This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. High resolution structural data (Larsen et al., 1996) and functional assays of site-directed mutant forms of yeast enolase (Poyner et al., 1996) indicate that Lys 345 serves as the catalytic base in the first step of the catalytic cycle of the enzyme. Lys 345 is positioned near the C-2 proton of the substrate, 2-phosphoglycerate (2-PGA), and mutant forms of enolase in which Lys 345 is changed to another residue have catalytic activities reduced by between 4 and 5 orders of magnitude relative to the wild type enzyme. Moreover, these mutant enzymes fail to catalyze exchange of the C-2 proton in D2O (Poyner et al., 1996). The crystallographically determined structure (Larsen et al., 1996) indicates that acidification of this carbon acid is accomplished by coordination of the carboxylate group to both required magnesium ions and a H-bonding interaction of one carboxylate oxygen to the ammonium group of Lys 396. It is well established that the pKa values of functional groups of amino acid side chains can be perturbed significantly in enzymic complexes (Highbarger et. al., 1996; Mcintosh et al., 1996), and interpretations of pH rate profiles are frequently problematic (Kyte, 1995). Nevertheless, understanding the energetics of the ionization at C-2 of 2-PGA in catalysis by enolase requires knowledge of the pKa of the side chain of Lys 345.
The aim of this project is to determine the pKa of a gamma-thialysine form of Lys 345 in yeast enolase and its complexes with substrates by direct titration using 15N or 13C NMR with isotopically labeled forms of thialysine.
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