An allosteric model for ribonuclease.
Ontology highlight
ABSTRACT:
SUBMITTER: Walker EJ
PROVIDER: S-EPMC1165468 | biostudies-other | 1975 Jun
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Evidence is presented from three experimental systems to support the allosteric model of Walker et al. (1975) (Biochem. J. 147, 425-433) which explains the substrate-concentration-dependent transition observed in the RNAase (ribonuclease)-catalysed hydrolysis of 2':3'-cyclic CMP (cytidine 2':3'-cyclic monophosphate). 1. Kinetic studies of the initial rate of hydrolysis of 2':3'-cyclic CMP show that the midpoint of the transition shifts to lower concentrations of 2':3'-cyclic CMP in the presence of the substrate analogues 3'-CMP, 5'-CMP, 3'-AMP, 3'-UMP and Pi; 2'-CMP and 2'-UMP do not cause such a shift. 2. Trypsin-digestion studies show that a conformational change in RNAase to a form less susceptible to tryptic inactivation is induced in the presence of the substrate analogues 3'-CMP, 5'-CMP, 3'-AMP, and 3'-UMP. 2'-CMP, 2'-AMP and 2'-UMP do not induce this conformational change. 3. Equilibrium-dialysis experiments demonstrate the multiple binding of molecules of 3'-CMP, 3'-AMP and 5'-AMP to a molecule of RNAase. 2'-CMP binds the ratio 1:1 over the analogue concentration range studied.
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Project description:A method for the preparation of allosteric ribonuclease from bovine pancreas is described. The effects of freeze-drying ribonuclease from acid and alkaline solutions on plots of velocity versus substrate concentration for the hydrolysis of 2':3'-cyclic CMP are examined. Comparison of these plots with the plots obtained with severeal commercial enzyme preparations indicates that the conformation of the enzyme is dependent on the method of preparation. Aging experiments demonstrate that further conformational changes occur at different rates, depending on the methods of storage. Results suggest that the allosteric behaviour of ribonuclease has not always been observed with commercial preparations, owing to variations in methods of preparation and storage of the enzyme.
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Project description:The allosteric model for ribonuclease activity by Walker, Ralston & Darvey [(1975) Biochem.J. 147, 425--433; (1976) Biochem.J. 153, 329--337] involves the binding of a large number of molecules of substrate or substrate analogue to a series of allosteric sites on the enzyme. In the present paper, the nature of these allosteric interactions is investigated. The effects of ionic strength pH carbamoylation of lysine to homocitrulline and of deamidation of glutamine and asparagine on plots of velocity versus substrate concentration are examined and evidence is presented that the allosteric transition involves an electrostatic interaction between the negatively charged substrate molecules and the cationic groups on the enzyme.
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Project description:The paper focussed on a step-by-step structural modification of a cycloheptathiophene-3-carboxamide derivative recently identified by us as reverse transcriptase (RT)-associated ribonuclease H (RNase H) inhibitor. In particular, its conversion to a 2-aryl-cycloheptathienoozaxinone derivative and the successive thorough exploration of both 2-aromatic and cycloheptathieno moieties led to identify oxazinone-based compounds as new anti-RNase H chemotypes. The presence of the catechol moiety at the C-2 position of the scaffold emerged as critical to achieve potent anti-RNase H activity, which also encompassed anti-RNA dependent DNA polymerase (RDDP) activity for the tricyclic derivatives. Benzothienooxazinone derivative 22 resulted the most potent dual inhibitor exhibiting IC50s of 0.53 and 2.90??M against the RNase H and RDDP functions. Mutagenesis and docking studies suggested that compound 22 binds two allosteric pockets within the RT, one located between the RNase H active site and the primer grip region and the other close to the DNA polymerase catalytic centre.
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