Structure-Function Studies of Enzymes from Ribose Metabolism

In the pentose phosphate pathway, carbohydrates such as glucose and ribose are degraded with production of reductive power and energy. Another important function is to produce essential pentoses, such as ribose 5-phosphate, which later can be used in biosynthesis of nucleic acids and cofactors.This thesis presents structural and functional studies on three enzymes involved in ribose metabolism in Escherichia coli.Ribokinase is an enzyme that phosphorylates ribose in the presence of ATP and magnesium, as the first step of exogenous ribose metabolism. Two important aspects of ribokinase function, not previously known, have been elucidated. Ribokinase was shown to be activated by monovalent cations, specifically potassium. Structural analysis of the monovalent ion binding site indicates that the ion has a structural rather than catalytic role; a mode of activation involving a conformational change has been suggested. Product inhibition studies suggest that ATP is the first substrate to bind the enzyme. Independent Kd measurements with the ATP analogue AMP-PCP support this. The results presented here will have implications for several enzymes in the protein family to which ribokinase belongs, in particular the medically interesting enzyme adenosine kinase.Ribose 5-phosphate isomerases convert ribose 5-phosphate into ribulose 5-phosphate or vice versa. Structural studies on the two genetically distinct isomerases in E. coli have shown them to be fundamentally different in many aspects, including active site architecture. However, a kinetic study has demonstrated both enzymes to be efficient in terms of catalysis…


General aspects of metabolism
The pentose phosphate pathway
Ribose uptake in Escherichia coli
Enzymes discussed in the thesis: Ribokinase and Ribose 5-phosphate
Protein expression, purification and crystallization
RpiA and RpiB
Steady state kinetics
RpiA and RpiB
Treatment of data
Dead end inhibition
Product inhibition
Fluorescence spectroscopy
Crystallographic methods
RK from E. coli
Overall structure of RK
Active site and substrate binding
Solved RK structures
Proposed mechanism
Results: RK; paper I and IV
Activation by monovalent ions
Various aspects of RK structure
Functional studies on wt RK
Verification of the monovalent ion binding site: RK mutant S294K function and structure
Ion binding
Binding of nucleotides and phosphates
Aspects of RK kinetic mechanism
Ribose 5-phosphate isomerases from E. coli
The reaction
Results: RpiA and RpiB; Paper II, III, V
Structure of RpiA
Structure of RpiB
Structural analysis of the apo enzymes
Kinetics and inhibition
Structure of RpiA with bound inhibitor
Docking experiments
Genome searches
Summary in Swedish

Author: Andersson, C. Evalena

Source: Uppsala University Library

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