The long-term objective of this project is directed towards an understanding of the factors involved in the catalytic properties of enzymes.
The specific aim of this proposal involves the elucidation of the mechanism of bacterial luciferase from Vibrio harveyi. This enzyme catalyzes the production of visible light from reduced flavin mononucleotide, molecular oxygen, and a long chain aldehyde. The other products are oxidized flavin mononucleotide, water, and a long chain acid. The primary aims of the proposed research are as follows: 1. Determine the chemical structures of all intermediates the transition states that are involved in the production of light at the active site of luciferase. 2. Construct a free energy profile for the luciferase reaction mechanism by measuring all of the rate constant for the interconversion of all intermediates. 3. Determine the roles of specific amino acid residues at the active site of luciferase that are required for binding and catalysis. These objectives will be obtained by utilizing stopped-flow kinetic techniques to measure the rates of formation and decomposition of the various intermediates. These studies will be conducted as a function of pH, temperature, amino acid substitutions, and modifications in the chemical structures of the substrates. High field NMR studies will be used to establish the structures of the intermediates. Site-directed and random mutants will be used to help establish specific amino acids in specific steps in the reaction sequence and to alter the rate constants for the interconversion of specific intermediates. Heavy atom isotope effects at the reaction centers will be used in the construction of the free energy profile. Isotope effects will also be used to correlate stopped-flow spectral changes with the cleavage of bonds at specific sites.
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