The structural and dynamical basis of enzyme action, primarily of proteolytic enzymes, will be investigated by (i) cryokinetic methods to stabilize true enzyme reaction intermediates; (ii) electron nuclear double resonance spectroscopy to determine the structure and conformation of substrates free in solution and bound in enzymic active sites of catalytically competent reaction intermediates; (iii) molecular graphics and modeling to assign stereochemical relationships of enzyme-substrate interactions based on spectroscopically determined data; (iv) interpreta- tion of structural data according to stereoelectronic principles; and (v) assessment of the relationships of dynamical motion of protein residues to enzyme catalytic function by computer based molecular dynamics simulations. The methods are designed to determine local structure of enzyme active site residues and of substrates in kinetically competent reaction intermediates, to assess the influence of protein dynamical motion in torsionally altering substrate structure to allow tight, specific binding in the active site, and to assess the structural and electronic basis of enzyme action on a detailed molecular level. The methods to be applied will yield data to assign three-dimensional structure and conformation of catalytically competent reaction intermediates of enzymes in a manner that cannot be achieved at present with other physical methods.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM021900-18
Application #
3270795
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1978-09-01
Project End
1995-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
18
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Chicago
Department
Type
Schools of Medicine
DUNS #
225410919
City
Chicago
State
IL
Country
United States
Zip Code
60637
Mustafi, D; Sosa-Peinado, A; Makinen, M W (2001) ENDOR structural characterization of a catalytically competent acylenzyme reaction intermediate of wild-type TEM-1 beta-lactamase confirms glutamate-166 as the base catalyst. Biochemistry 40:2397-409
Atanasov, B P; Mustafi, D; Makinen, M W (2000) Protonation of the beta-lactam nitrogen is the trigger event in the catalytic action of class A beta-lactamases. Proc Natl Acad Sci U S A 97:3160-5
Sosa-Peinado, A; Mustafi, D; Makinen, M W (2000) Overexpression and biosynthetic deuterium enrichment of TEM-1 beta-lactamase for structural characterization by magnetic resonance methods. Protein Expr Purif 19:235-45
Horvath, M P; Copeland, R A; Makinen, M W (1999) The second derivative electronic absorption spectrum of cytochrome c oxidase in the Soret region. Biophys J 77:1694-711
Makinen, M W (1998) Electron nuclear double resonance determined structures of enzyme reaction intermediates: structural evidence for substrate destabilization. Spectrochim Acta A Mol Biomol Spectrosc 54A:2269-81
Makinen, M W; Mustafi, D (1995) The vanadyl ion: molecular structure of coordinating ligands by electron paramagnetic resonance and electron nuclear double resonance spectroscopy. Met Ions Biol Syst 31:89-127
Mustafi, D; Nakagawa, Y (1994) Characterization of calcium-binding sites in the kidney stone inhibitor glycoprotein nephrocalcin with vanadyl ions: electron paramagnetic resonance and electron nuclear double resonance spectroscopy. Proc Natl Acad Sci U S A 91:11323-7
Mustafi, D; Makinen, M W (1994) Catalytic conformation of carboxypeptidase A. Structure of a true enzyme reaction intermediate determined by electron nuclear double resonance. J Biol Chem 269:4587-95
Wells, G B; Mustafi, D; Makinen, M W (1994) Structure at the active site of an acylenzyme of alpha-chymotrypsin and implications for the catalytic mechanism. An electron nuclear double resonance study. J Biol Chem 269:4577-86
Makinen, M W; Troyer, J M; van der Werff, H et al. (1989) Dynamical structure of carboxypeptidase A. J Mol Biol 207:201-16

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