The long term objectives of the research supported by this grant are to determine the chemical mechanisms by which the coenzyme forms of vitamin B1 (thiamin pyrophosphate, TPP) and vitamin B6 (pyridoxal-5'-phosphate, PLP) act in reactions that cannot be explained by the conventional mechanisms known for these coenzyme. The role of 2-acetyl-TPP in the metabolism of pyruvate catalyzed by the pyruvate dehydrogenase (PDH) complex will be determined using chemical methods. Several inherited diseases of pyruvate metabolism are caused by defects in the PDH complex. The standard mechanistic paradigm for the coenzymatic action of PLP involves the stabilization of carbanionic intermediates by the coenzyme. However, in a major focus of research supported by this grant, the action of PLP in the lysine 2,3-aminomutase-catalyzed conversion of lysine of beta-lysine PLP is proposed to act by stabilizing a radical intermediate, rather than a carbanion,. This is a new chemical mechanism for PLP with other aminomutases, such as beta-lysine mutase and arginine 2,3,-aminomutase. The aminomutases catalyze 1,2-amino group migrations that are important in amino acid metabolism and the biosynthesis of antibiotics such as mycomycin and Blasticidin I. Research supported by this grant will test the new mechanistic hypothesis by application of spectroscopic and chemical methods. The aminomutase reactions are also related to vitamin B12; in fact beta-lysine aminomutase is an adenosylcobalamin-dependent enzyme. Lysine 2,3-aminomutase catalyzes a chemically similar 1,2-amino group rearrangement, but does so without a vitamin B12 coenzyme, despite the fat that nearly all rearrangements of this chemical type in living cells involve adenosylcobalamin as an essential coenzyme. The cofactors of lysine 2,3,-aminomutase are iron-sulfur cluster, cobalt(II) and S- adenosylmethionine (AdoMet). Currently available information indicates that AdoMet and the metal cofactors interact chemically to generate an adenosyl- cofactor that has chemical properties in common with adenosycobalamin.a The research supported by this grant will seek to characterize this cofactor by the application of spectroscopic and chemical methods. Thus the central thrust of most of research is the elucidation of new chemical mechanisms in the biological actions of the coenzymatic forms of vitamins B1,B6, and B12. A biochemical problem of recent origin is that of determining the chemical functions of proteins discovered and characterized by genetic methods. A new method for identifying endogenous ligands for such proteins is needed. Such method is being developed for this purposed.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK028607-18
Application #
2734004
Study Section
Special Emphasis Panel (NSS)
Program Officer
Laughlin, Maren R
Project Start
1981-07-01
Project End
1999-06-30
Budget Start
1998-07-10
Budget End
1999-06-30
Support Year
18
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Other Domestic Higher Education
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Chen, Yung-Han; Maity, Amarendra N; Frey, Perry A et al. (2013) Mechanism-based inhibition reveals transitions between two conformational states in the action of lysine 5,6-aminomutase: a combination of electron paramagnetic resonance spectroscopy, electron nuclear double resonance spectroscopy, and density functional J Am Chem Soc 135:788-94
Chen, Yung-Han; Maity, Amarendra N; Pan, Yu-Chiang et al. (2011) Radical stabilization is crucial in the mechanism of action of lysine 5,6-aminomutase: role of tyrosine-263? as revealed by electron paramagnetic resonance spectroscopy. J Am Chem Soc 133:17152-5
Ruzicka, Frank J; Frey, Perry A (2010) Kinetic and spectroscopic evidence of negative cooperativity in the action of lysine 2,3-aminomutase. J Phys Chem B 114:16118-24
Maity, Amarendra N; Hsieh, Chih-Pin; Huang, Ming-Hui et al. (2009) Evidence for conformational movement and radical mechanism in the reaction of 4-thia-L-lysine with lysine 5,6-aminomutase. J Phys Chem B 113:12161-3
Tang, Kuo-Hsiang; Mansoorabadi, Steven O; Reed, George H et al. (2009) Radical triplets and suicide inhibition in reactions of 4-thia-D- and 4-thia-L-lysine with lysine 5,6-aminomutase. Biochemistry 48:8151-60
Schwartz, Phillip A; Lobrutto, Russell; Reed, George H et al. (2007) Probing interactions from solvent-exchangeable protons and monovalent cations with the 1,2-propanediol-1-yl radical intermediate in the reaction of dioldehydrase. Protein Sci 16:1157-64
Frey, Perry A; Hegeman, Adrian D; Reed, George H (2006) Free radical mechanisms in enzymology. Chem Rev 106:3302-16
Hinckley, Glen T; Frey, Perry A (2006) An adaptable spectroelectrochemical titrator: the midpoint reduction potential of the iron-sulfur center in lysine 2,3-aminomutase. Anal Biochem 349:103-11
Lees, Nicholas S; Chen, Dawei; Walsby, Charles J et al. (2006) How an enzyme tames reactive intermediates: positioning of the active-site components of lysine 2,3-aminomutase during enzymatic turnover as determined by ENDOR spectroscopy. J Am Chem Soc 128:10145-54
Lepore, Bryan W; Ruzicka, Frank J; Frey, Perry A et al. (2005) The x-ray crystal structure of lysine-2,3-aminomutase from Clostridium subterminale. Proc Natl Acad Sci U S A 102:13819-24

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