Pentavalent Organo-Vanadates as Transition State Analogues for Phosphoryl Transfer Reactions

Abstract

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 with natural and nonnatural amino acid residues at position 41. These variants differ by 10 5 -fold in their k cat /K m values for 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 constant for the enzymic complexes with substrate or product, indicating 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.

Genetic Selection for Critical Residues in Ribonucleases

“…52,72 Lys41 Several substitutions at residue 41 inactivate RNase A. Lys41 has been shown previously to be critical for ribonucleolytic activity, as it donates a hydrogen bond to the pentavalent transition state. 51,73 K41A RNase A has a k cat /K M value that is 10 4 -fold lower than that of the wild-type enzyme. 74 Yet, Lys41 can be replaced by arginine with only a 10 2 -fold decrease in catalytic activity.…”

The Chemistry and Biochemistry of Vanadium and the Biological Activities Exerted by Vanadium Compounds

“…49 Recently, the potency of the vanadate-nucleoside complex was investigated with several mutants of ribonuclease and these investigators confirmed the conclusion that the vanadiumnucleoside is not a "true and perfect" transition analogue. 50 Although small structural differences existing in the mutants were not considered, which could diminish the observed potency of the vanadate-nucleoside complex as an inhibitor, the fact that vanadate-nucleosides are likely to be potent, but not perfect, transition state analogues is likely to be confirmed. Indeed, considerations regarding what types of phosphorylase reactions V-complexes may be most effective against have been extensively considered.…”

A comparison of vanadate to a 2′–5′ linkage at the active site of a small ribozyme suggests a role for water in transition-state stabilization

“…Specifically, these amino acids interacted with vanadate in a manner that was incongruous with mechanistic evidence supporting a concerted, imidazole-mediated acid/base mechanism in which Lys41 ameliorates negative charge buildup on the nonbridging oxygens. Later studies offered explanations citing the apparent plasticity of the ''oxyvanadate framework'' (Ladner et al 1997) and fundamental differences in the vanadate electronics compared with phosphate (Messmore and Raines 2000). The salient and admonitory conclusion is that the bound vanadate was stabilized by the active site of RNase A, but through interactions irrelevant to a widely accepted view of the transition state.…”