Vanadate can often bind to phosphoryl transfer enzymes to form a trigonal-bipyramidal structure at the active site. The enzyme-vanadate dissociation constants in these enzymes are much lower than those for phosphate. Therefore, enzyme-bound vanadate moieties are often considered as transition state analogues. To test whether the enzyme-vanadate complex is a true transition state analogue beyond the simple geometry and binding affinity arguments and whether the bond orders of the VO bonds in the complex approach those of the PO bonds in the transition state, the binding properties of vanadate in the Yersinia protein-tyrosine phosphatase (PTPase) and its T410A, D356N, W354A, R409K, and D356A mutants have been studied by steady-state kinetic measurements and by difference Raman measurements. The results of the kinetic measurements show no correlation between K1 and kcat or kcat/Km in these mutants. In addition, our analysis of the Raman data shows that the bond order change of the nonbridging V--..O bonds in the vanadate complexes does not correlate with the kinetic parameters in a number of PTPase variants as predicted by the transition state binding paradigm. Furthermore, the ionization state of the bound vanadate moiety is not invariant across the PTPase variants studied, and the average bond order of the nonbridging V--..O bonds decreased by 0.06-0.07 valence unit in the wild type and all of the mutant PTPases, either in dianionic or in monoanionic form. Thus the complex would resemble an associative transition state, contrary to the previously determined dissociative structure of the transition state. Therefore, it is concluded that vanadate is not a true transition state analogue for the PTPase reactions.
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