RESUMO
The study of internal mobility in enzymes is of considerable importance for the understanding of their catalytic function, which cannot be adequately described as a property of a rigid protein. [13C]NMR spectroscopy permits simultaneous and selective observation of spectral lines from carbon atoms in many different residues in the enzyme with the chemical shift and relaxation parameters sensitive to structure, conformation and local motion. The changes in internal mobility in bovine carbonic anhydrase B (carbonate hydrolase, EC 4.2.1.1) in the native form and at various stages of denaturation are studied. Measurements of the relaxation parameters (T1, T1 rho) and of the NOE of 13C nuclei in the native protein showed that the extensive beta-sheet together with groups in the active center has a considerable internal librational mobility with tau G about 10(-11) s. This librational mobility is fairly uniform for all the alpha-carbons in the native enzyme. The use of a semiempirical modification of the motional theory proposed by Woessner allows to use simultaneously all the relaxation parameters measured in order to determine reliable values of the various correlation times.
