I. Introduction
The chemical reaction of enzymatic transamination was
discovered about 50 years ago. In this chemical reaction, an
amino group ios transferred to a keto acid to form new amino
acids. Since enzymatic transamination was first discovered,
more than 60 transaminases have been identified. The two
common types of transaminases are alanine aminotransferase
(ALT) and aspartate aminotransferase (AST). Aspartate
aminotransferase cataylzes the reaction in which aspartate and
alpha-ketoglutatrate is inter-converted into oxaloacetate and
glutamate.
AST is most commonly used and measured clinically as a part
of diagnostic liver function test, to determine liver health.
However, the liver is not always the source of AST when it
appears in blood tests. For example, when the level of AST is
higher than ALT levels, it should be considered that the
enzymes are coming from a muscle source rather than the liver.
While AST and ALT are able to be used in liver diagnostic
tests, there is the reminder that the enzymes are not good
measures because of they do not accurately represent the
synthetic ability of the liver and they could be coming from
tissue sources other than the liver.
II. General Structure
Aspartate aminotransferase is a dimer with a
molecular weight of 88,000, with two polypeptide chains
composed of 396 amino acids. The JMol displayed molecule is
the monomer version of this dimer. The active-site residues
are indetical to those found in eukaryotes. The complete
structure of aspartate aminotransferase is made up of two
subunits, each with two domains. There is a
large domain
containing the
Lysine (K258),
which forms the Schiff base with the PLP in the
wild-type enzyme. However, the lysine in this molecule has been changed to another amino acid. The
small domain
interacts with the alpha-carboxyl group of the
substrate when the substrate is bound. The large domain and
the small domain are connected by the
46-� helix.
III. Active Site Mutation
This molecule has a mutation at Lysine
(K258) which had been changed to
alanine.
In the wild-type,lysine 258 forms the Schiff base with
the pyridoxal phosphate. The enzyme is still able to
bind PLP and PMP, but is not able to bind them
covalently.
IV. Tertiary Structure of K258A Mutant
The structure of the mutant is very similar
to that of the wild-type. Features of the monomer include
the large coenzyme
binding domain, the small
domain, and an
extended N-terminal tail.
The large domain is made up of seven-stranded mixed
six-parallel, one antiparallel) (
pleated sheets (*pleated sheets for
small domain are also highlighted)
and the N-terminal of the long
helix.
The small domain is composed of (
four parallel helices(*helices
for large domain are also highlighted)
, a small segment of mixed pleated sheets, and the C-terminal
end of the long helix. There is no significant change seen in
the secondary or tertiary structure caused by the absence of
the lysine 258 sidechain.
V. Active Site Region
The active site of aspartate aminotransferrase K258A
is split up into five different regions, each of those five
regions having their own specific interactions.
The position of the substrate is stabilized by
the interactions of the alpha- and distal-carboxylate groups
with
Arg 386
and Arg 292 (which is in the large domain of the other subunit of the dimer). The phosphate group of the cofactor is situated at the the helix formed by
N-terminus of
residues 108-122. A network of hydrogen bonds
originate from the neighboring residues includes the
side-chain OH's of
Ser 255
, Tyr 70 from the other subunit, and
Thr 109
, and the main chain NH's of
Gly 108
and
Thr 109. A salt bridge is formed
between
Arg 266
and the phosphate group of the cofactor. The
pyridoxal ring of the PMP cofactor interacts with
Asp 222
and
Tyr 225. Because there is no interaction with Lys 258 in the
mutant, the PLP form of the mutant is much less active towards
aspartic acid as a substrate than the wild-type enzyme.
VI. References
Amino acid metabolism. (n.d.).
Retrieved December 10, 2018, from
http://watcut.uwaterloo.ca/webnotes/Metabolism/AminoAcids.html
Aspartate Aminotransferase. (n.d.).
Retrieved December 9, 2018, from
http://www.worthington-biochem.com/cgot/
Douglas L. Smith, Steven C. Almo,
Michael D. Toney, and Dagmar Ringe; 2.8-.ANG.-resolution
crystal structure of an active-site mutant of aspartate
aminotransferase from Escherichia coli, Biochemistry 1989 28
(20), 8161-8167, DOI: 10.1021/bi00446a030
Okamoto, A., Hirotsu, K., Higuchi, T.,
Kuramitsu, S., & Kagamiyama, H. 1991. Three-dimensional
Structure of Aspartate Aminotransferase from Escherichia
coli. Enzymes Dependent on Pyridoxal Phosphate and Other
Carbonyl Compounds As Cofactors, 107-109.
doi:10.1016/b978-0-08-040820-0.50024-8
Steven C. Almo, Douglas L. Smith, Avis
T. Danishefsky, Dagmar Ringe; The structural basis for the
altered substrate specificity of the R292D active site
mutant of aspartate aminotransferase from E.coli, Protein
Engineering, Design and Selection, Volume 7, Issue 3, 1
March 1994, Pages 405�412,
https://doi.org/10.1093/protein/7.3.405
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