Kinetics of T7 DNA polymerase conformational changes during nucleotide incorporation

The putative first intracellular and third extracellular linkers are known to play important roles in defining the transport properties of the type IIa Na+-coupled phosphate cotransporter (Kohler, K., I.C. Forster, G. Stange, J. Biber, and H. Murer. 2002b. J. Gen. Physiol. 120:693–705). To investigate whether other stretches that link predicted transmembrane domains are also involved, the substituted cysteine accessibility method (SCAM) was applied to sites in the predicted first and fourth extracellular linkers (ECL-1 and ECL-4). Mutants based on the wild-type (WT) backbone, with substituted novel cysteines, were expressed in Xenopus oocytes, and their function was assayed by isotope uptake and electrophysiology. Functionally important sites were identified in both linkers by exposing cells to membrane permeant and impermeant methanethiosulfonate (MTS) reagents. The cysteine modification reaction rates for sites in ECL-1 were faster than those in ECL-4, which suggested that the latter were less accessible from the extracellular medium. Generally, a finite cotransport activity remained at the end of the modification reaction. The change in activity was due to altered voltage-dependent kinetics of the Pi-dependent current. For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V ≤ −80 mV, whereas the WT exhibited a linear voltage dependency. After cys modification, this mutant displayed a supralinear voltage dependency in the same voltage range. The opposite behavior was documented for cys substitution at Met-533 in ECL-4. Modification of cysteines at two other sites in ECL-1 (Ile-136 and Phe-137) also resulted in supralinear voltage dependencies for hyperpolarizing potentials. Taken together, these findings suggest that ECL-1 and ECL-4 may not directly form part of the transport pathway, but specific sites in these linkers can interact directly or indirectly with parts of NaPi-IIa that undergo voltage-dependent conformational changes and thereby influence the voltage dependency of cotransport.

Download Full-text

Positive co-operative binding at two weak lysine-binding sites governs the Glu-plasminogen conformational change

Biochemical Journal ◽

10.1042/bj2850419 ◽

1992 ◽

Vol 285 (2) ◽

pp. 419-425 ◽

Cited By ~ 34

Author(s):

U Christensen ◽

L Mølgaard

Keyword(s):

Ligand Binding ◽

Binding Site ◽

Conformational Change ◽

Conformational Changes ◽

Binding Sites ◽

High Specificity ◽

Stopped Flow ◽

Protein Fluorescence ◽

Lysine Residues ◽

Kinetics Of

The kinetics of a series of Glu-plasminogen ligand-binding processes were investigated at pH 7.8 and 25 degrees C (in 0.1 M-NaCl). The ligands include compounds analogous to C-terminal lysine residues and to normal lysine residues. Changes of the Glu-plasminogen protein fluorescence were measured in a stopped-flow instrument as a function of time after rapid mixing of Glu-plasminogen and ligand at various concentrations. Large positive fluorescence changes (approximately 10%) accompany the ligand-induced conformational changes of Glu-plasminogen resulting from binding at weak lysine-binding sites. Detailed studies of the concentration-dependencies of the equilibrium signals and the rate constants of the process induced by various ligands showed the conformational change to involve two sites in a concerted positive co-operative process with three steps: (i) binding of a ligand at a very weak lysine-binding site that preferentially, but not exclusively, binds C-terminal-type lysine ligands, (ii) the rate-determining actual-conformational-change step and (iii) binding of one more lysine ligand at a second weak lysine-binding site that then binds the ligand more tightly. Further, totally independent initial small negative fluorescence changes (approximately 2-4%) corresponding to binding at the strong lysine-binding site of kringle 1 [Sottrup-Jensen, Claeys, Zajdel, Petersen & Magnusson (1978) Prog. Chem. Fibrinolysis Thrombolysis 3, 191-209] were observed for the C-terminal-type ligands. The finding that the conformational change in Glu-plasminogen involves two weak lysine-binding sites indicates that the effect cannot be assigned to any single kringle and that the problem of whether kringle 4 or kringle 5 is responsible for the process resolves itself. Probably kringle 4 and 5 are both participating. The involvement of two lysine binding-sites further makes the high specificity of Glu-plasminogen effectors more conceivable.

Download Full-text

Kinetic and structural analysis of the ultrasensitive behaviour of cyanobacterial ADP-glucose pyrophosphorylase

Biochemical Journal ◽

10.1042/bj3500139 ◽

2000 ◽

Vol 350 (1) ◽

pp. 139-147 ◽

Cited By ~ 11

Author(s):

Diego F. GÓMEZ CASATI ◽

Miguel A. AON ◽

Alberto A. IGLESIAS

Keyword(s):

Conformational Changes ◽

Tertiary Structure ◽

Fluorescence Emission ◽

Maximal Activity ◽

Size Exclusion ◽

Exclusion Chromatography ◽

Adp Glucose Pyrophosphorylase ◽

Kinetics Of ◽

Allosteric Regulators

The kinetic and (supra)molecular properties of the ultrasensitive behaviour of ADP-glucose pyrophosphorylase (AGPase) from Anabaena PCC 7120 (a cyanobacterium) were exhaustively studied. The response of the enzyme toward the allosteric activator 3-phosphoglycerate (3PGA) occurs with ultrasensitivity as a consequence of the cross-talk with the inhibitor Pi. Molecular ‘crowding’renders AGPase more sensitive to the interplay between the allosteric regulators and, consequently, enhances the ultrasensitive response. In crowded media, and when orthophosphate is present, the activation kinetics of the enzyme with 3PGA proceed with increased co-operativity and reduced affinity toward the activator. Under conditions of ultrasensitivity, the enzyme's maximal activation takes place in a narrow range of 3PGA concentrations. Moreover, saturation kinetics of the enzyme with respect to its substrates, glucose 1-phosphate and ATP, were different at low or high 3PGA levels in crowded media. Only under the latter conditions did AGPase exhibit discrimination between low or high levels of the activator, which increased the affinity toward the substrates and the maximal activity reached by the enzyme. Studies of fluorescence emission of tryptophan residues, fourth-derivative spectroscopy and size-exclusion chromatography indicated that the ultrasensitive behaviour is correlated with intramolecular conformational changes induced in the tertiary structure of the homotetrameric enzyme. The results suggest a physiological relevance of the ultrasensitive response of AGPase in vivo, since the enzyme could be subtly sensing changes in the levels of allosteric regulators and substrates, and thus determining the flux of metabolites toward synthesis of storage polysaccharides.

Download Full-text

The kinetics of conformational changes of α2 -macroglobulin determined by time resolved X-ray solution scattering

FEBS Letters ◽

10.1016/0014-5793(94)80267-x ◽

1994 ◽

Vol 337 (2) ◽

pp. 171-174 ◽

Cited By ~ 5

Author(s):

Hideo Arakawa ◽

Takuji Urisaka ◽

Hirotsugu Tsuruta ◽

Yoshiyuki Amemiya ◽

Hiroshi Kihara ◽

...

Keyword(s):

Conformational Changes ◽

Time Resolved ◽

X Ray ◽

Α2 Macroglobulin ◽

Kinetics Of

Download Full-text

DNA Polymerase and dRP-lyase activities of polymorphic variants of human Pol ι

Biochemical Journal ◽

10.1042/bcj20200491 ◽

2021 ◽

Vol 478 (7) ◽

pp. 1399-1412

Author(s):

Evgeniy S. Shilkin ◽

Anastasia S. Gromova ◽

Margarita P. Smal ◽

Alena V. Makarova

Keyword(s):

Dna Damage ◽

Dna Polymerase ◽

Polymerase Activity ◽

Altered Expression ◽

Dna Polymerase Iota ◽

Nucleotide Incorporation ◽

Lyase Activity ◽

Polymorphic Variants ◽

Y Family

Y-family DNA polymerase iota (Pol ι) is involved in DNA damage response and tolerance. Mutations and altered expression level of POLI gene are linked to a higher incidence of cancer. We biochemically characterized five active site polymorphic variants of human Pol ι: R71G (rs3218778), P118L (rs554252419), I236M (rs3218784), E251K (rs3218783) and P365R (rs200852409). We analyzed fidelity of nucleotide incorporation on undamaged DNA, efficiency and accuracy of DNA damage bypass, as well as 5′-deoxyribophosphate lyase (dRP-lyase) activity. The I236M and P118L variants were indistinguishable from the wild-type Pol ι in activity. The E251K and P365R substitutions altered the spectrum of nucleotide incorporation opposite several undamaged DNA bases. The P365R variant also reduced the dRP-lyase activity and possessed the decreased TLS activity opposite 8-oxo-G. The R71G mutation dramatically affected the catalytic activities of Pol ι. The reduced DNA polymerase activity of the R71G variant correlated with an enhanced fidelity of nucleotide incorporation on undamaged DNA, altered lesion-bypass activity and reduced dRP-lyase activity. Therefore, this amino acid substitution likely alters Pol ι functions in vivo.

Download Full-text