Abstract:The interactions of vanadate and its complexes of uridine, 5,6-dihydrouridine, and methyl P-D-ribofuranoside with bovine pancreatic ribonuclease A (RNase A) (EC 3.1.27.5) were studied by 5 1~ NMR spectroscopy and enzyme kinetics. From kinetic studies, it was found that neither inorganic vanadate nor the methyl P-D-ribofuranoside-vanadate complex significantly inhibited the RNase A catalyzed hydrolysis of uridine 2',3'-cyclic monophosphate. The NMR binding studies were in full agreement with the kinetics studies and showed that neither inorganic vanadate nor the methyl P-D-ribofuranoside-vanadate complex was bound tightly by the enzyme. Approximate binding constants were (5.0 + 1.0) x M and (3.0 + 0.6) x M for the uridine-and 5.6-dihydrouridine-vanadate complexes, respectively. An induced-fit mechanism is suggested, in which the pyrimidine subsite of the active site of RNase A must be fully occupied for the enzyme to be able to tightly bind the transition state or transition state analog. Calculation of the binding energies of vanadate complexes in ribonuclease, phosphoglycerate mutase, and phosphoglucomutase revealed an excess of binding energy over the analogous phosphate derivative of about 25 kJ/mol for all enzymes, even though the binding constants themselves varied by about six orders of magnitude. This energy represents about 40% of that expected to be available for a trigonal-bipyramidal transition state and requires a reassessment of the role of vanadate as a transition state analogue for phosphate transfer.Key words: vanadate, ribonuclease, transition state, binding constants, phosphate analogues, kinetics.Resum6 : Faisant appel a la spectrscopie RMN du 5 1~ et de la cinCtique des enzymes, on a CtudiC les interactions entre le vanadate et ses complexes de I'uridine, de la 5,6-dihydrouridine et du P-D-ribofuranoside de mCthyle avec la ribunuclease A du pancrCas de boeuf (RNase A) (EC 3.1.27.5). Sur la base des Ctudes cinCtiques, on a trouvC que, vis-a-vis de I'hydrolyse du monophosphate 2',3'-cyclique de I'uridine de la RNase A, ni le vanadate inorganique ni le complexe du P-D-ribofuranoside de mCthyle-vanadate n'inhibent cette rCaction d'une f a~o n significative. Les Ctudes par RMN ont montrt qu'il ne se produit aucune fixation entre ces matCriaux et elles Ctaient en parfait accord avec les ttudes cinCtiques; elles montrent aussi que ni le vanadate inorganique ni le complexe du P-D-ribofuranoside de mCthyle-vanadate ne se complexent fortement avec I'enzyme. Les constantes apparentes de fixation ont respectivement de (5,O + 1.0) x M et (3,O + 0.6) x M pour les complexes de I'uridine et du 5,6-dihydrouridine avec le vanadate. On propose un mCcanisme induit d'ajustement dans lequel il est important que tout le sous site de la pyrimidine du site actif de la RNase A soit occupC pour que I'enzyme soit capable de bien fixer 1'Ctat de transition ou son analogue. Des calculs d'Cnergie de fixation des complexes du vanadate dans la ribonucltase, la phosphoglycCratemutase ou la phosphoglucomutase ont rCvC...

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