The enzyme 3-oxo-delta(5)-steroid isomerase (KSI) from Pseudomonas testosteroni is an example of a class of enzymes that catalyze reactions proceeding through enol(ate) intermediates. An understanding of the mechanism of these enzymes has long been the goal of mechanistic enzymologists. It is proposed to examine the experimental basis for two different theories of catalysis of enzymatic enolizations. (1) Low barrier hydrogen bonds can provide the necessary energy to account for catalysis. (2) Catalysis is enhanced by the minimization of transition state imbalance. Each of these theories will be tested by the use of mutant enzymes containing a variety of amino acids at position 14, including fluorotyrosine derivatives. Our believes that his recent observation that KSI catalyzes the ketonization of ring substituted 2-tetralones enables him to separate the two steps of the normal KSI reaction. Using the enols of 2-tetralones, it is possible to observe a simple proton transfer as the only catalytically important step, allowing structure-reactivity correlations to be used. Dr. Pollack will also examine the effect of mutation of these residues on the binding of an intermediate analog to the active site. This work will enable him to determine the Bronsted coefficients for both the binding constants and the kinetic constants. These results will be interpreted in terms of the type of hydrogen bonding that is involved in these processes. We will also investigate by NMR the nature of the hydrogen bonding interactions at the active site, and assign the low field protons in the NMR spectrum of KSI complexed with intermediate analogs. Isotopically labeled ligands will be used to probe the source of these low field peaks, and to try to determine whether they represent low barrier hydrogen bonds.
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