Adenosine kinase from L1210 cells. Purification and some properties of the enzyme.

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.

Section: Methodsmentioning

confidence: 72%

Adenosine kinase from human erythrocytes: kinetic studies and characterization of adenosine binding sites

Hawkins¹,

Bagnara²

1987

“…Palella et al (1980) studied the kinetic properties of highly purified human placental adenosine kinase and concluded that the reaction proceeds by an ordered bi-bi addition of substrate and release of products in which adenosine is the first substrate to bind and AMP is the last product released. Some properties of adenosine kinase purified from LI210 cells have been reported (Chang et al, 1980). In a further study of this kinase we have found that the enzyme is phosphorylated and that the phosphorylated kinase can transfer phosphate to adenosine in the absence of MgCl2 and ATP; this activity, which interfered with the kinetic and isotope exchange study.…”

mentioning

confidence: 86%

“…Substrate binding, isotope exchange, and kinetic studies suggested that the enzyme catalyzes the reaction by means of a two-site ping-pong mechanism with the phosphorylated enzyme as an obligatory intermediate. Among -A.denosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) has been partially purified from a variety of mammalian sources (Caputto, 1951;Lindberg et al, 1967;Schnebli et al, 1967; Murray, 1968;Lindberg, 1969;Divekar & Hakala, 1971; Henderson et al, 1972; Shimizu et al, 1972;Namm & Leader, 1973;Schmidt et al, 1974;DeJong, 1977) and purified to apparent homogeneity from brewer's yeast (Leibach et al, 1971), rabbit liver (Miller et al, 1979), rat brain (Yamada et al, 1980), murine leukemia L1210 cells (Chang et al, 1980), and human liver (Yamada et al, 1981); 3600-fold purification of the kinase of human placenta was also reported (Andres & Fox, 1979). Adenosine kinase and adenosine deaminase are key enzymes in the metabolism of adenosine and its analogues.…”

mentioning

confidence: 99%

Kinetic studies of adenosine kinase from L1210 cells: a model enzyme with a two-site ping-pong mechanism

Chang¹,

Cha²,

Rw³

et al. 1983

Purified adenosine kinase from L1210 cells displayed substrate inhibition by high concentrations of adenosine (Ado), ATP, and MgCl2. When incubated with ATP and MgCl2, the enzyme was phosphorylated, and the phosphorylated kinase transferred phosphate to adenosine in the absence of ATP and MgCl2. Substrate binding, isotope exchange, and kinetic studies suggested that the enzyme catalyzes the reaction by means of a two-site ping-pong mechanism with the phosphorylated enzyme as an obligatory intermediate. Among many possible pathways within this mechanism probably a random-bi ordered-bi route is the preferred sequence in which the two substrates, adenosine and MgATP, bind in a random order to form the ternary complex MgATP . E . Ado followed by the sequential dissociation of MgADP and AMP. Dissociation constants of various enzyme-substrate and enzyme-product complexes and the first-order rate constant of the rate-limiting step were estimated.

“…but not from HEp-2 carcinoma or murine leukemia cells phosphorylates dAdo and ara-A (Schnebli et al, 1967;Chang et al, 1980). Similarly, partially purified dCyd kinase from calf thymus (Durham & Ives, 1970; Krenitsky et al, 1976) but not from human myeloid or murine leukemia cell phosphorylates dAdo and dGuo (Coleman et al, 1975;Cheng et al, 1977;Brockman et al, 1980).…”

mentioning

confidence: 99%

Identification of purine deoxyribonucleoside kinases from human leukemia cells: substrate activation by purine and pyrimidine deoxyribonucleosides

Sarup¹,

Fridland²

1987

Cell extracts from human leukemic T lymphoblasts and myeloblasts were chromatographed on DEAE-cellulose columns to separate purine deoxyribonucleoside, deoxyadenosine (dAdo) and deoxyguanosine (dGuo), phosphorylating activities. Three distinct purine deoxyribonucleoside kinases, a deoxycytidine (dCyd) kinase, an adenosine (Ado) kinase, and a deoxyguanosine (dGuo) kinase (the latter appears to be localized in mitochondria), were resolved. dCyd kinase contained the major phosphorylating activity for dAdo, dGuo, and 9-beta-D-arabinofuranosyladenine (ara-A). Ado kinase represented a second kinase for dAdo and ara-A while a third kinase for dAdo was found in mitochondria. dCyd kinase was purified about 2000-fold with ion-exchange, affinity, and hydrophobic chromatographies. On gel electrophoresis, both dCyd and dAdo phosphorylating activities comigrated, indicating that the activities are associated with the same protein. The enzyme showed a broad pH optimum ranging from pH 6.5 to pH 9.5. Divalent cations Mg2+, Mn2+, and Ca2+ stimulated dCyd kinase activity; Mg2+ produced the maximal activity. dCyd kinase from either lymphoid or myeloid cells showed broad substrate specificity. The enzyme used several nucleoside triphosphates, but ATP, GTP, and dTTP were the best phosphate donors. dCyd was the best nucleoside substrate, since dCyd kinase had an apparent Km of 0.3, 85, 90, and 1400 microM for dCyd, dAdo, dGuo, and ara-A, respectively. The enzyme exhibited substrate activation with both pyrimidine and purine deoxyribonucleosides, suggesting that there is more than one substrate binding site on the kinase. These studies show that, in lymphoblasts and myeloblasts, purine deoxyribonucleosides and their analogues are phosphorylated by dCyd kinase, Ado kinase, and dGuo kinase.