Regulation of human M2 pyruvate kinase
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Date
29/06/2015Author
Mitchell, Alice Rose
Mitchell, Rosie
Metadata
Abstract
Pyruvate kinase catalyses the final step in glycolysis and is responsible for net ATP
production. There are four pyruvate kinase isoforms expressed in humans; LPYK, RPYK,
M1PYK and M2PYK. The allosteric enzyme M2PYK plays an important role in cancer
cell metabolism and is subject to complex regulation by numerous naturally occurring
small-molecule metabolites. Post-translational modifications have also been found to play
a key role in the regulation of M2PYK, among these cysteine oxidation. This thesis
describes the production and characterisation of M2PYK cysteine point mutants in order
to investigate the mechanism of regulation by cysteine modification.
From a total of ten cysteines present in M2PYK, five were chosen for mutation based on
a combination of the results from the cysteine oxidation prediction program (COPP) web
interface and published experimental evidence for cysteine modification of M2PYK.
Eight point mutants of these five cysteines were produced and characterised. Low
resolution gel filtration of all the mutants shows that mutation of these cysteines has an
effect on tetramer:dimer:monomer equilibrium of M2PYK suggesting that cysteine
modifications could regulate M2PYK activity by affecting oligomeric state. Activity
assays show that none of the cysteine point mutations are sufficient to protect M2PYK
from oxidation by H2O2 indicating that more than one cysteine is involved in the
regulation of M2PYK by oxidation.
Nitric oxide (NO) imbalance has recently emerged as playing a key role in numerous
diseases including cancer. NO regulates the function of target proteins through the
addition of a nitroso moiety from NO-derived metabolites to a reactive cysteine, a
process known as protein S-nitrosylation. M2PYK has been found to be S-nitrosylated in
vivo. Using the biotin-switch assay in vitro combined with mass spectrometry I have
shown that a likely candidate for the target of S-nitrosylation of M2PYK is C326.
This thesis also describes the structures of two cysteine point mutants; M2PYK C424A
and M2PYK C358S. The structures show that these mutations have very little effect on
the overall conformation of M2PYK with only very subtle localised changes. The
structure of the mutant M2PYK C358S shows some interesting features including
varying occupation of the active site resulting in differing conformations of the B
domains within the same tetramer, and an unusual B factor distribution which could be
indicative of a perturbation in cooperativity within the tetramer caused by the mutation.