Date of Award
Doctor of Philosophy (PhD)
Biochemistry and Molecular Biology
Smith, Kerry S
Marcotte, Jr , William R
Morris , James C
Temesvari , Lesly A
Acetate, a short-chain fatty acid that plays a key role in all domains of life, can be utilized as a carbon source or excreted as a product of metabolism. Acetate kinase (ACK), a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as the phosphoryl donor. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya. A partially purified ACK which can utilize pyrophosphate (PPi) as the phosphoryl donor in the acetyl phosphate-forming direction was previously identified in Entamoeba histolytica, the causative agent of amoebic dysentery. Here I describe my research on the E. histolytica PPi-dependent ACK.
My biochemical and kinetic characterization of the E. histolytica ACK revealed several properties that are unique to this enzyme. First, the E. histolytica enzyme is capable of utilizing acyl substrates longer than butyrate and can utilize acyl substrates as long as hexanoate in the direction of acetyl phosphate synthesis. Second, this ACK can only utilize PPi as a phosphoryl donor and cannot utilize ATP. The kinetic parameters for substrate affinity indicate that the enzyme prefers both an acetate and propionate in the acetyl phosphate-forming direction; however, the low kcat raises question whether this direction of the reaction is physiological. The enzyme operates preferentially in the direction of acetate/PPisynthesis as the rate of catalysis and overall catalytic efficiency are 1,102-fold and 240,351-fold higher, respectively. Studies of the mechanism of this enzyme indicates that the PPi-ACK follows a sequential mechanism, supporting a direct in-line phosphoryl transfer mechanism previously reported in the well characterized Methanosarcina thermophila ATP-dependent ACK.
A modified hydroxamate assay was developed for measuring ACKs in the direction of acetate formation. A coupled enzyme assay has been used to measure ATP production in the direction for the prokaryotic ACKs, but there was not a method available for measuring a PPi-forming ACK in this direction. The assay described here allowed for the determination of kinetic parameters in the acetate-forming direction for the E. histolytica ACK and can not only be used to measure ACK activity of both ATP-dependent and PPi-dependent acetate kinases in the direction of acetate formation, but could also prove useful for other enzymes that utilize activated acyl substrates such as acetyl adenylate and acetyl-CoA.
Investigation of the residues in the putative PPi-ACK active site in E. histolytica revealed that while residues conserved across the ACK family are present in the active site, the enzyme has a number of significant active site changes in comparison to its counterpart, the M. thermophila ATP-dependent ACK. The residues His117, His172, Val87, Thr201, Thr15, Arg274, and Asp272 appear to be essential for catalysis in the direction of acetate synthesis. Furthermore, His117 appears to be critically important for binding acetyl phosphate and therefore important in acetate/PPi synthesis reactions.
RNA interference of E. histolytica ACK was unable to be carried out due to problems with transfection of plasmid DNA into the parasite. However, data garnered from cellular extracts indicates that E. histolytica has a PPi-dependent ACK but not ATP-dependent ACK. Activities of the eukaryotic ACK partner enzymes phosphotransacetylase and xylulose 5-phosphate/fructose 6- phosphate phosphoketolase were absent in cellular extracts. The absence of bacterial ACK partner enzymes in Entamoeba suggests that a novel acetyl phosphate generating enzyme is the partner for the PPi-ACK in this protist. This speculative partner enzyme could generate acetyl phosphate from an intermediate of the pentose phosphate pathway or the end product of a yet to be identified catabolic pathway.
Fowler, Matthew, "Investigations of the PPi-dependent Acetate Kinase from the Parasite Entamoeba histolytica" (2011). All Dissertations. 777.