INFLUENCE OF VANADIUM(V) COMPLEXES ON THE CATALYTIC ACTIVITY OF RIBONUCLEASE-A - THE ROLE OF VANADATE COMPLEXES AS TRANSITION-STATE ANALOGSTO REACTIONS AT PHOSPHATE

The interactions of vanadate and its complexes of uridine, 5,6-dihydro uridine, and methyl beta-D-ribofuranoside with bovine pancreatic ribon uclease A (RNase A) (EC 3.1.27.5) were studied by V-51 NMR spectroscop y and enzyme kinetics. From kinetic studies, it was found that neither inorganic vanadate nor the methyl beta-D-ribofuranoside-vanadate comp lex significantly inhibited the RNase A catalyzed hydrolysis of uridin e 2',3'-cyclic monophosphate. The NMR binding studies were in full agr eement with the kinetics studies and showed that neither inorganic van adate nor the methyl beta-D-ribofuranoside-vanadate complex was bound tightly by the enzyme. Approximate binding constants were (5.0 +/- 1.0 ) x 10(-7) M and (3.0 +/- 0.6) x 10(-6) M for the uridine- and 5,6-dih ydrouridine-vanadate complexes, respectively. An induced-fit mechanism is suggested, in which the pyrimidine subsite of the active site of R Nase A must be fully occupied for the enzyme to be able to tightly bin d the transition state or transition state analog. Calculation of the binding energies of vanadate complexes in ribonuclease, phosphoglycera te mutase, and phosphoglucomutase revealed an excess of binding energy over the analogous phosphate derivative of about 25 kJ/mol for all en zymes, even though the binding constants themselves varied by about si x orders of magnitude. This energy represents about 40% of that expect ed to be available for a trigonal-bipyramidal transition state and req uires a reassessment of the role of vanadate as a transition state ana logue for phosphate transfer.