Analysis of the inhibitor binding to the nucleoside site of phosphorylase |
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Authors: | Isabel Mérida Margarita Menéndez José Laynez Francisco Garcia Blanco |
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Institution: | 1. Instituto de Quimica-Fisica Rocasolano, CSIC, Serrano 119, Madrid 6, Spain;2. Departamento de Quimica-Fisica, Facultad de Ciencias, Universidad de Córdoba, Córdoba, Spain |
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Abstract: | The binding of inhibitors to site I of rabbit muscle phsphorylase has beenstudied kinetically and thermodynamically for caffeine, adenine and adenosine. The effect of ligands on the tertiary structure has been investigated by studying the protection against 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) titration of the slow-reacting sulphydryl groups of the enzyme. Calorimetric and cysteinyl protection data taken together suggest that these inhibitors bind to both sites N and I even under conditions of saturation by glucose. Calorimetric results show that inhibitor binding to sites I and N at 25°C is driven enthalpically, although both and of interaction are significant. We conclude that attractive dispersion forces ought to be the main ones responsible for inhibitor binding to site I. AMP-activated phosphorylase is inhibited by both caffeine and adenine by cooperative and exclusive binding to the inactive conformation. The binding of the substrate (phosphate) and AMP when adenine is present was found to be exlusive to the active conformation, whereas non-exclusive binding of the activator was observed when caffeine was added. |
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Keywords: | Enzymes inhibitor binding site I thermodynamics conformational states |
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