This proposal;l is concerned with delineation of the remaining structural and mechanistic questions on pyruvate decarboxylase (PDC, E.C.4.1.1.1), perhaps the simplest nonoxidative decarboxylase requiring thiamin diphosphate (ThDP, the vitamin B1 coenzyme, of fundamental significance in human metabolism). The brewers'yeast enzyme was purified as fully active alpha4 and beta4 homotetramers and the alpha4 structure was crystallized by the AI and collaborators at the VA Hospital in Pittsburgh. A 2.4 electron density map of this enzyme is more than 95% complete, including the coenzyme binding site. With this map already in hand, for the first time on this representative of a large group of alpha-keto acid decarboxylases, rational experiments can be designed to probe: a. the conformation of the enzyme-bound coenzyme in the absence of substrate, as well as in some of the three putative covalent substrate-coenzyme complexes; b. the activation towards catalysis (in the absence and presence of substrate activation) of the two aromatic rings (thiazolium and 4-aminopyramidine), and test of the hypothesis that both rings (not only the thiazolium) are participants in catalysis; c. the environment of the coenzyme and the function of amino acids surrounding it in catalysis, for example determining if surrounding hydrophobic amino acids provide a """"""""solvent effect"""""""" thereby lowering the transition state energies of a number of key steps, as suggested by model studies ; and d. structural bases and structural as well as mechanistic consequences of substrate activation (using among other tools pyruvamide, a nondecarboxylatable substrate surrogate that is capable of shifting the enzyme from a low to a high activity form). Most prominent among the tools to address these questions will be (all of these are """"""""in hand"""""""" as of writing): a. x-ray crystallographic methods (this part of the research is a collaborative effort with Furey, Sax and coworkers at the VA Hospital Biocrystallography Lab/Univ. of Pittsburgh); b. site-directed mutagenesis of amino acids found near the catalytic and regulatory sites based on the x-ray crystallographic and mechanistic information; c. further elucidation of the chemistry and enzyme-bound environment of the enamine, one of the three ThDP-substrate covalent complexes on PDC; 4. modeling based on the x-ray coordinates to help refine mechanistic models, to help design even more insightful experiments, and to compare the three dimensional models of two enzymes with very high sequence homology to PDC's (pyruvate oxidase and acetolactate synthetase) that have identical mechanisms through pyruvate decarboxylation, but diverge significantly thereafter. Undoubtedly, the fundamental questions to be resolved by the proposed research will be of profound interest and significance to many other research groups working on thiamin around the globe.

Project Start
1998-07-01
Project End
1999-12-31
Budget Start
Budget End
Support Year
15
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Rutgers University
Department
Type
DUNS #
130029205
City
Newark
State
NJ
Country
United States
Zip Code
07102
Baykal, Ahmet; Chakraborty, Sumit; Dodoo, Afua et al. (2006) Synthesis with good enantiomeric excess of both enantiomers of alpha-ketols and acetolactates by two thiamin diphosphate-dependent decarboxylases. Bioorg Chem 34:380-93
Mattson, Brandi J; Williams, Sharon E; Rosenblatt, Jay S et al. (2003) Preferences for cocaine- or pup-associated chambers differentiates otherwise behaviorally identical postpartum maternal rats. Psychopharmacology (Berl) 167:1-8
Gilchrist, Alan L; Annan Jr, Vidal (2002) Articulation effects in lightness: historical background and theoretical implications. Perception 31:141-50
Vathy, Ilona; Komisaruk, Barry R (2002) Differential effects of prenatal morphine exposure on analgesia produced by vaginocervical stimulation or systemic morphine administration in adult rats. Pharmacol Biochem Behav 72:165-70
Mattson, B J; Williams, S; Rosenblatt, J S et al. (2001) Comparison of two positive reinforcing stimuli: pups and cocaine throughout the postpartum period. Behav Neurosci 115:683-94
Duque, A; Balatoni, B; Detari, L et al. (2000) EEG correlation of the discharge properties of identified neurons in the basal forebrain. J Neurophysiol 84:1627-35
Komisaruk, B R; Rosenblatt, J S; Barona, M L et al. (2000) Combined c-fos and 14C-2-deoxyglucose method to differentiate site-specific excitation from disinhibition: analysis of maternal behavior in the rat. Brain Res 859:262-72
Caba, M; Komisaruk, B R; Beyer, C (1998) Analgesic synergism between AP5 (an NMDA receptor antagonist) and vaginocervical stimulation in the rat. Pharmacol Biochem Behav 61:45-8
Sansone, G R; Bianca, R; Cueva-Rolon, R et al. (1997) Cardiovascular responses to vaginocervical stimulation in the spinal cord-transected rat. Am J Physiol 273:R1361-6
Burroughs, L F; Fiber, J M; Swann, J M (1996) Neuropeptide Y in hamster limbic nuclei: lack of colocalization with substance P. Peptides 17:1053-62

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