Browsing by Subject "enzyme kinetics"
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Item A Structural and Kinetic Study into the Role of the Quaternary Shift in Bacillus stearothermophilus Phosphofructokinase(2011-10-21) Mosser, Rockann ElizabethBacillus stearothermophilus phosphofructokinase (BsPFK) is a homotetramer that is allosterically inhibited by phosphoenolpyruvate (PEP), which binds along one dimer-dimer interface. The substrate, fructose-6-phosphate (F6P), binds along the other dimer-dimer interface. The different functional forms BsPFK can take when in the presence of F6P and PEP can be described by the following diproportionation equilibrium: XE + EA <--> XEA + E where XE is the enzyme bound to PEP, EA is the enzyme bound to F6P, E represents the apo enzyme, and XEA is the ternary complex formed when both substrate and inhibitor are bound. Currently in the Protein Data Bank (PDB) there are two relevant forms of wild-type BsPFK, the EA form and the X'E form, which represents the enzyme bound to the PEP analog, phosphoglycolate (PGA). When comparing the EA and the X'E structures, a 7? rotation about the substrate-binding interface is observed and is termed the quaternary shift. The current study uses methyl TROSY NMR to examine the different liganded states of BsPFK, and for the first time structural data for the XEA species is shown. In addition, crystallography was used to obtain the first apo structure of BsPFK. To distinguish between changes associated with the quaternary shift and those associated with the intra-subunit tertiary changes, the variant D12A BsPFK was studied using kinetics, crystallography, and NMR. Crystal structures of apo and PEP bound forms of D12A BsPFK both indicate a shifted structure similar to the X'E form of wild-type. Kinetic studies of D12A BsPFK, when compared to wild-type, show a 50-fold diminished F6P binding affinity, 100-fold enhanced binding affinity, and a similar coupling constant. A conserved hydrogen bond between D12 and T156 takes place across the substrate binding interface in the EA form of BsPFK. The variant T156A BsPFK shows similar binding, coupling, and structural characteristics to D12A BsPFK. PEP still inhibits these variants of BsPFK despite the fact that the enzymes are in the quaternary shifted position prior to PEP binding. Therefore the quaternary shift of BsPFK primarily perturbs ligand binding but does not directly contribute to heterotropic allosteric inhibition.Item Variability of Specificity Determinants in the O- Succinylbenzoate Synthase Family(2012-12-10) Wang, Chenxi 1986-Understanding how protein sequence, structure and function coevolve is at the core of functional genome annotation and protein engineering. The fundamental problem is to determine whether sequence variation contributes to functional differences or if it is a consequence of evolutionary divergence that is unrelated to functional specificity. To address this problem, we cannot merely analyze sequence variation between homologous proteins that have different functions. For comparison, we need to understand the factors that determine sequence variation in proteins that have the same function, such as a set of orthologous enzymes. Here, we address this problem by analyzing the evolution of functionally important residues in the o-succinylbenzoate synthase (OSBS) family. The OSBS family consists of several hundred enzymes that catalyze a step in menaquinone (Vit. K2) synthesis. Based on phylogeny, the OSBS family can be divided into eight major subfamilies. We assayed wild-type OSBS enzyme activities. The results show that the enzymes from ?-Proteobacteria subfamily 1 and Bacteroidetes have relatively low values, the enzyme from Cyanobacteria subfamily 1 is intermediate, and the values for the proteins from the Actinobacteria and Firmicutes subfamilies are relatively high. We are using computational and experimental methods to identify functionally important amino acids in each subfamily. Our data suggest that each subfamily has a different set of functionally important residues, even though the enzymes catalyze the same reaction. These differences may have accumulated because different mutations were required in each subfamily to compensate for deleterious mutations or to adapt to changing environments. We assessed the roles of these amino acids in enzyme structure and function. Our method achieved 70% successful rate to identify positions that play important roles in one family but not another. The residues P119 and A329 play important role in D. psychrophila but not in T.fusca OSBS. We also observed two class switch mutations in T.fusca, P11 and P22. The mutations at these two position have a similar kinetic parameters as wild-type D. psychrophila OSBS.