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dc.contributor.advisorGoodwin, Douglas
dc.contributor.authorMoore, Robert
dc.date.accessioned2009-07-06T14:29:01Z
dc.date.available2009-07-06T14:29:01Z
dc.date.issued2009-07-06T14:29:01Z
dc.identifier.urihttp://hdl.handle.net/10415/1766
dc.description.abstractEnzymes are a highly diverse set of macromolecules, and, given their size and biocatalytic significance, each is an extremely complex system to study. As such, many assumptions must be made to simplify these systems to a manageable level to study. Unfortunately, the more complex the system, the more simplifications need to be introduced. Most simplifications entail two different types of assumptions: structural assumptions, and parametric assumptions. Since catalysis is generally limited to a small region of the enzyme (the active site), most non-active site structures are ignored if there is no evidence for substrate binding or allosteric control. In regards to parametric analysis, the assumption is that as long as one variable is held constant, that variable will have the same effect on a system regardless of changes to other variables. Catalase-peroxidases provide an ideal system to analyze these assumptions. Although having an active site identical to monofunctional peroxidases, catalase-peroxidases have significant catalase activity. Differentiation between which catalytic cycle is utilized appears to be linked to the pH of the environment. The commonly held assumption was that by varying the pH at substrate-saturating conditions, the pH optima of the two activities could be determined. Here, that assumption is dissected by varying pH and substrate concentrations simultaneously. This revealed substrate-dependent inhibition that had resulted in misidentification of the pH optima and possible misinterpretations of structural data. Another assumption was that by only providing the enzyme with the substrates required for one activity, the two different catalytic cycles could be studied and understood separately. Here, by placing the system in an environment where both activities could occur, it became clear that the two activities are synergistic and result in broadening the catalase pH range of the enzyme. Furthermore, the synergy of the two catalytic cycles rather than competition emphasized that the classical representation of catalase-peroxidase activity could not be true, leading to the proposal of a new mechanism. Previous studies have shown that an entire domain absent in monofunctional peroxidases is necessary for any catalysis in catalase-peroxidases. Here, by creating variants of residues 25 A (and further) away from the active site in two hydrogen bonding networks at the interface of the two domains, the significance of these non-active site networks is shown to be as great as some of the active site structures.en
dc.rightsEMBARGO_NOT_AUBURNen
dc.subjectChemistry and Biochemistryen
dc.titleTowards the Understanding of Complex Biochemical Systems: the Significance of Global Protein Structure and Thorough Parametric Analysisen
dc.typedissertationen
dc.embargo.lengthNO_RESTRICTIONen_US
dc.embargo.statusNOT_EMBARGOEDen_US


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