CHED 209 |
| Lactate dehydrogenase (LDH) is an oxidoreductase essential to adenosine triphosphate (ATP) synthesis under anaerobic conditions. It catalyzes a reversible reaction in which pyruvate is reduced via oxidation of NADH resulting in the formation of NAD+ and lactate. Blood levels of LDH are an important indicator of internal tissue damage, especially for skeletal muscle and the liver. LDH a tetrameric enzyme composed of two different subunits, M and H type. M subunits comprise isozymes often subjected to anaerobic conditions while H subunits are predominant in isozymes generally found under aerobic conditions. LDH-A (M4 isozyme) was purified from chicken breast muscle using standard techniques. LDH activity was assayed spectrophotometrically using established protocols. Crystals were obtained using commercially available random screens via vapor diffusion methods. Diffraction quality chicken LDH-A crystals have been grown from two screening conditions. Crystals were flash cooled and shipped to the Stanford Synchrotron Radiation Laboratory, where 1.9A native data were collected. The native structure has been solved by molecular replacement using porcine LDH-A as the probe. Crystals of chicken LDH-A in complex with NADH and pyruvate have been grown, and data has been collected at resolutions of 2.0A and 1.9A, respectively. Structure determination is in progress. Crystals of chicken LDH-A in complex with lactate and oxalate have been grown and data collection at SSRL is in progress. Evolutionary alterations in the flexibility of the molecule and patterns of amino acid sequence conservation suggest that the active site of LDH-A should be viewed as an extended unit that involves most, if not all, of the enzyme's structure for which substrate binding induces folding of a loop region over the active site. It is the goal of this study, in addition to determining a previously unsolved structure, to ascertain the veracity of this hypothesis. |
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Undergraduate Research Poster Session: Biochemistry
2:30 PM-4:30 PM, Monday, 11 September 2006 Moscone Center -- Hall D, Poster
Division of Chemical Education |