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2004
LSU-HHMI Summer Undergraduate Research Program |
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 Nathan
R. Ranney Lakesha F. Johnson (SUBR/LBRN), Grover L. Waldrop,
Biological Sciences
Functional Genomics of Carboxyltransferase
Acetyl-CoA carboxylase (ACC) catalyzes the first committed step
in the biosynthesis of long-chain fatty acids in all living
organisms. The Escherichia Coli form of the enzyme consists
of biotin carboxylase, carboxyltransferase, and a biotin carboxyl
carrier protein. The biotin carboxyl carrier protein has the
biotin moiety covalently attached and acts as a substrate for
biotin carboxylase and carboxyltransferase. This research primarily
focused on carboxyltransferase, a heterotetramer containing
a 35kD a subunit and a 31kD ß subunit. The objective of
the research was to kinetically characterize mutants of carboxyltransferase.
A blast search was used to identify several conserved residues
in this protein. On the a subunit, two amino acids—Lys142
and Arg145—were found to be strictly conserved. These
residues were each mutated to alanine. Steady-state kinetic
and inhibition studies were then performed on each mutant—K142A
and R145A. The Km values of K142A and R145A for both biocytin
and malonyl CoA did not vary greatly from the previously determined
values for wild-type carboxyltransferase. This suggests that
Lys142 and Arg145 are not involved in the binding of substrates.
However, the mutations did significantly decrease the Vmax value
for the reaction, indicating that Lys142 and Arg145 are involved
in catalysis of the reaction. Inhibition studies revealed that
the mutations also caused the kinetic mechanism to change from
an ordered to a random addition of substrates. Future work will
include the kinetic characterization of mutants of the ß
subunit of carboxyltransferase.
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