Raymond L. Blakley

Regulation of ribonucleotide reductase activity and its possible exploitation in chemotherapy

Abstract Although 2′-deoxyadenosine inhibits the growth of many organisms it is not inhibitory for Lactobacillus leichmannii. This is consistent with the presence of a powerful trans-N-deoxyribosylase in this organism, as well as the absence of any inhibitory effects of dATP on its ribonucleotide reductase. Specific activating effects of deoxyribonucleoside triphosphates on the reduction of ribonucleotides catalyzed by the L. leichmannii reductase did not act to equalize the production of pyrimidine and purine deoxyribonucleotides when GTP, ATP, CTP and UTP were reduced simultaneously in vitro. Under all conditions investigated dGTP and dATP were formed at a much greater rate than dCTP and dUTP. Deoxytubercidin has an ED50 for CCRF-CEM cells considerably lower than that for deoxyadenosine and only slightly higher than that for deoxyadenosine in the presence of an inhibitor of adenosine deaminase. This observation suggests that other analogs of deoxyadenosine may efficiently inhibit mammalian cell proliferation via conversion to dATP analogs which inhibit mammalian ribonucleotide reductase.

Allosterism, regulation and cooperativity: The case of ribonucleotide reductase of Lactobacilus leichmannii

Abstract The binding of modifiers and substrates to the ribonucleotide reductase from Lactobacillus leichmannii has been studied with the use of enzyme highly purified by a modified preparative procedure. This monomeric enzyme represents a simpler system than classical allosteric enzymes since the study of allosterism, regulation, and conformational responses in the latter is complicated by interaction between subunits. Analysis of earlier kinetic results indicates that ribonucleotide substrates bind weakly to the regulatory site (KD for GTP, 4 m m ) compared with binding to the catalytic site (KD for GTP 0.24 m m ), and in so doing produce substrate activation. The apparent rate constant for product formation when GTP is not bound to the regulatory site is 31% of that observed when GTP occupies the regulatory site. Part of this effect is due to tighter binding of coenzyme when substrate is bound to the regulatory site, a result confirmed by direct measurement of adenosylcobalamin binding. The effect of modifiers such as dGTP which accelerate the reduction of specific substrates is also in part due to increased coenzyme binding as measured both kinetically and by equilibrium dialysis and ultrafiltration methods. The modifiers also seem to have an obligatory role in the enzyme mechanism. Without their presence neither formation of the radical-pair intermediate from adenosylcobalamin nor associated reactions (coenzyme degradation, coenzyme H-exchange with water) occur under the experimental conditions observed. When modifier concentration dependence for these reactions is compared with estimates of equilibrium constants for dissociation of modifiers and substrates from complexes with the regulatory and catalytic sites it appears likely that binding at the regulatory site is responsible. Calculation of coenzyme saturation under the experimental conditions indicates, however, that insignificant amounts of coenzyme are bound to the enzyme except when the regulatory site is occupied, and this effect adequately explains the necessity for modifiers. Binding of dGTP does not alter the circular dichroism of the enzyme in the far-ultraviolet region (200 to 250 nm) but causes significant changes in the aromatic region, and therefore appears to perturb tryptophyl and tyrosyl residues though not necessarily directly. Measurements have been made of the fractional change in sedimentation coefficient due to dGTP binding to the enzymes by the use of an adjustable Raleigh mask and double sector cell in the Beckman model E ultracentrifuge. Values for Δs/ s of −1.14% at 22° and −1.16% at 37° were obtained. The former, after correction for the fractional increase due to the calculated effect of dGTP on the buoyant weight of the protein, gives a value of (Δs/ s corr = −2.84% . If this is due to increase in hydrodynamic volume of the protein alone, it corresponds to an 8.8% increase. If it were entirely due to increased departure from spherical shape it would correspond to a change in axial ratio from 2.9 to 3.2. In the presence of saturating concentrations of ATP and dihydrolipoate and the maximum permissible concentration of a coenzyme analogue Δs/ s was −0.33%, the corrected value being about −1.99% at 37°. These conformational responses are as great as determined for multimeric enzymes such as in the effect of CTP on aspartate transcarbamylase, and are consistent with relay of the regulatory signal over a considerable intersite distance.

A 13C nuclear magnetic resonance study of the interaction of ligands with arginine residues in dihydrofolate reductase

/sup 13/C NMR spectra of Streptococcus faecium dihydrofolate reductase containing (/sup 13/C-guanidino) arginine and ligand complexes with the labeled enzyme are reported. The spectrum of the native enzyme shows 5 well-resolved resonances (the enzyme contains 8 Arg) with a chemical shift range of 1.2 ppM. Addition of ligands to the enzyme produces distinct changes in the enzyme spectrum, and demonstrates that /sup 13/C NMR of labeled protein can be used in studies of protein-ligand interactions. An example of the use of /sup 13/C-depleted material is also presented.

Comparative effects of selected antifolates on transforming human lymphocytes and on established human lymphoblastic cell lines

Abstract When 15 nM methotrexate was added to the medium in which human peripheral lymphocytes stimulated with phytohemagglutinin were incubated, it caused a 50 per cent decrease in the maximum number of blasts produced, in the number of cells in mitosis and in the incorporation of [ 3 H]deoxyuridine into DNA. However, [ 3 H]thymidine incorporation into DNA was increased by methotrexate concentrations up to 0.5 mM. When 50 nM methotrexate was present continuously, blast formation, mitosis and deoxyuridine incorporation were virtually abolished, but if this concentration was present only during the induction phase (the first 24 hr), the subsequent effect on blast proliferation was slight. In contrast, 24-hr exposure during the proliferative phase (days 3–5) severely affected blast proliferation. The effects of methotrexate were largely reversible by thymidine, but hypoxanthine or purine nucleosides had no significant effect so that the metabolic block appears to be entirely at thymidylate synthetase under the experimental conditions. The inhibitory effects of ten other antifolates on transforming lymphocytes were determined and, with one exception, their relative effectiveness was found to be as predicted from inhibitory effects on highly purified bovine dihydrofolate reductase. The growth of four established lines of human lymphoblastic cells was inhibited to essentially the same extent by six of the antifolates, and these cells were only slightly less sensitive to the antifolates than were the transforming normal lymphocytes.

Detection by electron spin resonance of an exchange-coupled Cob(II)alamin...free radical pair species generated by anaerobic photolysis of polycrystalline adenosylcobalamin.

Summary Laser induced photolysis of highly purified polycrystalline adenosylcobalamin has been studied under anaerobic conditions. Electron spin resonance of photolyzed samples of adenosylcobalamin were studied versus a convenient new variable: the degree of hydration of the crystalline state. Photolysis of partially dehydrated samples of adenosylcobalamin leads to a reproducible, new type of electron spin resonance signal which is interpreted as an exchange coupled Co(II)…free radical species. We conclude that this new species may be the Cob(II)alamin…5′-deoxyadenosyl radical pair.

Molecular Basis for the Interaction of Polyglutamates of Folic Acid and its Analogs with Dihydrofolate Reductase

Fluorimetric titration has been used to measure the dissociation constants for the complexes of folate, pteroyltriglutamate and pteroylheptaglutamate with dihydrofolate reductase purified from Lactobacillus casei, Streptococcus faecium (isoenzyme 2) and bovine liver. Effects of pH, temperature, salt concentration and second ligands have been examined. The method is shown to be unsuitable for methotrexate complexes. The polyglutamates do not bind more tightly than folate to the S. faecium reductase under any conditions examined, but bind somewhat more tightly than folate to the L. casei reductase at low pH (less than 7) and to the bovine liver enzyme at pH 7-9. Increasing concentrations of KC1 decrease the binding of all three ligands to the L. casei and bovine liver enzymes. Increasing pH markedly raises the dissociation constants for all complexes of the L. casei reductase, but has only slight effects on the complexes of the S. faecium reductase. Complexes of the bovine enzyme are affected to an intermediate degree by pH, but the folate complex is affected much more than those of the polyglutamates. Model building studies have been performed with a three-dimensional model of the complex of L. casei reductase with NADPH and methotrexate. Additional glutamyl groups were added in gamma-linkage to the glutamate moiety of the complexed methotrexate. A proposed mode of binding of the pteroyl polyglutamates is discussed and sequence comparisons are used to predict residues that might be involved in polyglutamate binding by reductase from other sources.