Synthesis and kinetic study of transition state analogs for ribonuclease T1

Based on the proposal that ribonucleases cleave the RNA phosphodiester bond with a mechanism involving pentacovalent phosphorous as transition state, complexes of guanosine and inosine with vanadate-(IV, V), molybdate-(VI), tungstate-(VI), chromate-(VI) and hexacyanochromate-(III) were synthesized and probed as inhibitors of recombinant ribonuclease T1, obtained from an E. coli. overproducing strain. The apparent dissociation constants of these inhibitors and RNase T1, as determined by Michaelis-Menten kinetics, vary between 0.5-0.9 microM and indicate very strong binding, 100- to 1000-fold stronger than the binding of guanosine (Kd = 545 microM) and inosine (Kd = 780 microM), and 50-100-fold stronger than the binding of the product 3' GMP (Kd = 55 microM). Therefore the synthesized inhibitors may be considered as genuine transition state analogs for the enzyme.