The reaction catalyzed by adenosine kinase purified from human erythrocytes proceeds via a classical ordered sequential mechanism in which adenosine is the first substrate to bind to and AMP is the last product to dissociate from the enzyme. However, the interpretation of the steady-state kinetic data is complicated by the finding that while AMP acts as a classical product inhibitor at concentrations greater than 5 mM, at lower concentrations AMP can act as an apparent activator of the enzyme under certain conditions. This apparent activation by AMP is proposed to be due to AMP allowing the enzyme mechanism to proceed via an alternative reaction pathway that avoids substrate inhibition by adenosine. Quantitative studies of the protection of the enzyme afforded by adenosine against both spontaneous and 5,5'-dithiobis(2-nitrobenzoic acid)-mediated oxidation of thiol groups yielded "protection" constants (equivalent to enzyme-adenosine dissociation constant) of 12.8 microM and 12.6 microM, respectively, values that are more than an order of magnitude greater than the dissociation constant (Kia = 0.53 microM) for the "catalytic" enzyme-adenosine complex. These results suggest that adenosine kinase has at least two adenosine binding sites, one at the catalytic center and another quite distinct site at which binding of adenosine protects the reactive thiol group(s). This "protection" site appears to be separate from the nucleoside triphosphate binding site, and it also appears to be the site that is responsible for the substrate inhibition caused by adenosine.

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The intracellular contents of nucleotides and 5-phosphoribosyl l-pyrophosphate (P-Rib-PP) in cells of Escherichia coli were quantitatively determined under various conditions of growth. When the growth rate was limited by the carbon source available in the growth medium the intracellular content of each of ATP, CTP, G!rP, UTP, ADP and GDP was proportional to the growth rate. The intracellular content of P-Rib-PP showed a similar, though stronger dependence, suggesting that a t the slower growth rates, the availability of P-Rib-PP could become limiting for nucleotide biosynthesis.Several treatments which are known to specifically stimulate the rate of net RNA synthesis in E. coli had a common effect in causing a transient depletion of intracellular ribonucleoside triphosphates. For each treatment, the depletion of triphosphates coincided with transiently elevated levels of P-Rib-PP, despite increases in the ADP : ATP ratio, an effect which might be expected to inhibit P-Rib-PP synthetase activity in E . coli (Atkinson and Fall [15], Klungssyr et al.[lS]). Explanations of this apparent anomaly might be made on the basis of the properties of P-Rib-PP synthetase from 8almonella typhimurium, (Switzer, [22], Switzer and Sogin, [21]). One explanation is that the enzyme is regulated by total concentrations of nucleotides so that decreases in nucleoside triphosphate concentrations result in stimulation of the reaction. Another is that the decrease in nucleoside triphosphate concentrations releases from chelation a pulse of free Ng2+ which stimulates the synthetase. And thirdly, the concentration of the substrate, ribose 5-phosphate, might be initially high enough to exert its inhibitory effect on the enzyme, so that stimulation of the reaction can occur if the substrate concentration falls.Evaluation of the regulatory mechanisms which have been proposed for control of nucleotide metabolism in. vivo in bacteria requires a knowledge of the intracellular concentrations of various substrates and possible effectors of enzyme activity, and how these concentrations vary with changes in the conditions to which the bacteria are subjected. Few reliable quantitative data of this type are currently available, and consequently, the functioning in vivo of many postulated regulatory mechanisms has little supporting evidence.We have used the thin-layer chromatographic techniques developed by Randerath and coworkers [l, 21 to

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It has been reported that supplying preformed purine and pyrimidine bases and nucleosides to growing cells of Escherichia coli and other organisms results in the preferential utilization of these precursors for nucleotide biosynthesis, but the factors responsible for inhibition of the de novo pathways in vivo are not known. We have surveyed the effects of addition of purine and pyrimidine bases and nucleosides on the intracellular contents of nucleotides and 5-phosphoribosyl I-pyrophosphate (P-Rib-PP) of Escherichia coli in an attempt to determine what some of these factors might be. The results show that initially adenine is converted mainly to ATP, guanine mainly to GTP and uracil mainly to UTP, while hypoxanthine is converted to both ATP and GTP.The corresponding nucleosides have similar effects except for adenosine, whose effects are similar to those of hypoxanthine and inosine. All purine precursors, except for guanosine, deplete the intracellular content of P-Rib-PP to loo/, or less of its normal value. This finding indicates that the limitation in the concentration of P-Rib-PP for the first reaction of the de novo pathway is an important factor in the inhibition of that pathway when a preformed purine is available to support a salvage pathway of purine nucleotide biosynthesis. Our results also suggest that the decreased availability of P-Rib-PP may also cause immediate transient decreases observed in the levels of CTP and UTP, after the addition of a preformed purine precursor.Ura,cil as a pyrimidine precursor causes only a slight depletion of P-Rib-PP while uridine causes an initial increase in the P-Rib-PP content. It is concluded, therefore, that an increase in feedback inhibition, caused by elevated concentrations of uridine nucleotides, may be a major factor causing the inhibition of de novv biosynthesis of pyrimidine nucleotides when preformed pyrimidines are available, or that the inhibition may not be fully effective until repression and growth have diluted out the enzymes for the de novo biosynthesis.In most microorganisms, purine and pyrimidine nucleotides may be synthesized either from simpler metabolites via the de novo pathways, or from preformed bases and nucleosides when these precursors are available [1,2]. While much effort has been spent to elucidate the various enzymatic reactions of these pathways and the factors controlling the synthesis of the individual enzymes (repression-derepression) and their activities (feedback inhibition and allosteric activation) it cannot be said that the process of regulation is well understood for any complete Abbreviations. P-Rib-PP, 5-phosphoribosyl l-pyrophosphate ; PEI-cellulose, polyethyleneimine-cellulose.Enzymes. Adenine phosphoribosyltransferase (EC 2.4.2.7) aspartate transcarbamylase (EC 2.1.3.2) ; P-Rib-PP amidotransferase (EC 2.4.2.14) ; oarbamyl phosphate synthethase (EC 2.7.2.5); CTP synthetase (EC 6.3.4.2); GMP reductase (EC 1.6.6.8); orotate phosphoribosyltransferase (EC 2.4.2.10); P-Rib-PP synthetase (EC 2.7.6.1) uracil phosphoribosyltrans...