Pentavalent organo-vanadates as transition state analogues for phosphoryl transfer reactions

Pentavalent organo-vanadates have been put forth as transition-state analogues for a variety of phosphoryl transfer reactions. in particular, uridine 2',3'-cyclic vanadate (U>v) has been proposed to resemble the transition state during catalysis by ribonuclease A (RNase A). Here, this hypothesis is tested. Lys41 of RNase A is known to donate a hydrogen bond to a nonbridging phosphoryl oxygen in the transition state during catalysis. Site-directed mutagenesis and semisynthesis were used to create enzymes having natural and nonnatural amino acid residues at position 41. These variants differ by 10(5)-fold in their k(cat)/K-m values fur catalysis, but <40-fold in their K-i values for inhibition of catalysis by U>v. Plots of logK(i) vs log(K-m/k(cat)) for three distinct substrates [poly(cytidylic acid), uridine 3'-(p-nitrophenyl phosphate), and cytidine 2',3'-cyclic phosphate] have slopes that range from 0.25 and 0.36. These plots would have a slope of unity if U>v were a perfect transition state analogue. Values of K-i for U>v correlate weakly with the equilibrium dissociation constants for the enzymic complexes with substrate or product, which indicates that U>v bears some resemblance to the substrate and product as well as the transition state. Thus, U>v is a transition state analogue for RNase A, but only a marginal one. This finding indicates that a pentavalent organo-vanadate cannot necessarily be the basis for a rigorous analysis of the transition state for a phosphoryl transfer reaction.