Phosphoryl Group Transfer Research Articles

The extramolecular chemical approach to enzyme analogues.

result and others obtained with the various analogues suggest that, while enzyme-substrate hydrogen bonding may play a role in the A-kinase catalysed phosphoryl group transfer reactions, the a-N-methylated and depsi-containing peptide 1 analogues react at relative rates which require additional explanations. Among the explanations for our results that must be considered are the possibilities that the substitutions of N-methyl groups in the amide bonds cause disruptive peptide-enzyme steric interactions or that intrapeptide steric interactions could prevent a particular peptide analogue from assuming the conformation recognizable by A-kinase. The nature of such steric interactions has been probed in the second aspect of our work (Bramson et al., 1987) which is briefly described herein. As already mentioned, by means of studies utilizing n.m.r. spectroscopy evidence has been obtained that A-kinase probably binds Leu-Arg-Arg-Ala-Ser-Leu-Gly, peptide 1, in one of two extended coil conformations (A or B, described by Rosevear et al., 1984). We have considered, therefore, the possibility that the relative reactivities of a series of N-methylated peptides based on the structure of peptide 1 might be related to the ease with which each peptide can assume the A or B Conformation. The N-methylated peptides which we examined are those illustrated in Table 1 and also the additional ones illustrated in Table 2. Utilizing computer graphics, on the basis of estimates of the magnitude of the steric interactions that would be induced by N-methylation of the amide groups in peptide 1 derivatives locked in either conformation, the ability of each peptide analogue to form a particular conformation was predicted. We found that there was a good correlation between the catalytic activity of A-kinase in the phosphorylation of the N-methylated peptides and the ability of each peptide to form conformation A but not B. To test these findings further we probed the possibility that the reactivity of a relatively unreactive N-methylated peptide might be partially restored by a second change which allowed the peptide to assume conformation A. The finding that such restoration could be achieved together with our other results suggests that when peptide 1 is bound in the enzymic active site it has a conformation which resembles structure A much more closely than structure B (Bramson et al., 1987). In the third aspect of our work we have probed whether when other protein kinases are observed to catalyse the phosphorylation of the same peptide sequences as does A-kinase, the protein kinases utilize the same conformation of the peptide in their reactions (Thomas et al., 19873). We have employed, therefore, the reactions of the conformationally restricted N-methylated peptides which have been utilized as substrates for A-kinase in examining the conformational requirements of the cyclic GMP-dependent protein kinase (G-kinase). The G-kinase is homologous in sequence to A-kinase (Taiko et al., 1984) and has comparable substrate specificities (Lincoln & Corbin, 1977). Our kinetic results with the N-methylated peptides show that, despite the ability of the G-kinase to bind the peptides in a conformation resembling that of conformation A, the Genzyme is more tolerant of backbone methylation than in A-kinase. As a result, while the reactivity of the G-kinase with the prototypic peptide substrate Leu-Arg-Arg-Ala-SerLeu-Gly, peptide 1, is about 10-fold less than the reactivity with the A-kinase, as assessed from the relative magnitudes of k,,,/K,, when the parent peptide was N-methylated at the amide group of the Ser’ residue, the resultant peptide substrate was at least 700-fold more reactive with G-kinase than with A-kinase. Our observations with the peptide which is N-methylated at the phosphorylatable serine residue of peptide 1 show that backbone methylation can represent an approach to making peptide substrates which are selective for a particular kinase (Thomas et al., 19873). This finding encourages us to pursue the possibility that N-methylation of peptides might provide a method for targeting peptidebased inhibitors to selected protein kinases. Partial support of this research by National Institutes of Health Grant GM 32204 is gratefully acknowledged.

Role of enzyme-peptide substrate backbone hydrogen bonding in determining protein kinase substrate specificities.

As part of a search for peptides that have specificity for selected protein kinases, the possibility that adenosine cyclic 3',5'-phosphate dependent protein kinase (A-kinase) recognizes the hydrogen-bonding potential of its peptide substrates was investigated. A-Kinase catalyzes the phosphorylation of five N alpha-methylated and four depsipeptide derivatives of Leu-Arg-Arg-Ala-Ser-Leu-Gly (peptide 1) at rates that differ by at least 7 orders of magnitude. These peptide 1 analogues each lack the ability to donate a hydrogen bond at selected positions in the peptide chain. If a particular amide hydrogen of a peptide amide is involved in hydrogen bonding, which is important for enzyme recognition, the prediction is that peptides which contain an ester or a N-methylated bond at that position in peptide 1 will be comparatively poor substrates. In contrast, if a depsipeptide has a reactivity comparable to that of peptide 1 but the analogous N-methylated peptide has a poor reactivity with A-kinase, the result might indicate that the N-methyl group causes unfavorable steric effects. The depsipeptide that lacks a Leu6 amide proton is a good substrate for A-kinase, but the corresponding N-methylated peptide is phosphorylated far less efficiently. This result and others presented in this paper suggest that although enzyme-substrate hydrogen bonding may play some role in A-kinase catalysis of phosphoryl group transfer, other explanations are necessary to account for the relative reactivities of N alpha-methylated and depsi-containing peptide 1 analogues.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutations in the active site of Escherichia coli phosphofructokinase.

The enzyme-catalysed transfer of a phosphoryl group from ATP is an important reaction in a wide variety of biological processes. We demonstrate here the essential function of an aspartate group in the catalysis of phosphoryl transfer by Escherichia coli phosphofructokinase, and the minor role of an arginine residue. We have used oligonucleotide-directed mutagenesis to replace two amino-acid residues which X-ray analysis has shown to be close to the transferred phosphoryl group and we have analysed the forward and back reactions of the mutant enzymes by steady-state kinetics. Changing Asp 127 to Ser reduced the turnover number by a factor of 18,000 in the forward direction and 3,100 in the back reaction, and the Michaelis constant for fructose 1,6-bisphosphate in the reverse reaction by a factor of 45. This shows that this aspartate is a key residue in the rate enhancement by the enzyme, probably acting as a base in the reaction mechanism, and that it also destabilizes the product complex. Changing Arg 171 to Ser reduced the turnover numbers by about 3.4, showing that this arginine has only a minor effect on the catalysis.

Mechanism of endogenous phosphorylation of microtubule proteins during GTP-induced microtubule assembly and implications for stability of the assembled structures

Cycle-purified microtubule protein from mammalian brain incorporated [ 32P]P i upon incubation with [γ- 32P]GTP under the conditions used to promote assembly. This phosphorylation also occurred in the same proteins when phosphorylated with [γ- 32P]ATP and was only slightly stimulated by cAMP. GTP was a much less effective substrate than ATP. The transfer of phosphoryl groups from [γ- 32P]GTP to endogenous proteins followed a linear time-course and was stimulated by low concentrations of ATP and, more efficiently, by ADP. These data are in agreement with the predictions derived from a mechanism of phosphorylation by which [γ- 32P]GTP does not act as a phosphoryl donor for the protein kinase activity but, instead, only as a repository of high group transfer potential phosphoryl groups used to make [γ- 32P]ATP, from contaminating ADP, by means of the nucleoside diphosphate kinase activity. Using 100 mM fluoride, which suppressed protein phosphorylation without inhibiting the nucleoside diphosphate kinase activity, formation of [γ- 32P]ATP was detected. Fluoride was also able to protect microtubules from a slow depolymerization which was found to occur during long-term incubation of microtubules. This indicates that the phosphorylation observed in the presence of GTP is sufficient to destabilize microtubules.

Inhibition of adenosine and thymidylate kinases by bisubstrate analogs.

Potential bisubstrate analogs, in which the 5'-hydroxyl group of adenosine was joined to the phosphoryl group acceptor by polyphosphoryl bridges of varying length (ApnX, where n is the number of phosphoryl groups and X is the nucleoside moiety of the acceptor), were tested as inhibitors of human liver adenosine kinase and of thymidylate kinase from peripheral blast cells of patients with acute myelocytic leukemia. Adenosine kinase was most strongly inhibited by P1,P4-(diadenosine 5')-tetraphosphate (Kd = 30 nM) and P1,P5-(diadenosine 5')-pentaphosphate (Kd = 73 nM). Thymidylate kinase was most strongly inhibited by P1-(adenosine 5')-P5-(thymidine 5')-pentaphosphate (Kd = 120 nM) and by P1(adenosine 5')-P6-(thymidine 5')-hexaphosphate (Kd = 43 nM). In these enzymes, as in adenylate and thymidylate kinases, strongest inhibition was achieved in compounds containing one or two more phosphoryl groups than the substrates combined. These results support the view that nucleoside and nucleotide kinases mediate direct transfer of phosphoryl groups from ATP to acceptors, rather than acting by a double displacement mechanism.

Creatine kinase-catalyzed ATP-phosphocreatine exchange:: Comparison of 31P-NMR saturation transfer technique and radioisotope tracer methods

Unidirectional fluxes from ATP to phosphocreatine, catalyzed by the MM isoenzyme of creatine kinase, were measured by both the 31P-NMR saturation transfer technique and radioisotope tracer ([γ- 32P]ATP) method. It was found that at 30–37°C and pH 7.4, over a wide range of[phosphocreatine]/[creatine] (from 0.2 to 5.0) ratios, both methods gave the same results, showing that magnetization transfer allows determination of real fluxes under ‘physiological’ conditions. However, at[PCr]/[Cr] ratios higher than 5 ([ADP] free < 30 μM) or at lower temperatures (t < 15°C,[PCr]/[Cr] ∼ 1), the fluxes assessed by saturation transfer were somewhat faster than those detected by the radioisotope tracer method. These data imply that under physiological conditions phosphoryl group transfer is actually the rate-determining step of the creatine kinase reaction. In contrast, at high[PCr]/[Cr] ratios or at lower temperatures, control may be shifted from phosphoryl group transfer or distributed among other steps of the reaction.

Energy transduction coupling mechanisms in multiredox center proteins

The data obtained for the physicochemical parameters of Desulfovibrio gigas cytochrome c 3, a small tetraheme electron transfer protein, are analyzed in terms of its possible use as an electron/proton/ phosphoryl group transfer potential coupling device.

Mevalonate-5-diphosphate decarboxylase: stereochemical course of ATP-dependent phosphorylation of mevalonate 5-diphosphate.

Chicken liver mevalonate-5-diphosphate decarboxylase catalyzes the reaction of mevalonate 5-diphosphate (MVADP) with ATP to produce isopentenyl diphosphate, ADP, CO2, and inorganic phosphate. The overall reaction involves an anti elimination of the tertiary hydroxyl and carboxyl groups. To investigate the mechanism for transfer of the terminal phosphoryl group of ATP to the C-3 oxygen of MVADP, we have carried out the reaction using stereospecifically labeled (Sp)-adenosine 5'-O-(3-thio[3-17O2,18O]triphosphate) [( gamma-17O2,18O]ATP gamma S) in place of ATP. The configuration of the [17O,18O]thiophosphate produced was found to be Rp, corresponding to overall inversion of configuration at phosphorus in the thiophosphoryl group transfer step. This result is consistent with the direct transfer of the thiophosphoryl group from (Sp)-[gamma-17O2,18O]ATP gamma S to MVADP at the active site. Our result does not indicate the involvement of a covalent thiophosphoryl-enzyme on the reaction pathway.

ChemInform Abstract: Bonding in Phosphoryl (‐PO32‐) and Sulfuryl (‐SO32‐) Group Transfer Between Nitrogen Nucleophiles as Determined from Rate Constants for Identity Reactions.

ChemInform Abstract: Bonding in Phosphoryl (‐PO₃^2‐) and Sulfuryl (‐SO₃^2‐) Group Transfer Between Nitrogen Nucleophiles as Determined from Rate Constants for Identity Reactions.

The phosphoenolpyruvate-dependent fructose-specific phosphotransferase system in Rhodopseudomonas sphaeroides. Energetics of the phosphoryl group transfer from phosphoenolpyruvate to fructose.

Energy coupling to fructose transport in Rhodopseudomonas sphaeroides is achieved by phosphorylation of the membrane-spanning fructose-specific carrier protein, EFruII. The phosphoryl group of phosphoenolpyruvate is transferred to EFruII via the cytoplasmic component SF (soluble factor). The standard free enthalpy of hydrolysis of the two phosphorylated proteins has been estimated from isotope exchange measurements in chemical equilibrium. The delta G degrees for SF-P is -60.5 kJ/mol. The standard free enthalpy for hydrolysis of EII-P is -37.9 kJ/mol, but -45.2 kJ/mol when SF is still complexed to it, as in the overall reaction. Therefore the standard free enthalpy of hydrolysis of SF X EII-P is 70% of the standard free enthalpy of hydrolysis of P-enolpyruvate. The measurements reveal two regulation sites in the system. First, the phosphorylation of SF is inhibited by pyruvate when the concentration ratio of pyruvate/P-enolpyruvate becomes too high. Second, a low concentration of internal fructose prevents the phosphorylation of the carrier by the internal fructose-1-P pool when the concentration of the latter becomes too high or the phosphorylation rate by P-enolpyruvate too slow. Furthermore comparison of the isotope exchange and the overall phosphotransferase reaction kinetics leads to the conclusion that binding of fructose to the carrier is a slow step relative to the phosphoryl group transfer from EFruII to fructose.

The use of N-methylated peptides and depsipeptides to probe the binding of heptapeptide substrates to cAMP-dependent protein kinase.

Peptide 1, Leu-Arg-Arg-Ala-Ser-Leu-Gly, is an excellent substrate for cAMP-dependent protein kinase. While the importance of both arginines for effective enzyme-substrate interactions has been shown, it has not been known whether the kinase will catalyze phosphorylation of substrates which contain other than peptide bonds. We report that analogs of peptide 1 which contain depsi linkages replacing selected amide bonds are good protein kinase substrates. Therefore, with the possible exception of the serine amide proton, no peptide 1 amide hydrogens are involved in peptide-peptide or peptide-enzyme hydrogen bonding crucial to defining the high substrate activity of this peptide. It is thus unlikely that peptide 1 is bound by the protein kinase while in an alpha-helical or a beta-turn structure. Three peptides were found to be very poor substrates for protein kinase, those containing N-methyl amino acids in place of Ser5 or Leu6 and a peptide containing Pro in place of Leu6. These peptides are poor substrates for the enzyme possibly because they are unable to adopt a conformation necessary for catalysis of phosphoryl group transfer to occur or due to steric effects in the enzymatic active site.

Bonding in phosphoryl (-PO32-) and sulfuryl (-SO3-) group transfer between nitrogen nucleophiles as determined from rate constants for identity reactions

Determination des constantes de vitesse des reactions bimoleculaires de pyridines avec des pyridinophosphonates et pyridinosulfonates dans des solutions aqueuses

A simple procedure for selective inversion of NMR resonances for spin transfer enzyme kinetic measurements

The unidirectional fluxes through enzyme-catalyzed reactions operating at equilibrium can be measured using saturation or inversion transfer NMR spectroscopy. The procedure requires selective saturation or inversion of the magnetization of one species (A) which is exchanging with others (B). To date selective excitation has been via a separate frequency synthesizer channel or multiple-pulse sequences such as DANTE. We apply here a far simpler pulse sequence for selective inversion of spins which generates the so-called d-ordered state. The pulse sequence is π 2 x (η A )-τ 1- π 2 ±x -τ 2- π 2 ±x,±y - acquisition , where τ1 = 1 (2|;η A − η B | ), the subscripted ± x and ± y refer to rotating frame axes along which the rf irradiation is applied and τ 2 is a variable delay time. The application of this procedure to measurement of the rate of phosphoryl group transfer catalyzed by phosphoglyceromutase is demonstrated.

Biomimetic study of intramolecular phosphoryl group transfer in thioureas under acid/base and stereoelectronic control

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTBiomimetic study of intramolecular phosphoryl group transfer in thioureas under acid/base and stereoelectronic controlC. Blonski, M. B. Gasc, A. F. Hegarty, A. Klaebe, and J. J. PerieCite this: J. Am. Chem. Soc. 1984, 106, 24, 7523–7529Publication Date (Print):November 1, 1984Publication History Published online1 May 2002Published inissue 1 November 1984https://doi.org/10.1021/ja00336a036RIGHTS & PERMISSIONSArticle Views84Altmetric-Citations8LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (770 KB) Get e-Alerts Get e-Alerts

Physico-chemical principles of cAMP-dependent protein phosphorylation: Catalysis of phosphoryl group transfer to nucleophilic agents

The specificity of pig brain protein kinase towards high- and low-M r ‘analogs’ of protein substrate was studied. Solvolysis of the phosphoenzyme intermediate by various nucleophilic agents is shown. The transition state structure of the phosphoryl transfer reaction is discussed.

Effective charge on oxygen in phosphoryl (-PO32-) group transfer from an oxygen donor

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTEffective charge on oxygen in phosphoryl (-PO32-) group transfer from an oxygen donorNicholas Bourne and Andrew WilliamsCite this: J. Org. Chem. 1984, 49, 7, 1200–1204Publication Date (Print):April 1, 1984Publication History Published online1 May 2002Published inissue 1 April 1984https://doi.org/10.1021/jo00181a011RIGHTS & PERMISSIONSArticle Views340Altmetric-Citations82LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (673 KB) Get e-Alerts Get e-Alerts

ChemInform Abstract: THE QUESTION OF CONCERTED OR STEPWISE MECHANISMS IN PHOSPHORYL GROUP (‐PO32‐) TRANSFER TO PYRIDINES FROM ISOQUINOLIN‐N‐PHOSPHONATE

Chemischer InformationsdienstVolume 14, Issue 35 Organoelement Compounds ChemInform Abstract: THE QUESTION OF CONCERTED OR STEPWISE MECHANISMS IN PHOSPHORYL GROUP (-PO32-) TRANSFER TO PYRIDINES FROM ISOQUINOLIN-N-PHOSPHONATE N. BOURNE, N. BOURNESearch for more papers by this authorA. WILLIAMS, A. WILLIAMSSearch for more papers by this author N. BOURNE, N. BOURNESearch for more papers by this authorA. WILLIAMS, A. WILLIAMSSearch for more papers by this author First published: August 30, 1983 https://doi.org/10.1002/chin.198335273AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume14, Issue35August 30, 1983 RelatedInformation

31P-NMR spectrum of phosphocreatine: Deuterium-induced splitting of the signal

It has been found in experiments with high resolution 31P-NMR spectroscopy (200 MHz) that the phosphocreatine peak is splitted into two different peaks in the mixtures of H 2O and D 2O and is single but with different chemical shifts in pure H 2O and D 2O. This phenomenon is explained by substitution of protons of guanidino group in phosphocreatine by deuterium. The effect of splitting disappeared at extreme pH values (>8.5 or <4.0) and at temperatures higher than 45°C due to accelerated proton-deuterium exchange. Creatine kinase added to phosphocreatine solution also lowered its temperature of peaks' collapse by 5°–10°C. A saturation (spin) transfer method was used to show that the phosphoryl group transfer to ADP in creatine kinase active center is slower with deuterium-substituted phosphocreatine than with H-phosphocreatine. The data are taken to show the importance of the proton transfer step in the creatine kinase reaction mechanism and acceleration of phosphocreatine proton-deuterium exchange by creatine kinase.

The question of concerted or stepwise mechanisms in phosphoryl group (-PO32-) transfer to pyridines from isoquinolin-N-phosphonate

The question of concerted or stepwise mechanisms in phosphoryl group (-PO32-) transfer to pyridines from isoquinolin-N-phosphonate

Synthesis and accumulation of an extremely stable high-energy phosphate compound by muscle, heart, and brain of animals fed the creatine analog, 1-carboxyethyl-2-iminoimidazolidine (homocyclocreatine)

A new creatine analog, l-carboxyethyl-2-iminoimidazolidine (homocyclocreatine), has been synthesized and compared with other synthetic analogs of creatine as a substrate for creatine kinase under both in vitro and in vivo conditions. Reactivity with rabbit muscle creatine kinase at 2 m m and pH 7.0 occurred in the order: creatine > cyclocreatine (1-carboxymethyl-2-iminoimidazolidine) > N-ethylguanidinoacetate > N-propylguanidinoacetate > guanidinoacetate > N-methyl-3-guanidinopropionate > 3-guanidinopropionate > homocyclocreatine. Homocyclocreatine was 10,000-fold less active than creatine. In the reverse direction at 0.2 m m and pH 7.0: creatine- P > N-ethylguanidinoacetate- P > cyclocreatine- P ⪢ homocyclocreatine- P. Homocyclocreatine- P was 200,000-fold less active than creatine- P. The phosphoryl group transfer potential of homocyclocreatine- P was estimated to be 2 kcal/mol lower than that of creatine- P. Chicks fed 5% homocyclocreatine for 16 days synthesized and accumulated homocyclocreatine- P in breast muscle (32 μmol/g wet wt), leg muscle (24 μmol/g), heart (7 μmol/g), intestine (8.5 μmol/g), and brain (2.4 μmol/g). During ischemia homocyclocreatine- P was utilized by muscle much more slowly for the regeneration of ATP than was creatine- P or cyclocreatine- P. Our results suggest that in tissues of homocyclocreatine-fed animals subjected to a sudden large increase in work load or to ischemia, the residual creatine- P system would rapidly equilibrate with the adenylate system at the new lower cytosolic phosphorylation potential, whereas in the same cytosol the (homocyclocreatine- P)/(homocyclocreatine) ratio would exhibit a hysteresis or memory effect and reflect for a considerable period of time the earlier higher (ATP)/(free ADP) ratio rather than the actual lower (ATP)/(free ADP) ratio.