Nucleotide stereochemistry in the formyltetrahydrofolate synthetase reaction

In a recent study, we have shown that N10-formyltetrahydrofolate synthetase prefers (Sp)-MgATP beta S over the Rp isomer in the forward reaction. In this report the stereochemistry of ATP beta S produced from prochiral ADP beta S in the reverse reaction was determined. The ATP beta S product was purified and tested as a substrate for hexokinase (preference for the Rp isomer), adenylate kinase (preference for the Sp isomer) and N10-formyltetrahydrofolate synthetase. A comparison of kinetic constants for the product and the authentic Sp and Rp isomers shows that the product is the Sp diastereomer. 31P NMR was also used to identify the product as (Sp)-ATP beta S.     

Adenosine 3', 5'-monophosphorothioate (Rp-isomer) induces down-regulation of surface cyclic AMP receptors without receptor activation in Dictyostelium discoideum

cAMP induces the activation and subsequent desensitization of adenylate cyclase in Dictyostelium discoideum. cAMP also induces down-regulation of surface cAMP receptors. Desensitization of adenylate cyclase is composed of a rapidly reversible component (adaptation) and a slowly reversible component related to down-regulation of surface cAMP receptors (Van Haastert, P.J.M. (1987) J. Biol. Chem. 262, 7700-7704). The agonistic and antagonistic activities of the cAMP derivative adenosine 3',5'-monophosphorothioate ((Rp)-cAMPS) for these responses were investigated. (Rp)-cAMPS competes with cAMP for binding to different receptor forms with an apparent Ki = 5 microM. (Rp)-cAMPS does not activate adenylate cyclase and antagonizes the cAMP-induced activation with an apparent Ki = 5 microM. (Rp)-cAMPS induces down-regulation of surface cAMP receptors with EC50 = 5 microM. (Rp)-cAMPS induces desensitization of adenylate cyclase, which is not rapidly reversible. These results indicate that desensitization of adenylate cyclase by (Rp)-cAMPS is due to down-regulation of surface cAMP receptors and not to adaptation. We conclude that down-regulation of surface cAMP receptors does not require their activation or modification involved in adaptation.     

Synergistic inhibition of hepatic glycogenolysis in the presence of insulin and a cAMP antagonist

The effects of insulin on the ability of the specific intracellular cAMP-dependent protein kinase antagonist, the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate, to inhibit glycogenolysis induced by the Sp diastereomer was studied in hepatocytes isolated from fed rats. Addition of the cAMP agonist, (Sp)-cAMPS, to hepatocytes resulted in a concentration-dependent increase in glycogenolytic glucose production concomitant with the cAMP-dependent activation of phosphorylase and inhibition of glycogen synthase. Activity curves were shifted to the right in the presence of the cAMP antagonist, (Rp)-cAMPS. Preincubation of the hepatocytes with a maximally effective concentration of insulin did not affect the concentration of (Sp)-cAMPS required for half-maximal activation of phosphorylase but did result in a 10-fold shift in the concentration of (Sp)-cAMPS required for half-maximal inactivation of glycogen synthase. Preincubation of hepatocytes with a combination of the cAMP antagonist, (Rp)-cAMPS, and insulin resulted in synergistic inhibition of (Sp)-cAMPS-induced phosphorylase activation, glycogen synthase inactivation, and glycogenolytic glucose production. Since neither phosphorothioate diastereomer was hydrolyzed significantly during the course of the experiments, the synergistic effects of insulin are postulated to be working through a mechanism subsequent to the phosphodiesterase activation step.     

In Vitro Phosphorylation of Microtubule-Associated Protein 2: Differential Effects of Cyclic AMP Analogues

Microtubules purified from brain tissue contain endogenous cyclic AMP (cAMP)-dependent protein kinase activity, and microtubule-associated protein 2 (MAP2) is the major substrate. Beef brain microtubules were prepared and used as a model system to study the differential effects of rationally selected cyclic nucleotide analogues on microtubule receptor protein kinase. Data are presented to indicate that the following molecular interactions are essential for activation of the phosphorylation of MAP2: (a) hydrogen bond formation toward the 2', 3', or 5' position, (b) interaction with phosphorus, and (c) no hydrogen bonds but hydrophobic interactions at the base moiety. Thus, the activation mechanism of the type II protein kinase associated with brain microtubules resembles the mechanism found in protein kinases of other systems. In addition, we have studied the effect of the two diastereomers of adenosine 3',5'-monophosphorothioate (cAMPS). The (Sp)-cAMPS isomer was found to activate MAP2 protein kinase, whereas the (Rp)-cAMPS isomer had no activating effect. In contrast, this compound was able to inhibit cAMP-stimulated MAP2 phosphorylation and thus acts as an antagonist of the Sp diastereomer and cAMP. Hence, this analogue provides a useful means to clarify further the effect of cAMP-dependent phosphorylation on functional properties in microtubules in general.     

Stereochemical course of the reaction catalyzed by guanylate cyclase from bovine retinal rod outer segments

The stereochemical course of the reaction catalyzed by guanylate cyclase from bovine retinal rod outer segments was investigated using phosphorothioate analogs of GTP as chiral probes. (Sp)-Guanosine 5'-O-(1-thiotriphosphate) (Sp-GTP alpha S) is a substrate, whereas (Rp)-GTP alpha S is a competitive inhibitor (K1 = 0.1 mM), but not a substrate. (Sp)-GTP alpha S is converted into (Rp)-guanosine 3':5'-monophosphorothioate, showing that the reaction proceeds with inversion of configuration at the alpha-phosphorus atom. Km and Vmax for (Sp)-GTP alpha S (at low [Ca2+], 20 nM) are 3.7 mM and 1.1 nmol/min/mg of rhodopsin, respectively, compared with 1.1 mM and 23.1 nmol/min/mg of rhodopsin for GTP. Vmax for the cyclization of (Sp)-GTP alpha S, as for GTP, increases 10-20-fold when the calcium level is lowered. This activity change is centered at approximately 90 nM and has a Hill coefficient of 4.8. The configuration of the metal-substrate complex was determined by measuring the effectiveness of the Sp and Rp isomers of GTP alpha S and guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) in the presence of Mg2+ or Mn2+. (Sp)-GTP alpha S is a substrate with either Mg2+ or Mn2+, whereas (Rp)-GTP beta S is a substrate with only Mn2+. These findings suggest that the substrate is a metal-beta, gamma-bidentate complex with delta screwsense. We also found that the cyclization reaction catalyzed by the membrane-bound guanylate cyclase from sea urchin sperm proceeds with inversion of configuration at the alpha-phosphorus atom. The stereochemical course of the reactions catalyzed by all prokaryotic and eukaryotic adenylate cyclases and guanylate cyclases studied thus far is the same.     

The interaction of phosphorothioate analogues of ATP with phosphomevalonate kinase. Kinetic and 31P NMR studies

The diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S), adenosine 5'-O-(2-thiotriphosphate) (ATP beta S), and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) could act as substrates for phosphomevalonate kinase in the presence of Mg2+ and Cd2+ as activating divalent metal cations. The Sp diastereomer of ATP alpha S was the preferred substrate regardless of the metal ion used, consistent with the metal ion not binding to the alpha-phosphate. With ATP beta S, the Sp diastereomer was the preferred substrate with Mg2+, and the Rp diastereomer was the preferred substrate with Cd2+. The reversal of specificity establishes that the metal is chelated through the beta-phosphate in the active site of the phosphomevalonate kinase reaction. A comparison of the Vmax values as a function of substitution of oxygen by sulfur showed the order for Mg2+ to be: ATP greater than ATP alpha S(Sp) greater than ATP alpha S(Rp) greater than ATP beta S(Sp) greater than ATP gamma S greater than ATP beta S(Rp). With Cd2+ as the activating metal ion, the order was: ATP greater than ATP alpha S(Sp) greater than ATP alpha S(Rp) greater than ATP beta S(Rp) greater than ATP gamma S greater than ATP beta S(Sp). It is concluded that the chelate structure of metal ATP substrate in the phosphomevalonate kinase reaction is the delta, beta, gamma-bidentate complex. 31P NMR measurements and radioassay with [2-14C] phosphomevalonate were used to measure the equilibrium of the reaction catalyzed by phosphomevalonate kinase with ATP and phosphorothioate analogues of ATP as the phosphoryl group donor. The order as a phosphate donor as determined by both methods in the phosphomevalonate kinase reaction is ATP beta S greater than ATP alpha S greater than ATP greater than ATP gamma S. Except for ATP gamma S, the equilibrium is shifted in the direction of formation of ADP alpha S and ADP beta S relative to ADP formation. Thus, ATP beta S rather than ATP would be effective for the synthesis of diphosphomevalonate. The phosphomevalonate kinase reaction could also be used to synthesize mevalonate 5-(2-thiodiphosphate) using ATP gamma S as the phosphoryl group donor.     

P alpha-chiral phosphorothioate analogues of bis(5''-adenosyl)tetraphosphate (Ap4A); their enzymatic synthesis and degradation.

Synthesis of Sp and Rp diastereomers of Ap4A alpha S has been characterized in two enzymatic systems, the lysyl-tRNA synthetase from Escherichia coli and the Ap4A alpha, beta-phosphorylase from Saccharomyces cerevisiae. The synthetase was able to use both (Sp)ATP alpha S and (Rp)ATP alpha S as acceptors of adenylate thus yielding corresponding monothioanalogues of Ap4A,(Sp) Ap4A alpha S and (Rp)Ap4A alpha S. No dithiophosphate analogue was formed. Relative synthetase velocities of the formation of Ap4A,(Sp) Ap4A alpha S and (Rp)Ap4A alpha S were 1:0.38:0.15, and the computed Km values for (Sp)ATP alpha S and (Rp)ATP alpha S were 0.48 and 1.34 mM, respectively. The yeast Ap4A phosphorylase synthesized (Sp)Ap4A alpha S and (Rp)Ap4A alpha S using adenosine 5'-phosphosulfate (APS) as source of adenylate. The adenylate was accepted by corresponding thioanalogues of ATP. In that system, relative velocities of Ap4A, (Sp)Ap4A alpha S and (Rp)Ap4A alpha S formation were 1:0.15:0.60. The two isomeric phosphorothioate analogues of Ap4A were tested as substrates for the following specific Ap4A-degrading enzymes: (asymmetrical) Ap4A hydrolase (EC 3.6.1.17) from yellow lupin (Lupinus luteus) seeds hydrolyzed each of the analogues to AMP and the corresponding isomer of ATP alpha S; (symmetrical) Ap4A hydrolase (EC 3.6.1.41) from E. coli produced ADP and the corresponding diastereomer of ADP alpha S; and Ap4A phosphorylase (EC 2.7.7.53) from S. cerevisiae cleaved the Rp isomer only at the unmodified end yielding ADP and (Rp)ATP alpha S whereas the Sp isomer was degraded non-specifically yielding a mixture of ADP, (Sp)ADP alpha S, ATP and (Sp)ATP alpha S. For all the Ap4A-degrading enzymes, the Rp isomer of Ap4A alpha S appeared to be a better substrate than its Sp counterpart; stereoselectivity of the three enzymes for the Ap4A alpha S diastereomers is 51, 6 and 2.5, respectively. Basic kinetic parameters of the degradation reactions are presented and structural requirements of the Ap4A-metabolizing enzymes with respect to the potential substrates modified at the Ap4A-P alpha are discussed.     

The role of cAMP in nerve growth factor-promoted neurite outgrowth in PC12 cells

Nerve growth factor (NGF)-mediated neurite outgrowth in rat pheochromocytoma PC12 cells has been described to be synergistically potentiated by the simultaneous addition of dibutyryl cAMP. To elucidate further the role of cAMP in NGF-induced neurite outgrowth we have used the adenylate cyclase activator forskolin, cAMP, and a set of chemically modified cAMP analogues, including the adenosine cyclic 3',5'-phosphorothioates (cAMPS) (Rp)-cAMPS and (Sp)-cAMPS. These diastereomers have differential effects on the activation of cAMP-dependent protein kinases, i.e., (Sp)-cAMPS behaves as a cAMP agonist and (Rp)-cAMPS behaves as a cAMP antagonist. Our data show that the establishment of a neuritic network, as observed from PC12 cells treated with NGF alone, could not be induced by either forskolin, cAMP, or cAMP analogues alone. The presence of NGF in combination with forskolin or cAMP or its agonistic analogues potentiated the initiation of neurite outgrowth from PC12 cells. The (Sp)-cAMPS-induced stimulation of NGF-mediated process formation was successfully blocked by the (Rp)-cAMPS diastereomer. On the other hand, NGF-stimulated neurite outgrowth was not inhibited by the presence of the cAMP antagonist (Rp)-cAMPS. We conclude that the morphological differentiation of PC12 cells stimulated by NGF does not require cAMP as a second messenger. The constant increase of intracellular cAMP, caused by either forskolin or cAMP and the analogues, in combination with NGF, not only rapidly stimulated early neurite outgrowth but also exerted a maintaining effect on the neuronal network established by NGF.     

Phosphorothioate analogues of 2',5'-oligoadenylate. Enzymatic synthesis, properties, and biological activities of 2',5'-phosphorothioates from adenosine 5'-O-(2-thiotriphosphate) and adenosine 5'-O-(3-thiotriphosphate)

The chiral and achiral phosphorothioate analogues of 2',5'-oligoadenylates (2-5A) have been enzymatically synthesized from the Sp and Rp isomers of adenosine 5'-O-(2-thiotriphosphate) [(Sp)-ATP beta S and (Rp)-ATP beta S, respectively] and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) by 2-5A synthetase from L929 cells and lysed rabbit reticulocytes. These 2',5'-phosphorothioate analogues were separated, purified, and structurally characterized. While ATP gamma S and (Sp)-ATP beta S were as efficient substrates for the 2-5A synthetase as was ATP, (Rp)-ATP beta S was more than 50-fold less efficient a substrate. The beta- and gamma-phosphorothioates were more resistant to enzymatic hydrolysis than was authentic 2-5A. Compared to 2-5A, there were marked differences in the biological activities of the 2',5'-phosphorothioates as determined by (i) binding to 2-5A-dependent endoribonuclease (RNase L), (ii) activation of RNase L to hydrolyze RNA, and (iii) inhibition of protein synthesis in intact L929 cells. These studies extend previous reports on the elucidation of the stereochemical requirements of 2-5A synthetase and RNase L [Karikó, K., Sobol, R. W., Jr., Suhadolnik, L., Li, S. W., Reichenbach, N. L., Suhadolnik, R. J., Charubala, R., & Pfleiderer, W. (1987) Biochemistry (first of three papers in this issue); Karikó, K., Li, S. W., Sobol, R. W., Jr., Suhadolnik, R. J., Charubala, R., & Pfleiderer, W. (1987) Biochemistry (second of three papers in this issue)] with the phosphorothioate analogues of 2-5A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanistic studies on deoxyribonucleic acid dependent ribonucleic acid polymerase from Escherichia coli using phosphorothioate analogues. 1. Initiation and pyrophosphate exchange reactions.

The diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) and adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) can replace adenosine triphosphate (ATP) in the initiation reaction catalyzed by deoxyribonucleic acid (DNA) dependent ribonucleic acid (RNA) polymerase from Escherichia coli. In both cases, the Sp diastereomer is a better initiator than the Rp isomer. The diasteromers of 3'-uridyl 5'-adenosyl ,O-phosphorothioate [Up(S)A] can replace UpA in the primed initiation reaction catalyzed by RNA polymerase; however, the Rp diastereomer is a better initiator than the Sp isomer. By using ATP or CpA as initiator and UTP alpha S, isomer A, as substrate, we determined the stereochemical courses of both the initiation and primed initiation reactions, respectively, with T7 DNA template and found them to proceed with inversion of configuration. Determination of the stereochemical course of the pyrophosphate exchange reaction catalyzed by RNA polymerase provides evidence that this reaction is the reverse of the phosphodiester bond-forming reaction.     

Structures of the mono- and divalent metal nucleotide complexes in the myosin ATPase

The structure of both the mono- and the divalent metal nucleotide complexes active in the myosin subfragment 1 ATPase has been determined using the phosphorothioate analogs of ATP in the presence of various cations. Both the Sp and the Rp diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) were substrates in the presence of Mg2+, Ca2+, Mn2+, Co2+, Zn2+, and Cd2+ as well as with NH4+ and T1+. The Sp/Rp activity ratios obtained were largely independent of the cation. The simplest explanation of these results is that both mono- and divalent cations do not coordinate to the alpha-phosphate group. With adenosine 5'-O-(2-thiotriphosphate) (ATP beta S), essentially only the Sp diastereomer was active with Mg2+ with Sp/Rp ratio of greater 3000. As the divalent metal ion was varied in the series given above, this ratio was progressively lowered to the value of 0.2 found with Cd2+. Similar changes in stereoselectivity were seen with monovalent cations. Thus, with NH4+, an Sp/Rp ratio of 8 was observed, whereas with T1+, this figure was reduced to 0.04. These data indicate that both mono- and divalent cations coordinate to the beta-phosphate group of the nucleoside triphosphate substrate. These results obtained with ATP alpha S and ATP beta S suggest that myosin uses the mono- or divalent cation delta, beta, gamma-bidentate nucleotide chelate as substrate.     

Inhibitory action of certain cyclophosphate derivatives of cAMP on cAMP-dependent protein kinases

A series cAMP derivatives with modifications in the adenine, ribose and cyclophosphate moiety were screened for their binding affinity for the two types of cAMP-binding sites in mammalian protein kinase type 1. In addition, the activation of the kinase by these analogs was monitored. The binding data indicate that cAMP is bound to both sites in a comparable manner: the adenine appears to have no hydrogen-bond interactions with the binding sites, whereas the ribose may be bound by three hydrogen bonds involving the 2', 3' and 5' positions of cAMP. The binding data are not conclusive about the nature of the interaction with the exocyclic oxygen atoms on phosphorus, though a charge interaction seems to be absent. The cAMP molecule seems to be bound in the syn conformation. The results of activation experiments show that modifications in the adenine and ribose moiety do not affect the maximal activation level, while alteration of the two exocyclic oxygen atoms may result in a reduced maximal activation level and in one case, (Rp)-adenosine 3', 5'-monophosphorothioate [Rp-cAMPS], in total absence of activation even at concentrations at which the analog saturates both binding sites. Since occupancy of the cAMP-binding sites by this derivative apparently did not lead to activation of the enzyme, we examined whether this compound could antagonize the activation by cAMP. Indeed (Rp)-cAMPS was found to inhibit cAMP stimulated kinase activity at concentrations compatible to its binding affinity. Also with mammalian protein kinase type II (Rp)-cAMPS showed antagonistic activity, while with a cAMP-dependent protein kinase from Dictyostelium discoideum partial agonistic activity was observed. Previously a mechanism for activation of protein kinase type I was proposed involving a charge interaction between the equatorial exocyclic oxygen atom and the binding site [De Wit et. al. (1982) Eur. J. Biochem 122, 95-99]. This was based on measurements with impure preparations of (Rp)-cAMPS and the Rp and Sp isomers adenosine 3', 5'-monophosphodimethylamidate. cAMPN(CH3)2. The present work using highly purified compounds suggests the absence of a charge interaction, since the uncharged analog (Sp)-cAMPN(CH3)2 activates the kinase effectively. The data seem compatible with an activation model involving the formation of a covalent bond with phosphorus in both cAMP binding sites.     

A study of the mechanism of glucagon-induced protein phosphorylation in isolated rat hepatocytes using (Sp)-cAMPS and (Rp)-cAMPS, the stimulatory and inhibitory diastereomers of adenosine cyclic 3',5'-phosphorothioate

Maximal doses of glucagon increase the phosphorylation state of 12 cytosolic proteins in isolated hepatocytes from fasted rats (Garrison, J. C., and Wagner, J. D. (1982) J. Biol. Chem. 257, 13135-13143). Incubation of hepatocytes with lower concentrations of glucagon indicates that a hierarchy of substrates exists with the concentration of glucagon required for half-maximal increases in phosphorylation varying 5-15-fold. The proteins whose phosphorylation state is most sensitive to low concentrations of glucagon are pyruvate kinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, both of which play key roles in the regulation of gluconeogenesis. Treatment of hepatocytes with (Sp)-cAMPS, the stimulatory diastereomer of adenosine cyclic 3',5'-phosphorothioate, mimics the response seen with glucagon. When hepatocytes are pretreated with the cAMP antagonist, (Rp)-cAMPS, the phosphorylation response is abolished at low concentrations of glucagon, and the dose of glucagon required for half-maximal stimulation of phosphorylation is increased 5-10-fold. The (Sp)-cAMPS-stimulated increases in phosphorylation state are also blunted by (Rp)-cAMPS. These results provide direct pharmacological evidence for the activation of the cAMP-dependent protein kinase in response to glucagon in the intact cell. Although low doses of glucagon appear to stimulate protein phosphorylation via the cAMP-dependent protein kinase, high doses of glucagon also cause a small increase in the concentration of free intracellular Ca2+ in hepatocytes. The glucagon-stimulated increases in the level of Ca2+ can be mimicked by (Sp)-cAMPS and inhibited by pretreatment with (Rp)-cAMPS. These results suggest that glucagon can elevate intracellular Ca2+ via cAMP and the cAMP-dependent protein kinase.     

Effects of the specific cAMP antagonist, (Rp)-adenosine cyclic 3',5'-phosphorothioate, on the cAMP-dependent protein kinase-induced activity of hepatic glycogen phosphorylase and glycogen synthase

The cAMP-dependent protein kinase-induced effects on phosphorylase and glycogen synthase activities and glucose production were studied in hepatocytes isolated from fed rats in the presence of the diastereomers of adenosine cyclic 3',5'-phosphorothioate, (Sp)-cAMPS and (Rp)-cAMPS. Incubation of hepatocytes with (Sp)-cAMPS or glucagon, both of which lead to cAMP-dependent protein kinase activation, resulted in a concentration-dependent increase in glycogen phosphorylase activity and a decrease in glycogen synthase activity. Incubation of hepatocytes with the cAMP-dependent protein kinase antagonist, (Rp)-cAMPS, in the absence of an agonist, had no significant effect on phosphorylase or glycogen synthase activities. Incubation of hepatocytes with a half-maximally inhibitory concentration of (Rp)-cAMPS shifted the agonist-induced activation curves for phosphorylase and the agonist-induced inhibition curves for glycogen synthase to 5-fold higher concentrations for both (Sp)-cAMPS and glucagon. Phosphorylase activity was very sensitive to the rapid, concentration-dependent inhibition by (Rp)-cAMPS of agonist-induced activation of cAMP-dependent protein kinase. The effects on phosphorylase activity were observable in 30 s and were concentration-dependent with half-maximal inhibition at 10 microM, similar to that observed for cAMP-dependent protein kinase. In contrast, glycogen synthase activity was less sensitive to (Rp)-cAMPS inhibition of agonist-induced activation of cAMP-dependent protein kinase. The effects on glycogen synthase activity lagged behind those on phosphorylase activity and the concentration dependence did not parallel the cAMP-dependent protein kinase effect, but was shifted to higher concentrations of (Rp)-cAMPS with half-maximal inhibition at 60 microM. Glucose (10 to 40 mM) increased the sensitivity of glycogen synthase to (Rp)-cAMPS inhibition of cAMP-dependent protein kinase over a narrow range of agonist concentration, but had no significant effect throughout most of the agonist-induced activation range. Thus, the diastereomers, (Sp)- and (Rp)-cAMPS, influence glycogen metabolism and the glycogenolytic enzymes through their modulation of cAMP-dependent protein kinase levels.     

Stereochemical course of the reaction catalyzed by the cyclic GMP phosphodiesterase from retinal rod outer segments

The stereochemical course of hydrolysis catalyzed by the cyclic GMP phosphodiesterase from bovine retinal rod outer segments was determined. The Sp diastereomer of guanosine 3',5'-cyclic monophosphorothioate was hydrolyzed by cyclic GMP phosphodiesterase in H2(18)O to give [16O,18O]guanosine 5'-monophosphorothioate. This isotopomer was reacted with diphenyl phosphorochloridate to form the two diastereomers of P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate. The 31P NMR spectrum of this mixture of diastereomers was identical to that obtained from [16O,18O]guanosine 5'-monophosphorothioate resulting from the hydrolysis of the Rp diastereomer of guanosine 5'-p-nitrophenyl phosphorothioate by snake venom phosphodiesterase. This finding indicates that the 18O is bridging in the Rp diastereomer of the P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate and nonbridging in the Sp diastereomer. As the snake venom phosphodiesterase reaction is known to proceed with retention of configuration, it follows that hydrolysis by retinal rod cyclic GMP phosphodiesterase proceeds with inversion of configuration at the phosphorus atom.     

Inhibition of choriogonadotropin-activated steroidogenesis in cultured Leydig tumor cells by the Rp diastereoisomer of adenosine 3',5'-cyclic phosphorothioate

The diastereoisomers of adenosine 3',5'-cyclic phosphorothioate, (Sp)-cAMPS and (Rp)-cAMPS, have been previously shown to act as agonists and antagonists, respectively, in the activation of several mammalian cAMP-dependent protein kinases. In an effort to characterize further the involvement of cAMP in the activation of Leydig cell steroidogenesis by lutropin/choriogonadotropin (LH/CG), we examined the effects of these cyclic nucleotide analogues on a clonal strain of cultured murine Leydig tumor cells (designated MA-10). Our results show that (i) (Sp)-cAMPS activates and (Rp)-cAMPS inhibits the isolated cAMP-dependent protein kinase of the MA-10 cells; (ii) both analogues inhibit the isolated cAMP phosphodiesterase(s); (iii) (Sp)-cAMPS activates steroid biosynthesis in intact cells, but (Rp)-cAMPS does not; and (iv) (Rp)-cAMPS is a competitive inhibitor of the activation of steroidogenesis by (Sp)-cAMPS, 8-bromo-cAMP, human CG, cholera toxin, and forskolin. However, (Rp)-cAMPS is a more effective inhibitor when steroidogenesis is activated by (Sp)-cAMPS or 8-bromo-cAMP than when it is activated by human CG, cholera toxin, or forskolin. This difference appears to be related to the combined effects of (Rp)-cAMPS on the cAMP-dependent protein kinases and cAMP phosphodiesterase(s). We conclude that cAMP is a quantitatively important mediator of the activation of steroidogenesis by LH/CG even at low concentrations of hormone where an increase in steroid biosynthesis cannot be easily correlated with increased cAMP accumulation. Thus, our data indicate that if other second messengers are involved in the activation of steroidogenesis by LH/CG, they must do so by acting together with, rather than independently of, cAMP.     

Inhibition of cGMP-dependent protein kinase by (Rp)-guanosine 3',5'-monophosphorothioates

The activation of the cGMP-dependent protein kinase and cAMP-dependent protein kinase by the diastereomers of guanosine 3',5'-monophosphorothioate, (Sp)-cGMPS and (Rp)-cGMPS, and 8-chloroguanosine 3',5'-monophosphorothioate, (Sp)-8-Cl-cGMPS and (Rp)-8-Cl-cGMPS, was investigated using the peptide Kemptide as substrate. The (Sp)-diastereomers, which have an axial exocyclic sulfur atom, bound to the cGMP-dependent protein kinase and stimulated its phosphotransferase activity. In contrast, the (Rp)-isomers, which have an equatorial exocyclic sulfur atom, bound to the enzyme without stimulation of its activity. (Rp)-cGMPS and (Rp)-8-Cl-cGMPS antagonized the activation of the cGMP-dependent protein kinase with a Ki of 20 microM and 1.5 microM, respectively. (Rp)-cGMPS also antagonized the activation of cAMP-dependent protein kinase with a Ki of 20 microM. In contrast, (Rp)-8-cGMPS ws a weak inhibitor of the cAMP-dependent protein kinase with a Ki of 100 microM. (Rp)-8-Cl-cGMPS appears to be a rather selective inhibitor of the cGMP-dependent protein kinase and may be a useful tool for studying the role of cGMP in broken and intact cell systems.     

Structure of the metal-nucleotide complex in the acetate kinase reaction. A study with gamma-32P-labeled phosphorothioate analogs of ATP

The synthesis of the gamma-32P-labeled diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) and the Sp isomer of adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) by a modification of the Glynn and Chappell method (Glynn, I. M., and Chappell, J. T., (1964) Biochem. J. 90, 147-149) is described. These analogs were tested as substrates for acetate kinase in the presence of several divalent metal ions. Both isomers of ATP alpha S are substrates in the presence of Mg2+, Mn2+, Co2+, Zn2+, and Cd2+, the Sp isomer being preferred by a factor of between 4.8 (Mg2+) and 52.5 (Cd2+). Only the Rp isomer of ATP beta S is a substrate in the presence of Mg2+, and the Sp isomer becomes a better substrate in the presence of Mn2+, Co2+, and Zn2+; both isomers are equally good substrates in the presence of Cd2+. The change in specificity upon replacing Mg2+ by Cd2+ is greater than 1800 at beta-phosphorus and 10 at alpha phosphorus. These results provide a basis for proposing that the lambda screw sense configuration of the beta, gamma-bidentate MgATP complex is the substrate for acetate kinase. In the reverse reaction, both Sp and Rp isomers of ADP alpha S are substrates in the presence of all metal ions tested, the Sp isomer preferred by a factor between 12.3 (Mg2+) and 45.5 (Cd2+). In the presence of Mg2+, Mn2+, and Co2+, only the Rp isomer of ATP beta S is synthesized from prochiral ADP beta S, while a mixture of Rp and Sp isomers is synthesized in the presence of Zn2+ and Cd2+. These results are analogous to those for the forward reaction and suggest that the Mg.ADP complex which binds as a substrate in the reverse reaction, and is released as a product in the forward reaction, is the beta-monodentate. The classification of acetate kinase as an enzyme having a type I mechanism (Dunaway-Mariano, D. and Cleland, W. W. (1980) Biochemistry 19, 1506-1515) for kinases, is discussed.     

Metal ion dependence of phosphorothioate ATP analogues in the Bacillus stearothermophilus tyrosyl-tRNA synthetase reaction

Pre-steady-state kinetic analyses on the formation of tyrosyl adenylate from tyrosine and each of the four diastereomers of alpha- and beta-phosphorothioate adenosine triphosphates [ATP alpha S and ATP beta S; Eckstein, F., & Goody, R. (1976) Biochemistry 15, 1685-1691; Yee, D., Armstrong, V. W., & Eckstein, F. (1979) Biochemistry 18, 4116-4123] were performed in the presence of Mg2+, Co2+, and Cd2+ as the divalent metal ion cofactor. A modest preference of 5.5-fold in kappa 3/KA' (where kappa 3 is the rate constant for tyrosyl adenylate formation and KA' is the dissociation constant for ATP, or phosphorothioate ATP, from the E.Tyr.metal.ATP complex) for the Sp ATP alpha S diastereomer and the absence of an inversion of preference when the metal ion is changed suggest that there is a stereospecific enzyme-alpha-phosphate interaction and that there is no direct metal ion interaction with the alpha-phosphate. The extent of reaction of the ATP alpha S diastereomers (30-50%) implies that these analogues are more susceptible to the hydrolytic site reaction previously reported for this enzyme [Wells, T. N. C., & Fersht, A. R. (1986) Biochemistry 25, 1881-1886]. The strong preference in kappa 3/KA' for the RP ATP beta S diastereomer (16-fold for Mg2+ and 50-fold for Co2+) is indicative of a stereospecific interaction with the pro SP beta oxygen of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereochemistry of internucleotide bond formation by polynucleotide phosphorylase from Micrococcus luteus.

Polynucleotide phosphorylase catalyzes the formation of polynucleotides from the Sp diastereomer of adenosine 5'-O-(l-thiodiphosphate) ADPalphaS), whereas the Rp diastereomer is a competitive inhibitor. The absolute configuration of the phosphorothioate diester bond in the polymer was determined by copolymerizing ADPalpha S, Sp isomer with UDP and degrading the resulting copolymer with R Nase A and spleen phosphodiesterase to give, inter alia, uridine 2',-3'-cyclic phosphorothioate. The latter product was shown to be the endo isomer by high-performance liquid chromatography. No evidence for the presence of the exo isomer was obtained. It can thus be concluded that the Sp diastereomer of ADPalphaS polymerizes with inversion of configuration at phosphorus without racemization to give a phosphorothioate diester bond with the Rp configuration.