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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM038155-12A1
Application #
6130040
Study Section
Biochemistry Study Section (BIO)
Program Officer
Jones, Warren
Project Start
1987-08-01
Project End
2004-06-30
Budget Start
2000-07-01
Budget End
2001-06-30
Support Year
12
Fiscal Year
2000
Total Cost
$303,065
Indirect Cost
Name
University of Maryland Balt CO Campus
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Baltimore
State
MD
Country
United States
Zip Code
21250
Wilde, Thomas C; Blotny, Grzegorz; Pollack, Ralph M (2008) Experimental evidence for enzyme-enhanced coupled motion/quantum mechanical hydrogen tunneling by ketosteroid isomerase. J Am Chem Soc 130:6577-85
Pollack, Ralph M (2004) Enzymatic mechanisms for catalysis of enolization: ketosteroid isomerase. Bioorg Chem 32:341-53
Petrounia, I P; Blotny, G; Pollack, R M (2000) Binding of 2-naphthols to D38E mutants of 3-oxo-Delta 5-steroid isomerase: variation of ligand ionization state with the nature of the electrophilic component. Biochemistry 39:110-6
Henot, F; Pollack, R M (2000) Catalytic activity of the D38A mutant of 3-oxo-Delta 5-steroid isomerase: recruitment of aspartate-99 as the base. Biochemistry 39:3351-9
Pollack, R M; Thornburg, L D; Wu, Z R et al. (1999) Mechanistic insights from the three-dimensional structure of 3-oxo-Delta(5)-steroid isomerase. Arch Biochem Biophys 370:9-15
Petrounia, I P; Pollack, R M (1998) Substituent effects on the binding of phenols to the D38N mutant of 3-oxo-delta5-steroid isomerase. A probe for the nature of hydrogen bonding to the intermediate. Biochemistry 37:700-5
Thornburg, L D; Henot, F; Bash, D P et al. (1998) Electrophilic assistance by Asp-99 of 3-oxo-Delta 5-steroid isomerase. Biochemistry 37:10499-506
Qi, L; Pollack, R M (1998) Catalytic contribution of phenylalanine-101 of 3-oxo-Delta 5-steroid isomerase. Biochemistry 37:6760-6
Wu, Z R; Ebrahimian, S; Zawrotny, M E et al. (1997) Solution structure of 3-oxo-delta5-steroid isomerase. Science 276:415-8
Zawrotny, M E; Hawkinson, D C; Blotny, G et al. (1996) Mechanism of proton transfer in the isomerization of 5-androstene-3, 17-dione by 3-oxo-delta 5-steroid isomerase and its D38E mutant. Biochemistry 35:6438-42

Showing the most recent 10 out of 19 publications