Gwendolyn A. Sowa 1995-2000
St. Lukes Medical Center, Milwaukee, WI
Lafayette College 1993
Biochemistry PhD 1997
W W Cleland
Insight into the mechanism of enzymatic phosphoryl transfer through investigation of transition state structures
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Heavy atom isotope effects have been used to characterize the transition state structure of several phosphoryl transfer systems. The effects were measured by the competitive method, and thus are effects on Vm/Km. The dephosphorylation of p-nitrophenyl phosphate by Yersinia Protein-Tyrosine Phosphatase (PTPase) and by the rat PTP1 has been examined. Normal primary isotope effects, inverse isotope effects at the nonbridge oxygen atoms, and isotope effects of unity at the leaving group nitrogen atoms were observed for the enzyme catalyzed reactions. The magnitudes of these isotope effects are similar to the intrinsic values measured in solution, indicating that the chemical step is rate limiting for Vm/Km. These data reveal that the transition state for the enzyme catalyzed dephosphorylation of p-nitrophenyl phosphate is dissociative in character. Cleavage of the P-O bond and proton transfer from the general acid Asp to the leaving group are both far advanced in the transition state. Experiments with several general acid mutants show primary oxygen and secondary nitrogen isotope effects larger than with wild type enzyme, consistent with this conclusion.
Heavy atom isotope effects have been measured for the ribonuclease A catalyzed cleavage of the alternative substrate uridine 3’-m-nitrobenzyl phosphate. A normal primary isotope effect of 1.6% was observed for P-O bond cleavage. This value was pH independent, revealing that chemistry is rate limiting in this system. A normal secondary isotope effect of 0.5% was observed as the overall effect for both oxygen atoms in the nonbridge position. This result is direct evidence against a mechanism involving a phosphorane intermediate. The isotope effect measured on isomerization of this substrate to uridine 2’-m-nitrobenzyl phosphate, which proceeds through a monoanionic phosphorane intermediate, is unity. A large inverse isotope effect would be expected for formation of a neutral phosphorane due to protonation of both nonbridge oxygen atoms, and a large (2.5-4%) normal isotope effect would be expected for formation of an unstable dianionic phosphorane. Therefore, the observed normal nonbridge isotope effect of 0.5% for the ribonuclease A catalyzed reaction is consistent with a concerted mechanism with a transition state having slightly associative character.
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| Thesis Publications |
- Gerratana B, Sowa GA, Cleland WW. Characterization of the transition-state structures and mechanisms for the isomerization and cleavage reactions uridine 3’-m-Nitrobenzyl phosphate. J Am Chem Soc 122:12615-12621, 2000.
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