochemistry. 1994; 33: 8262-8271Crossref PubMed Scopus (432) Google Scholar), which is based on the fluorescence change of a coumarin-labeled phosphate-binding protein (PBP) from E. coli. The A197S mutant of PBP was prepared and labeled with N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC) as described by Brune et al. (30Brune M. Hunter J.L. Howell S.A. Martin S.R. Hazlett T.L. Corrie J.E. Webb M.R. Biochemistry. 1998; 37: 10370-10380Crossref PubMed Scopus (173) Google Scholar). Following this procedure we obtained protein, which was labeled to more than 93%, as determined from the absorbance at 280 and 430 nm. The Pi responsiveness of our preparation was analyzed by fluorescence titration using an LS-55 spectrofluorimeter (PerkinElmer Life Sciences). MDCC-PBP at 125 μm was titrated with a potassium phosphate solution containing the same MDCC-PBP concentration. Excitation and emission wavelength were 430 nm and 465 nm, respectively. Kinetics of Pi release were measured under single turnover conditions using the SFM400 stopped-flow instrument. The concentrations of hGBP1 and GTP were the same as in the quench flow experiments. MDCC-PBP was added to both nucleotide and hGBP1 solutions. The fluorescence was excited at 430 nm and monitored after passing through a 455 nm cut-off filter. The Pi concentration was obtained from the fluorescence signal after normalization to the Pi concentration calculated from a parallel rapid quench flow experiment. To minimize phosphate contamination, the fluorescence cuvettes and the stopped-flow apparatus were incubated for 30 min with 400 μm 7-methylguanosine and 1 unit/ml purine nucleoside phosphorylase before the measurements (29Brune M. Hunter J.L. Corrie J.E. Webb M.R. Biochemistry. 1994; 33: 8262-8271Crossref PubMed Scopus (432) Google Scholar). All experiments were carried out at least in duplicate in 50 mm Tris-HCl, pH 8.0, 5 mm MgCl2, 2 mm dithioerythritol at 25 °C unless indicated otherwise. Data Analysis—Analysis of single transients was performed by nonlinear regression, using the program Grafit (Erithacus Software). Single-turnover kinetics of GTP hydrolysis lead to three nucleotide time courses that were analyzed simultaneously by a global fit using the program Scientist (MicroMath). For this kind of analysis the differential equations derived from Scheme 2 were used yielding the values for k1, k2, and k3. Correspondingly, and with the same program, the kinetics of the multiple turnover experiments were analyzed according to Scheme 3, which results in the value for k4. Here, the constants as indicated in Scheme 3 were used as fixed values, and for the fast association and dissociation rate constant values of 100 μm–1 s–1 and 100 s–1 were used, respectively.SCHEME 3View Large Image Figure ViewerDownload Hi-res image Download (PPT) Multiturnover Kinetics—GTP hydrolysis was measured with the help of radioactively labeled GTP, and in a first approach we used [γ-32P]GTP. To resolve the early phase of GTP hydrolysis, we used a rapid quench flow apparatus that allows us to stop the reaction after a short time and to analyze for product (or intermediate) formation. First, GTP hydrolysis kinetics were measured by mixing hGBP1 with an excess of substrate, i.e. multiple turnover conditions. Concentrations of GTP were chosen such that association with hGBP1 is fast according to our results from binding kinetics (see below). A concentration of 350 μm GTP ensures that binding is completed to 99% after 5 ms. The dissociation constant of the GTP-bound hGBP1 dimer was estimated to lie below 1 μm (11Prakash B. Praefcke G.J. Renault L. Wittinghofer A. Herrmann C. Nature. 2000; 403: 567-571Crossref PubMed Scopus (249) Google Scholar) so that we can assume almost quantitative dimer formation in our experiments. Fig. 1 shows a typical multiple turnover experiment with the concentration of hGBP1 at 50 μm and [γ-32P]GTP at 350 μm. The time course of cleaved phosphate concentration, [Pi], normalized to the total enzyme concentration, [E]0, is shown, and two kinetic phases can clearly be distinguished. After a fast exponential rise, the onset of a linear steady state phase was observed. Three parameters in Equation 1 are needed to describe this observation, i.e. the size of the burst amplitude, A, the rate constant for the initial exponential phase, k, and the steady state rate constant, s; PiE0=A·1-e-k·t+s·t(Eq. 1) The burst amplitude of phosphate cleavage is 0.63 as a result of the fit and as indicated by the dashed line in Fig. 1. The values obtained for the rate constants are k = 0.74 s–1 and s = 0.25 s–1. This type of kinetics demands a minimum of two reaction steps as shown in Scheme 1. According to Scheme 1, there are three constants representing the rate of the first step, k1, and all of following steps (at least one), kf, and the concentration of catalytically active enzyme, [E]a. They define the three parameters of Equation 1 according to Equations 2, 3, 4 (31Fersht A. Structure and Mechanism in Protein Science. W. H. Freeman & Co., New York1999: 153-157Google Scholar). A=EaE0k1k1+kf2(Eq. 2) k=k1+kf(Eq. 3) s=EaE0k1·kfk1+kf(Eq. 4) Analysis of the phosphate transient in Fig. 1 on the basis of Scheme 1 by using Equations 1, 2, 3, 4 yields the rate constants k1 = 0.50 s–1 and kf = 0.27 s–1. Thus the first step is not much faster than the following leading to a burst amplitude smaller than 1. Surprisingly, the fit results in a value larger than 1 for the active enzyme fraction, [E]a/[E]0 = 1.4, which could be explained by a systematic error on the protein and/or nucleotide concentration.SCHEME 1View Large Image Figure ViewerDownload Hi-res image Download (PPT) At this stage of kinetic analysis only γ-phosphate is detected. Further potential steps in the catalytic mechanism like GDP hydrolysis and product release are dismantled in kf. With this type of experiment and the use of [γ-32P]GTP, formation of GMP was not possible to detect. Nevertheless, it is worthwhile to mention that no formation of pyrophosphate was observed in the course of these experiments. As a positive control in TLC, radioactive pyrophosphate generated from [γ-32P]GTP by incubation with phosphodiesterase was used. Single-turnover Kinetics—In a next step we studied the hydrolysis of GTP under single-turnover conditions where the kinetics of nucleotide release are not relevant. To this end we used [α-32P]GTP, which allows us to follow the transients of all nucleotides. The reaction is started by mixing equal volumes of 150 μm hGBP1 and 70 μm [α-32P]GTP in a rapid quench flow apparatus. Typical results of this kind of experiment are shown in Fig. 2. As already known not only GDP but also GMP is formed by hGBP1-catalyzed hydrolysis of GTP. In the experiments of the previous section only formation of phosphate ions was observed, and no pyrophosphate was seen. Here it is most evident that GDP is generated in a first step followed by cleavage of another phosphate ion leading to the formation of GMP. At the beginning of the time trace of GMP a clear lag phase is observed in Fig. 2, which is the result of the successive phosphate cleavage mechanism. In particular, the experiment at 37 °C in Fig. 2B shows that GDP is formed as an intermediate, which is for the most part hydrolyzed further and yields GMP as the major product. The data in Fig. 2 can be explained by Scheme 2, which includes two successive hydrolysis steps and an inactivation step that we assume to be dimer dissociation. Competition of the second step of hydrolysis and the inactivation accounts for the final ratios of the products GMP and GDP, which are 72:28 at 25 °C and 95:5 at 37 °C. Global fit analysis according to Scheme 2 and as described under “Experimental Procedures” yields the rate constants k1 = 0.43 s–1, k2 = 2.2 s–1, and k3 = 0.87 s–1 at 25 °C and k1 = 3.9 s–1, k2 = 8.03 s–1, and k3 = 0.40 s–1 at 37 °C. Most notably, the first step is slower than the following, which cannot explain the phosphate burst behavior. Substrate Binding Kinetics Using Stopped-flow—Elucidation of the mechanism of GTP hydrolysis presupposes knowledge about the binding kinetics of the substrate. As we have used nucleotide analogs carrying a fluorescence label in our earlier studies, we determined here the binding kinetics of GTP, i.e. the genuine substrate that we also used for the investigation of the hydrolysis above. For this purpose we followed the binding kinetics after mixing in a stopped-flow apparatus 2 equal volumes of 1.0 μm hGBP1 and 5.4 μm fluorescent mant-GMP (and in a second set of experiments 12 μm), including increasing concentrations of GTP. This competitive binding experiment yields the association rate constant of hGBP1 and GTP according to Equation 5 (27Nowak E. Goody R.S. Biochemistry. 1988; 27: 8613-8617Crossref PubMed Scopus (16) Google Scholar, 28John J. Sohmen R. Feuerstein J. Linke R. Wittinghofer A. Goody R.S. Biochemistry. 1990; 29: 6058-6065Crossref PubMed Scopus (344) Google Scholar). Equation 5 is simplified by the assumption that the dissociation rates of the nucleotides can be neglected. For mant-GMP, a value of koff = 1.3 s–1 was determined earlier at 20 °C (24Praefcke G.J. Kloep S. Benscheid U. Lilie H. Prakash B. Herrmann C. J. Mol. Biol. 2004; 344: 257-269Crossref PubMed Scopus (90) Google Scholar). kobs=konGTP·[GTP]+konmGMP·[mGMP](Eq. 5) Fig. 3A shows typical fluorescence time traces that yield the kobs values by fitting an exponential equation. The results are shown in Fig. 3B together with the linear fit. The ordinate yields the kmGMPon value for mant-GMP, which is 4.5 μm–1 s–1 in the first set of experiments and 5.7 μm–1 s–1 in the second. These values are in good agreement with our earlier measurement of this value, namely 5.3 μm–1 s–1 at 20 °C (24Praefcke G.J. Kloep S. Benscheid U. Lilie H. Prakash B. Herrmann C. J. Mol. Biol. 2004; 344: 257-269Crossref PubMed Scopus (90) Google Scholar). A value of kGTPon = 3.1 μm–1 s–1 (second set 3.0 μm–1 s–1) is obtained from these data for the association of hGBP1 and its substrate GTP. This is close to the kon value measured for mant-GTP (2.6 μm–1 s–1 at 20 °C (32Kunzelmann S. Praefcke G.J. Herrmann C. Methods Enzymol. 2005; 404: 512-527Crossref PubMed Scopus (41) Google Scholar)), and it shows that substrate binding is very fast compared with hydrolysis at the concentrations we have used above. Substrate Binding Kinetics Using Cold Substrate Chase—We determined the kinetics of GTP binding to hGBP1 by another independent method that does not necessitate the use of any fluorescent compounds. Cold substrate chase experiments allow quantification of the amount of bound GTP at individual time points by taking advantage of the enzymatic cleavage reaction. In these measurements mixtures of hGBP1 and [γ-32P]GTP were aged for short periods of time in a rapid quench flow device, and the binding reaction was stopped by the addition of a large excess of nonradioactive (cold) GTP. The mixtures were incubated for a few reciprocal turnover numbers (see above), i.e. 20 s, just enough to completely cleave the bound radioactive GTP. Then they were quenched in acid and analyzed for radioactive phosphate. The fraction of radioactive GTP, which has bound to the enzyme before the addition of cold substrate, is converted to product, whereas free labeled substrate is greatly diluted out and no longer participates in the binding and cleavage reactions. Thus, the amount of radioactive phosphate is proportional to the concentration of the enzyme-substrate complex formed during the first incubation period. Fig. 4 shows typical cold GTP chase experiments reflecting the association kinetics of 2.3 nm GTP and 20 and 40 μm hGBP1, respectively. Single exponential curve fitting yields the pseudo-first order rate constants kobs, which are 45 and 88 s–1 at 20 and 40 μm hGBP1, respectively. From these values the association rate constant kon = 2.2 μm–1 s–1 (1.7 μm–1 s–1 with a different protein preparation) is calculated, which is in good agreement with the result from the competitive stopped-flow experiments above. As the dissociation rate constant of the hGBP1-GTP complex is small compared with the kobs values, it cannot be determined accurately from these measurements. However, 100% Pi production is not reached (Fig. 4), which indicates that the GTP dissociation rate constant is of similar magnitude as the hydrolysis rate constant. Rate of Phosphate Release—In some nucleotide cleavage reactions the rate of phosphate release was found to be rate-limiting and responsible for the phosphate burst kinetics. For example, phosphate release in myosin ATPase turned out to be a crucial step triggering large conformational changes of the enzyme (33Geeves M.A. Holmes K.C. Adv. Protein Chem. 2005; 71: 161-193Crossref PubMed Scopus (298) Google Scholar, 34Trentham D.R. Eccleston J.F. Bagshaw C.R. Q. Rev. Biophys. 1976; 9: 217-281Crossref PubMed Scopus (221) Google Scholar). To address this issue we used a PBP that allows us to monitor in real time the increases of phosphate concentration because of dissociation from hGBP1 complexes. This assay is based on PBP covalently labeled with the fluorescent coumarin derivative MDCC, which binds phosphate ions rapidly (kon > 100 μm–1 s–1) and tightly (Kd ∼ 0.1 μm) accompanied by a large increase of its fluorescence intensity (29Brune M. Hunter J.L. Corrie J.E. Webb M.R. Biochemistry. 1994; 33: 8262-8271Crossref PubMed Scopus (432) Google Scholar). Fig. 5A shows a phosphate titration of our MDCC-PBP preparation (at 125 μm) demonstrating a linear response and an active site fraction of 95% under our experimental conditions. Phosphate release kinetics were studied with the help of a stopped-flow apparatus at 75 μm hGBP1 and 35 μm GTP. In addition, MDCC-PBP was present at 125 μm. The recorded fluorescence change is shown in Fig. 5B. From the single exponential fit to the data, a value of 0.41 s–1 was obtained for the rate constant matching the value of k1 measured above. Parallel to this experiment, a single-turnover experiment at the same GTP and hGBP1 concentrations was performed by rapid quench flow as described above. [α-32P]GTP was used and no was MDCC-PBP added, and the time courses of nucleotide concentrations were assayed as described. Quantitative comparison of phosphate and nucleotide concentrations necessitates multiplication of the GMP concentration by a factor of 2 as two phosphate ions stem from this nucleotide. The sum of the concentrations of GDP and GMP (times two) are shown in Fig. 5C together with the experimental fluorescence change from Fig. 5B. Here in Fig. 5C the maximum fluorescence value is normalized to a phosphate concentration of 59.5 μm (=1.7 × 35" @default.
• W2026692906 created "2016-06-24" @default.
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• W2026692906 creator A5057675654 @default.
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• W2026692906 date "2006-09-01" @default.
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• W2026692906 title "Transient Kinetic Investigation of GTP Hydrolysis Catalyzed by Interferon-γ-induced hGBP1 (Human Guanylate Binding Protein 1)" @default.
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