Investigation of acetate kinase, butyrate kinase, and caveolin
Abstract
Acetate kinase is a well conserved, ancient enzyme that is widespread in both the Bacterial and Archaea domains, yet its primary function is different in these two evolutionary branches. Bacteria primarily use acetate kinase for energy generation by converting ADP and acetyl phosphate into ATP and acetate. Archaea use the reverse of this reaction as the first step of methanogenesis, the sequestration and activation of acetate. Despite decades of kinetic investigation, it is unclear whether a phosphoenzyme is an intermediate in catalysis. To address this, a combination of MAD and MIR phasing was used to determine the structure of Methanosarcina thermophila acetate kinase to 1.8 Å. This protein is clearly a member of the actin/Hsc70/sugar kinase superfamily. The active site structure would support either a direct transfer of the phosphate or a triple-displacement mechanism, and therefore lends support to the proposed reaction schemes that allow the reaction to occur via either mechanism. The structure suggests future experiments to answer remaining enzymological questions. A butyrate kinase from Thermotoga maritima was cloned, purified and characterized. This enzyme is a member of the acetate kinase superfamily. It is the first butyrate kinase to show a preference for branched-chain carboxylic acids. The substrate specificity and operon position of this protein clearly define it as a branched-chain acyl kinase. Thus, T. maritima belongs to the exclusive group of bacteria that possess a more efficient pathway for the catabolism of branched chain amino acids. Butyrate kinase was determined to be a hexamer, and homogeneous solutions can be isolated and concentrated. Crystallization trials are in progress. Caveolin is a membrane-associated protein that oligomerizes and leads to the formation of caveolae, organelle-like invaginations of the plasma membrane of eukaryotes. It also binds to key signal transduction proteins in a regulated fashion, providing a scaffold for the concentration of ready-to-signal proteins, such as Ras, G-protein alpha subunits, and nitric oxide synthase. Aberrations in caveolin and caveolae formation have been directly linked to a number of human diseases, including cancers and muscular dystrophy. Several forms of recombinant caveolin-1 were purified and characterized with the goal of crystallizing this vital protein.
Degree
Ph.D.
Advisors
Hasson, Purdue University.
Subject Area
Biophysics|Molecular biology|Cellular biology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.