Characterization of a novel AICARFT inhibitor which potently elevates ZMP and has anti-tumor activity in murine models.

AICARFT is a folate dependent catalytic site within the ATIC gene, part of the purine biosynthetic pathway, a pathway frequently upregulated in cancers. LSN3213128 is a potent (16 nM) anti-folate inhibitor of AICARFT and selective relative to TS, SHMT1, MTHFD1, MTHFD2 and MTHFD2L. Increases in ZMP, accompanied by activation of AMPK and cell growth inhibition, were observed with treatment of LY3213128. These effects on ZMP and proliferation were dependent on folate levels. In human breast MDA-MB-231met2 and lung NCI-H460 cell lines, growth inhibition was rescued by hypoxanthine, but not in the A9 murine cell line which is deficient in purine salvage. In athymic nude mice, LSN3213128 robustly elevates ZMP in MDA-MB-231met2, NCI-H460 and A9 tumors in a time and dose dependent manner. Significant tumor growth inhibition in human breast MDA-MB231met2 and lung NCI-H460 xenografts and in the syngeneic A9 tumor model were observed with oral administration of LSN3213128. Strikingly, AMPK appeared activated within the tumors and did not change even at high levels of intratumoral ZMP after weeks of dosing. These results support the evaluation of LSN3213128 as an antineoplastic agent.

Modulation of the de novo purine nucleotide pathway as a therapeutic strategy in mitochondrial myopathy.

Mitochondrial myopathy (MM) is characterised by muscle weakness, exercise intolerance and various histopathological changes. Recently, a subset of MM has also been associated with aberrant activation of mammalian target of rapamycin complex 1 (mTORC1) in skeletal muscle. This aberrant mTORC1 activation promotes increased de novo nucleotide synthesis, which contributes to abnormal expansion and imbalance of skeletal muscle deoxyribonucleoside triphosphates (dNTP) pools. However, the exact mechanism via which mTORC1-stimulated de novo nucleotide biosynthesis ultimately disturbs muscle dNTP pools remains unclear. In this article, it is proposed that mTORC1-stimulated de novo nucleotide synthesis in skeletal muscle cells with respiratory chain dysfunction promotes an asymmetric increase of purine nucleotides, probably due to NAD+ deficiency. This in turn could disrupt purine nucleotide-dependent allosteric feedback regulatory mechanisms, ultimately leading to dNTP pools aberration. Pharmacological down-modulation of aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) activity is also proposed as a potential therapeutic strategy in MM exhibiting mTORC1-driven abnormal metabolic reprogramming, including aberrant dNTPs pools.

Identification of ATIC as a Novel Target for Chemoradiosensitization.

PURPOSE: Mutations in the gene encoding 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), a bifunctional enzyme that catalyzes the final 2 steps of the purine de novo biosynthetic pathway, were identified in a subject referred for radiation sensitivity testing. Functional studies were performed to determine whether ATIC inhibition was radiosensitizing and, if so, to elucidate the mechanism of this effect and determine whether small molecule inhibitors of ATIC could act as effective radiosensitizing agents. METHODS AND MATERIALS: Both small interfering RNA knockdown and small molecule inhibitors were used to inactivate ATIC in cell culture. Clonogenic survival assays, the neutral comet assay, and γH2AX staining were used to assess the effects of ATIC inhibition or depletion on cellular DNA damage responses. RESULTS: Depletion of ATIC or inhibition of its transformylase activity significantly reduced the surviving fraction of cells in clonogenic survival assays in multiple cancer cell lines. In the absence of ionizing radiation exposure, ATIC knockdown or chemical inhibition activated cell cycle checkpoints, shifting cells to the more radiosensitive G2/M phase of the cell cycle, and depleted cellular adenosine triphosphate but did not result in detectable DNA damage. Cells in which ATIC was knocked down or inhibited and then treated with ionizing radiation displayed increased numbers of DNA double-strand breaks and a delay in the repair of those breaks relative to irradiated, but otherwise untreated, controls. Supplementation of culture media with exogenous adenosine triphosphate ameliorated the DNA repair phenotypes. CONCLUSIONS: These findings implicate ATIC as an effective, and previously unrecognized, target for chemoradiosensitization and, more broadly, suggest that purine levels in cells might have an underappreciated role in modulating the efficiency of DNA damage responses that could be exploited in radiosensitizing strategies.

Structural Insights into Mycobacterium tuberculosis Rv2671 Protein as a Dihydrofolate Reductase Functional Analogue Contributing to para-Aminosalicylic Acid Resistance.

Mycobacterium tuberculosis (Mtb) Rv2671 is annotated as a 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate (AROPP) reductase (RibD) in the riboflavin biosynthetic pathway. Recently, a strain of Mtb with a mutation in the 5' untranslated region of Rv2671, which resulted in its overexpression, was found to be resistant to dihydrofolate reductase (DHFR) inhibitors including the anti-Mtb drug para-aminosalicylic acid (PAS). In this study, a biochemical analysis of Rv2671 showed that it was able to catalyze the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF), which explained why the overexpression of Rv2671 was sufficient to confer PAS resistance. We solved the structure of Rv2671 in complex with the NADP(+) and tetrahydrofolate (THF), which revealed the structural basis for the DHFR activity. The structures of Rv2671 complexed with two DHFR inhibitors, trimethoprim and trimetrexate, provided additional details of the substrate binding pocket and elucidated the differences between their inhibitory activities. Finally, Rv2671 was unable to catalyze the reduction of AROPP, which indicated that Rv2671 and its closely related orthologues are not involved in riboflavin biosynthesis.

AMPK Activation via Modulation of De Novo Purine Biosynthesis with an Inhibitor of ATIC Homodimerization.

5-Aminoimidazole-4-carboxamide ribonucleotide (known as ZMP) is a metabolite produced in de novo purine biosynthesis and histidine biosynthesis, but only utilized in the cell by a homodimeric bifunctional enzyme (called ATIC) that catalyzes the last two steps of de novo purine biosynthesis. ZMP is known to act as an allosteric activator of the cellular energy sensor adenosine monophosphate-activated protein kinase (AMPK), when exogenously administered as the corresponding cell-permeable ribonucleoside. Here, we demonstrate that endogenous ZMP, produced by the aforementioned metabolic pathways, is also capable of activating AMPK. Using an inhibitor of ATIC homodimerization to block the ninth step of de novo purine biosynthesis, we demonstrate that the subsequent increase in endogenous ZMP activates AMPK and its downstream signaling pathways. We go on to illustrate the viability of using this approach to AMPK activation as a therapeutic strategy with an in vivo mouse model for metabolic disorders.

The enzymatic activity of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase is enhanced by NPM-ALK: new insights in ALK-mediated pathogenesis and the treatment of ALCL.

Anaplastic large cell lymphoma represents a subset of neoplasms caused by translocations that juxtapose the anaplastic lymphoma kinase (ALK) to dimerization partners. The constitutive activation of ALK fusion proteins leads to cellular transformation through a complex signaling network. To elucidate the ALK pathways sustaining lymphomagenesis and tumor maintenance, we analyzed the tyrosine-kinase protein profiles of ALK-positive cell lines using 2 complementary proteomic-based approaches, taking advantage of a specific ALK RNA interference (RNAi) or cell-permeable inhibitors. A well-defined set of ALK-associated tyrosine phosphopeptides, including metabolic enzymes, kinases, ribosomal and cytoskeletal proteins, was identified. Validation studies confirmed that vasodilator-stimulated phosphoprotein and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) associated with nucleophosmin (NPM)-ALK, and their phosphorylation required ALK activity. ATIC phosphorylation was documented in cell lines and primary tumors carrying ALK proteins and other tyrosine kinases, including TPR-Met and wild type c-Met. Functional analyses revealed that ALK-mediated ATIC phosphorylation enhanced its enzymatic activity, dampening the methotrexate-mediated transformylase activity inhibition. These findings demonstrate that proteomic approaches in well-controlled experimental settings allow the definition of informative proteomic profiles and the discovery of novel ALK downstream players that contribute to the maintenance of the neoplastic phenotype. Prediction of tumor responses to methotrexate may justify specific molecular-based chemotherapy.

Mechanism of dTTP inhibition of the bifunctional dCTP deaminase:dUTPase encoded by Mycobacterium tuberculosis.

Recombinant deoxycytidine triphosphate (dCTP) deaminase from Mycobacterium tuberculosis was produced in Escherichia coli and purified. The enzyme proved to be a bifunctional dCTP deaminase:deoxyuridine triphosphatase. As such, the M. tuberculosis enzyme is the second bifunctional enzyme to be characterised and provides evidence for bifunctionality of dCTP deaminase occurring outside the Archaea kingdom. A steady-state kinetic analysis revealed that the affinity for dCTP and deoxyuridine triphosphate as substrates for the synthesis of deoxyuridine monophosphate were very similar, a result that contrasts that obtained previously for the archaean Methanocaldococcus jannaschii enzyme, which showed approximately 10-fold lower affinity for deoxyuridine triphosphate than for dCTP. The crystal structures of the enzyme in complex with the inhibitor, thymidine triphosphate, and the apo form have been solved. Comparison of the two shows that upon binding of thymidine triphosphate, the disordered C-terminal arranges as a lid covering the active site, and the enzyme adapts an inactive conformation as a result of structural changes in the active site. In the inactive conformation dephosphorylation cannot take place due to the absence of a water molecule otherwise hydrogen-bonded to O2 of the alpha-phosphate.

Methotrexate enhances the antianabolic and antiproliferative effects of 5-aminoimidazole-4-carboxamide riboside.

Because of its ability to mimic a low energy status of the cell, the cell-permeable nucleoside 5-aminoimidazole-4-carboxamide (AICA) riboside was proposed as an antineoplastic agent switching off major energy-consuming processes associated with the malignant phenotype (lipid production, DNA synthesis, cell proliferation, cell migration, etc.). Key to the antineoplastic action of AICA riboside is its conversion to ZMP, an AMP mimetic that at high concentrations activates the AMP-activated protein kinase (AMPK). Here, in an attempt to increase the efficacy of AICA riboside, we pretreated cancer cells with methotrexate, an antimetabolite blocking the metabolism of ZMP. Methotrexate enhanced the AICA riboside-induced accumulation of ZMP and led to a decrease in the levels of ATP, which functions as an intrasteric inhibitor of AMPK. Consequently, methotrexate markedly sensitized AMPK for activation by AICA riboside and potentiated the inhibitory effects of AICA riboside on tumor-associated processes. As cotreatment elicited antiproliferative effects already at concentrations of compounds that were only marginally effective when used alone, our findings on the cooperation between methotrexate and AICA riboside provide new opportunities both for the application of classic antimetabolic chemotherapeutics, such as methotrexate, and for the exploitation of the energy-sensing machinery as a target for cancer intervention.

Structural insights into the human and avian IMP cyclohydrolase mechanism via crystal structures with the bound XMP inhibitor.

Within de novo purine biosynthesis, the AICAR transformylase and IMP cyclohydrolase activities of the bifunctional enzyme ATIC convert the intermediate AICAR to the final product of the pathway, IMP. Identification of the AICAR transformylase active site and a proposed formyl transfer mechanism have already resulted from analysis of crystal structures of avian ATIC in complex with substrate and/or inhibitors. Herein, we focus on the IMPCH active site and the cyclohydrolase mechanism through comparison of crystal structures of XMP inhibitor complexes of human ATIC at 1.9 A resolution with the previously determined avian enzyme. This first human ATIC structure was also determined to ascertain whether any subtle structural differences, compared to the homologous avian enzyme, should be taken into account for structure-based inhibitor design. These structural comparisons, as well as comparative analyses with other IMP and XMP binding proteins, have enabled a catalytic mechanism to be formulated. The primary role of the IMPCH active site appears to be to induce a reconfiguration of the substrate FAICAR to a less energetically favorable, but more reactive, conformer. Backbone (Arg64 and Lys66) and side chain interactions (Thr67) in the IMPCH active site reorient the 4-carboxamide from the preferred conformer that binds to the AICAR Tfase active site to one that promotes intramolecular cyclization. Other backbone amides (Ile126 and Gly127) create an oxyanion hole that helps orient the formyl group for nucleophilic attack by the 4-carboxamide amine and then stabilize the anionic intermediate. Several other residues, including Lys66, Tyr104, Asp125, and Lys137', provide substrate specificity and likely enhance the catalytic rate through contributions to acid-base catalysis.

Human AICAR transformylase: role of the 4-carboxamide of AICAR in binding and catalysis.

We have prepared 4-substituted analogues of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to investigate the specificity and mechanism of AICAR transformylase (AICAR Tfase). Of the nine analogues of AICAR studied, only one analogue, 5-aminoimidazole-4-thiocarboxamide ribonucleotide, was a substrate, and it was converted to 6-mercaptopurine ribonucleotide. The other analogues either did not bind or were competitive inhibitors, the most potent being 5-amino-4-nitroimidazole ribonucleotide with a K(i) of 0.7 +/- 0.5 microM. The results show that the 4-carboxamide of AICAR is essential for catalysis, and it is proposed to assist in mediating proton transfer, catalyzing the reaction by trapping of the addition compound. AICAR analogues where the nitrogen of the 4-carboxamide was derivatized with a methyl or an allylic group did not bind AICAR Tfase, as determined by pre-steady-state burst kinetics; however, these compounds were potent inhibitors of IMP cyclohydrolase (IMP CHase), a second activity of the bifunctional mammalian enzyme (K(i) = 0.05 +/- 0.02 microM for 4-N-allyl-AlCAR). It is proposed that the conformation of the carboxamide moiety required for binding to AICAR Tfase is different than the conformation required for binding to IMP CHase, which is supported by inhibition studies of purine ribonucleotides. It is shown that 5-formyl-AICAR (FAICAR) is a product inhibitor of AICAR Tfase with K(i) of 0.4 +/- 0.1 microM. We have determined the equilibrium constant of the transformylase reaction to be 0.024 +/- 0.001, showing that the reaction strongly favors AICAR and the 10-formyl-folate cofactor. The coupling of the AICAR Tfase and IMP CHase activities on a single polypeptide allows the overall conversion of AICAR to IMP to be favorable by coupling the unfavorable formation of FAICAR with the highly favorable cyclization reaction. The current kinetic studies have also indicated that the release of FAICAR is the rate-limiting step, under steady-state conditions, in the bifunctional enzyme and channeling is not observed between AICAR Tfase and IMP CHase.

Inosine-5'-monophosphate analogues as inhibitors of human IMP cyclohydrolase and cellular growth.

The catalytic mechanism for the enzyme, IMP cyclohydrolase, may involve a reaction intermediate with negative charge in the 2-position of the purine ring (Szabados, E., Hindmarsh, E., Phillips, L., Duggleby, R.G. & Christopherson, R.I. (1994) Biochemistry 33, 14237-14245). Three analogues of IMP have been synthesised where fluorine, chlorine or bromine has been substituted in the 2-position on the purine ring. These analogues with an electronegative substituent may resemble a reaction intermediate for IMP cyclohydrolase; 2-fluoro IMP is a potent inhibitor of the enzyme with a Ki value of 0.19 microM, while 2-chloro IMP has a Ki of 1.9 microM and 2-bromo IMP is not inhibitory. However, IMP cyclohydrolase is not inhibited in human CCRF-CEM leukaemia cells exposed to 2-fluoro inosine although it is toxic to these cells with an IC50 value of 4.9 microM.

5-Aminoimidazole-4-carboxamide ribotide transformylase-IMP cyclohydrolase from human CCRF-CEM leukemia cells: purification, pH dependence, and inhibitors.

The bifunctional enzyme 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase-IMP cyclohydrolase has been purified 780-fold to apparent homogeneity from human CCRF-CEM leukemia cells, completed with chromatography on Affi-Gel Blue followed by AICAR-Sepharose 4B. Using a sensitive radioassay, IMP cyclohydrolase has a Ks value for 5-formamidoimidazole-4-carboxamide ribotide (FAICAR) at pH 7.4 of 0.87 +/- 0.11 microM. The following purine nucleotide derivatives were potent competitive inhibitors of IMP cyclohydrolase: 2-mercaptoinosine 5'-monophosphate (Ki = 0.094 +/- 0.024 microM), xanthosine 5'-monophosphate (Ki = 0.12 +/- 0.01 microM), 2-fluoroadenine arabinoside 5'-monophosphate (Ki = 0.16 +/- 0.02 microM), 6-mercaptopurine riboside 5'-monophosphate (Ki = 0.20 +/- 0.02 microM), adenosine N1-oxide 5'-monophosphate (Ki = 0.28 +/- 0.03 microM), and N6-(carboxymethyl)adenosine 5'-monophosphate (Ki = 1.7 +/- 0.42 microM). The pH dependencies of Vmax and Vmax/Ks values for IMP cyclohydrolase are consistent with a single ionizable amino acid residue (pKa = 7.57 +/- 0.09) of the enzyme which must be unprotonated for catalysis to occur and a residue (pKa = 7.57 +/- 0.14) which must be unprotonated for FAICAR to bind. The pKa values of 5.81 +/- 0.03 and 9.41 +/- 0.04 determined for FAICAR indicate that ionization of the substrate does not contribute significantly to the pH effects observed. Chemical modification of IMP cyclohydrolase provides evidence for arginine and cysteine residues at the active site, and roles for these residues in the mechanism of catalysis are proposed.

Modulation of deoxynucleotide metabolism by the deoxycytidylate deaminase inhibitor 3,4,5,6-tetrahydrodeoxyuridine.

Tetrahydrodeoxyuridine (dTHU) inhibits deoxycytidine deaminase and, after intracellular phosphorylation to the active 5'-monophosphate, also inhibits deoxycytidylate deaminase (dCMPD). Because in vitro studies have shown that dCMPD may regulate pyrimidine deoxynucleotide metabolism, the objective of this study was to investigate the effects of dTHU on deoxynucleotide metabolism in whole cells. Nearly complete inhibition of dCMPD, measured in intact CCRF-CEM cells by incorporation of [14C]dCyd into dTTP, occurred after a 45-min incubation with 100 microM dTHU. This was accompanied by an 8-fold dCTP pool expansion, although dATP, dTTP, dGTP, and ribonucleoside triphosphate pools were unaffected. Tetrahydrouridine, which inhibits deoxycytidine deaminase exclusively, had no effect on nucleotide pools. The dCTP pool expansion was directly proportional to the dTHU concentration (3-100 microM) and reached a maximum after 2 hr. Inhibition of ribonucleotide reductase by hydroxyurea completely prevented the dTHU-induced dCTP pool expansion, indicating that the substrate of dCMPD was derived from the ribonucleotide pool and that CDP was the predominant precursor of dCTP. dTHU-mediated inhibition of dCMPD appeared reversible. Exposure of cells to 100 microM dTHU followed by washing into fresh medium resulted in a linear decrease of the dCTP pool and an increase in the dTTP pool. The increased dCTP concentration after preincubation with dTHU was associated with an inhibition of deoxycytidine kinase, as indicated by a reduced capacity of cells to phosphorylate ara-C. dTHU is a useful new tool for investigating the role of dCMPD in the regulation of deoxynucleotide metabolism in whole cells.

The influence of phosphatidate bilayers on pig heart AMP deaminase. Crucial role of pH-dependent lipid-phase transition.

Phosphatidate bilayers composed of dilauroylphosphatidate, dimyristoylphosphatidate, dipalmitoylphosphatidate and dioleoylphosphatidate were prepared. Their interaction with AMP deaminase isolated from pig heart was investigated. Dioleoylphosphatidate bilayers were found to exert non-competitive inhibition on the AMP deaminase with a Ki of 15 x 10(-6) M. This inhibition is three orders of magnitude stronger than that exerted by orthophosphate. The phosphatidate species containing saturated fatty acids were either non-inhibitory or inhibited enzyme activity rather poorly. However, alkalinization of the medium from pH 6.5 to pH 7.9 led to the inhibition of pig heart AMP deaminase by dilauroylphosphatidate bilayers. This was accompanied by the fluidization of the saturated phosphatidate species, i.e. the lowering of their phase transition temperature in alkaline pH, as measured by light-scattering and fluorescence scans. The possible significance of these findings for the regulation of AMP deaminase activity in vivo by natural membranes is discussed.

A kinetic study of the inhibition of yeast AMP deaminase by polyphosphate.

Inorganic pyrophosphate and polyphosphates have acted as potent inhibitors of purified AMP deaminase (EC 3.5.4.6) from yeast: the activity fell to a definite limit with the increase in the concentration of the inhibitor. The effect of polyphosphate was largely on the maximal velocity of the enzyme with some decrease in affinity. The cooperative effect of AMP, analyzed in terms of a Hill coefficient, remained at 2 in the absence and presence of polyphosphate. Binding of polyphosphate to the enzyme showed no cooperativity. The inhibition of AMP deaminase by polyphosphate can be qualitatively and quantitatively accounted for by the partial mixed-type inhibition mechanism. Both the Ki value for the inhibitor and the breakdown rate of the enzyme-substrate-inhibitor complex are dependent on the chain length of polyphosphate, suggesting that the breakdown rate of the enzyme-substrate-inhibitor complex is regulated by binding of polyphosphate to a specific inhibitory site.

Inhibition of 5-aminoimidazole-4-carboxamide ribotide transformylase, adenosine deaminase and 5'-adenylate deaminase by polyglutamates of methotrexate and oxidized folates and by 5-aminoimidazole-4-carboxamide riboside and ribotide.

With the use of a continuous spectrophotometric assay and initial rates determined by the method of Waley [Biochem. J. (1981) 193, 1009-1012] methotrexate was found to be a non-competitive inhibitor, with Ki(intercept) = 72 microM and Ki(slope) = 41 microM, of 5-aminoimidazole-4-carboxamide ribotide transformylase, whereas a polyglutamate of methotrexate containing three gamma-linked glutamate residues was a competitive inhibitor, with Ki = 3.15 microM. Pentaglutamates of folic acid and 10-formylfolic acid were also competitive inhibitors of the transformylase, with Ki values of 0.088 and 1.37 microM respectively. Unexpectedly, the pentaglutamate of 10-formyldihydrofolic acid was a good substrate for the transformylase, with a Km of 0.51 microM and a relative Vmax. of 0.72, which compared favourably with a Km of 0.23 microM and relative Vmax. of 1.0 for the tetrahydro analogue. An analysis of the progress curve of the transformylase-catalysed reaction with the above dihydro coenzyme revealed that the pentaglutamate of dihydrofolic acid was a competitive product inhibitor, with Ki = 0.14 microM. The continuous spectrophotometric assay for adenosine deaminase based on change in the absorbance at 265 nm was shown to be valid with adenosine concentrations above 100 microM, which contradicts a previous report [Murphy, Baker, Behling & Turner (1982) Anal. Biochem. 122, 328-337] that this assay was invalid above this concentration. With the spectrophotometric assay, 5-aminoimidazole-4-carboxamide riboside was found to be a competitive inhibitor of adenosine deaminase, with (Ki = 362 microM), whereas the ribotide was a competitive inhibitor of 5'-adenylate deaminase, with Ki = 1.01 mM. Methotrexate treatment of susceptible cells results in (1) its conversion into polyglutamates, (2) the accumulation of oxidized folate polyglutamates, and (3) the accumulation of 5-aminoimidazole-4-carboxamide riboside and ribotide. The above metabolic events may be integral elements producing the cytotoxic effect of this drug by (1) producing tighter binding of methotrexate to folate-dependent enzymes, (2) producing inhibitors of folate-dependent enzymes from their tetrahydrofolate coenzymes, and (3) trapping toxic amounts of adenine nucleosides and nucleotides as a result of inhibition of adenosine deaminase and 5'-adenylate deaminase respectively.

Probing the infra-structure of thymidylate synthase and deoxycytidylate deaminase.

Methods are described for preparing and structurally analyzing two enzymes involved in the formation of dTMP, deoxycytidylate deaminase and thymidylate synthase. In the latter case, it has been possible through the use of recombinant DNA techniques with an amplification plasmid to obtain sufficient amounts of the E. coli and T4-phage synthases to complete the entire sequence of both enzymes by employing a combination of protein and DNA sequencing methods. A comparative analysis of the L. casei and E. coli synthases has revealed a 62% conservation of sequences but an even greater homology in their hydrophobic active site regions (82%), which are primarily hydrophobic in nature. The homology between these enzymes becomes apparent by deleting a 51 amino acid segment (residues 89-139) from the L. casei synthase, which accounts for the difference in size between these enzymes. Methods for obtaining the binding sites of both substrates are described, one being the activation of the carboxyls of folate with a water soluble carbodiimide and the other, the activation of dUMP by ultraviolet light. The DNA and protein sequence of the T4-phage synthase has recently been clarified by us and is in preparation. Of great interest is the finding by Purohit and Mathews (42), based on our sequence data for the synthase, that the gene segment for the carboxyl terminal end of dihydrofolate reductase overlaps with the amino end of the gene for thymidylate synthase. The complete amino acid sequence of T2-phage deoxycytidylate deaminase has been elucidated by conventional protein sequencing methods. The binding characteristics of this enzyme for its positive allosteric effectors and substrates, as determined by equilibrium dialysis, are consistent with the cooperative nature of its kinetic responses. Consistent with these findings was the demonstration that each of the enzyme's six subunits bound an equivalent amount of substrate or allosteric modifier. Similarly the deaminase showed a marked negative change in ellipticity at 280 nm in response to increasing concentrations of dCTP, changes which could be reversed by dTTP. From the information on the enzyme's primary sequence, it should be possible to define the substrate and allosteric binding regions within the deaminase with the appropriately activated compounds. A start in this direction has been initiated by the finding that dTTP is rapidly and apparently covalently fixed to the amino terminal cyanogen bromide peptide of the enzyme in the presence of ultraviolet light.

Theophylline effect on the cyclic AMP degrading multienzyme sequence.

Membrane-bound 3'.5'-cyclic nucleotide phosphodiesterase (EC 3.1.4.17) is closely associated physically with nucleotidase and deaminase, thus forming an enzyme cluster of unique catalytic behaviour [H. Wombacher, Archs. Biochem. Biophys. 201, 8 (1980)]. This multienzyme cluster, which was found in the microsomal fraction of beef adrenal cortex, catalyses the degradation of cyclic AMP, via AMP and adenosine, to inosine. The present study shows how theophylline, a well-known inhibitor of the phosphodiesterase, acts on the membrane-bound multienzyme sequence. The findings were as follows. Firstly, as expected, theophylline inhibited the phosphodiesterase competitively. In particular, the high-affinity enzyme was inhibited by mM concentrations of theophylline. Phosphodiesterase activity was tentatively ascribed to two enzymes, one with a low Km [0.3 microM], one with a high Km [60 microM]. Secondly, theophylline inhibited the nucleotidase activity to a great extent. A detailed kinetic analysis showed the inhibition to be hyperbolic noncompetitive (alpha = 1, beta = 0.35 and Ki = 0.25 mM). Thirdly, theophylline did not inhibit the deaminase activity of the multienzyme sequence. A model of theophylline inhibition is suggested explaining how an effector could modulate the kinetic behaviour of an enzyme cluster by acting at a single allosteric site. Finally, in view of the existence of the cyclic AMP degrading multienzyme sequence and the effect of theophylline on it, the possibility is discussed that physiologically active adenosine is derived from cyclic AMP.