Resolution of two deoxynucleoside kinases from Lactobacillus acidophilus R-26 by Blue Sepharose affinity chromatography.

Heterodimeric Deoxynucleoside Kinases of Lactobacillus acidophilus R-26: Functional Assignment of Subunits Using Limited Proteolysis Controlled by End-Product Inhibitors

“…Further investigation is necessary to see if these two findings are related. In contrast to broad specificity for the phosphate donor, each kinase shows almost absolute deoxynucleoside substrate specificity (Deibel & Ives, 1977a). The structural basis for this specificity must be found in the internal amino acid sequence of each kinase polypeptide.…”

“…(3) The binding sites for deoxynucleoside and ATP must be situated next to each other at each active site, according to its sequential kinetics (Deibel et al, 1977;Chakravarty et al, 1984). (4) The end-product inhibitor, dNTP, specifically and strongly inhibits the corresponding kinase activity by binding to each catalytic site (Deibel & Ives, 1977a;Ikeda et al, 1986). Presumably, the deoxynucleoside moiety of dNTP fits into the deoxynucleoside binding site, while the triphosphate portion of dNTP interacts with the triphosphate binding site for ATP, but in a reversed orientation relative to that of ATP (see the bottom panel of Figure 5) (Ikeda et al, 1986).…”

“…For N-terminal amino acid sequencing of separated subunits, the separating gel was cast 1 day before use and was subjected to preelectrophoresis in separating gel buffer containing 0.1 mM thioglycolatc (Moos et al, 1988). In order to locate the kinase activity peak on nondenaturing gels, one channel of the gel was cut into 2-mm slices and assayed as described previously (Deibel & Ives, 1977a).…”

“…0006-2960/94/0433-5328$04.50/0 (dNTP)-but apparently not at an allosteric binding site. The kinetic effects of each dNTP are identical to those of the respective multisubstrate inhibitor (Deibel & Ives, 1977a;Ikeda et al, 1986). Therefore, it has been proposed that the binding site for a dNTP is the active site; the simplest explanation consistent with all the evidence is that the deoxynucleoside moiety of dNTP fits into the deoxynucleoside binding site, while the triphosphate portion of dNTP interacts with the triphosphate binding site for ATP in a "reversed" orientation relative to that of ATP (Ikeda et al, 1986).…”

Amino-terminal nucleotide-binding sequences of a Lactobacillus deoxynucleoside kinase complex isolated by novel affinity chromatography

“…Lactobacillus acidophilus R-26 has two unique paired de-oxynucleoside kinases, namely, dCyd/dAdo kinase and dGuo/dAdo kinase (Deibel & Ives, 1977a). Kinetic studies have revealed that these two paired activities do not share a common active site in either case but exhibit positive allosteric interactions (Deibel et al, 1977;Chakravarty et al, 1984).…”