Park, Jinseo

Rhee, Sangkee

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676a Wednesday, February 19, 2014with a high and low affinity. In addition, the ITC experiments of L-PGDS
mutant proteins suggested that the active Cys65 residue is located at the high
affinity-binding site. The results of these experiments will be discussed in
this paper.
3423-Pos Board B151
The Evolutionary Constraints Imposed on Tyrosine Hydroxylation by its
Beginnings as a Phenylalanine Hydroxylase; Studies of the Polypeptide
Loop Determining the Substrate Specificity
Ewa Nowara.
St. Mary’s University, San Antonio, TX, USA.
Tyrosine hydroxylase (TyrH), phenylalanine hydroxylase (PheH) and trypto-
phan hydroxylase (TrpH) belong to the family of aromatic amino acid hydrox-
ylases (AAAH) and are genetically related. An ancient PheH probably was the
ancestor of the modern enzymes. Their active sites are very similar, however
their substrate specificities vary. They all hydroxylate phenylalanine; TyrH is
the only one that can make DOPA from tyrosine. Previous work identified
the aspartate at position 425 in TyrH, which lies in a very flexible and dynamic
polypeptide loop, to be critical for the enzyme’s achievement of this new activ-
ity. We started this project to examine whether alterations in the shape of this
polypeptide loop could make a stronger TyrH. The crystal structure of TyrH
shows D425 is at the end of this loop. We also knew that substituting it with
a glutamate (D425E) decreased its DOPA forming ability 10-fold. We sought
to find out if making the loop shorter but keeping the glu would make a
strong TyrH. Therefore, we deleted separately and together the glutamines
(Q424 and Q426) that are adjacent to D425. Of these three mutants, only
D425EDQ426 has comparable activity to WT. Previously reported Vmax
values are: WT, 150; D425ETyrH, 13.4. The value for D425EDQ426 is 118.
We determined the specific activity for the enzyme by finding the ratio of
Vmax/KM for tyrosine to Vmax/KM for phenylalanine and comparing this
to WT’s and previous mutants’ values. The value for D425EDQ426 was 3.3,
and for WT, 3.75, and for D425ETyrH, 0.34.
3424-Pos Board B152
Structural and Functional Basis for Substrate Specificity and Catalysis of
Levan Fructotransferase
Sangkee Rhee, Jinseo Park.
Seoul National University, Seoul, Korea, Republic of.
Levan is b-2,6-linked fructan and serves as reserve carbohydrate in some
plants and microorganisms. This fructan is usually utilized by enzymes such
as glycoside hydrolase (GH) for producing a fructose. Levan fructotransferase
(LFTase) of the GH32 family catalyzes an intramolecular fructosyl transfer re-
action and results in production of cyclic difructose dianhydride, thus exhibit-
ing a novel substrate-specificity. The mechanism by which LFTase carries out
these functions via the structural fold conserved in the GH32 family is un-
known. Here, we report the crystal structure of LFTase from Arthrobacter
ureafaciens in apo form and complex with sucrose and levanbiose. Despite
the structural similarity with members of the GH32 family, LFTase contains
the 1 and 2 subsites for a difructosaccharide in an active site. This feature
is unique among GH32 proteins and is facilitated by small side-chain residues
in the loop region of a catalytic domain, which is conserved in the LFTase
family. Together with functional analysis, our data provide a molecular basis
for the catalytic mechanism of LFTase and suggest functional variations from
other GH32 family proteins, notwithstanding the conserved structural
elements.
3425-Pos Board B153
Co Recombination in Human IDO and TDO - A Comparison
Karin Nienhaus, G. Ulrich Nienhaus.
Karlsruhe Institute of Technology, Karlsruhe, Germany.
The heme enzymes tryptophan 2,3-dioxygenase (TDO) and indoleamine
2,3-dioxygenase (IDO) catalyze the oxidative cleavage of the L-tryptophan
(L-Trp) pyrrole ring and the insertion of molecular oxygen to produce
N-formylkynurenine, the first and rate-limiting step in the kynurenine pathway.
The catalytic reaction involves the formation of a ternary complex including
the protein, the ligand (O2), which binds to the ferrous heme iron, and the sub-
strate (L-Trp). For optimal turnover of the enzyme, the protein scaffold has to
provide easy access for both O2 and L-Trp to the heme active site. Efficient O2
association may require transient docking sites to be accessible. As both oxy-
gen atoms are inserted into the C2 - C3 bond of the L-Trp pyrrole ring during
the reaction, efficient ligand escape is (most likely) not required. L-Trp binding
calls for a rather large ’access gate’. In addition, the reaction product N-formyl-
kynurenine must be expelled from the active site to allow the uptake of new
reagents.The molecular details of the catalytic mechanisms employed by TDO and IDO
have, as yet, not been elucidated. Therefore, we have investigated ligand
rebinding in ferrous IDO and TDO using flash photolysis at ambient tempera-
tures, replacing the physiological ligand O2 by CO, to disclose both similarities
of and differences between the enzymes. CO allows us to exploit its excellent
properties as a spectroscopic probe. Moreover, CO is not reactive towards
L-Trp, so that we can focus on ligand and substrate dynamics in the absence
of the ensuing enzymatic reaction.
3426-Pos Board B154
Molecular Dynamics Simulations of the Proton Transfer Reaction Between
the Catalytic Residues in HTLV-I Protease
Shuhua Ma, Natalie Petrillo, Kimberly Vogt.
Towson University, Towson, MD, USA.
In order to study the catalytic mechanism of Human T-cell leukemia virus type
I (HTLV-I) protease, it is important to determine the protonation states of the
two aspartic acid residues at its active site. Free energy simulations have been
conducted to study the proton transfer reaction between the catalytic residues of
HTLV-I protease using a combined quantum mechanical and molecular
mechanical (QM/MM) molecular dynamic simulation. Free energy profiles
for the reaction in apo-enzyme and in an enzyme-substrate complex will be pre-
sented. In addition, the structural features of HTLV-I protease will be compared
and discussed for different protonation states of the active site residues.
3427-Pos Board B155
Evidence for Functional Role of C-H$$$S Hydrogen Bond in Enzyme
Catalysis and Substrate Specificity: Type 1 Methionine Aminopeptidase
Anthony Addlagatta, Ravikumar Reddi.
Indian Institute of Chemical Technology, Hyderabad, Andhra Pradesh, India.
This study is about the functional role of a weak C-H$$$S hydrogen bond
between conserved cysteine in the S1 pocket of methionine aminopeptidase
(MetAP) and the substrate methionine during recognition and catalysis. Muta-
tion of this conserved cysteine leads to relaxed substrate specificity, reduced
stability and catalytic efficiency. Unexpectedly short distance (3.6 A˚) is noticed
between the cysteine S-atom from the enzyme and the carbon atom of the
substrate. pKa of this cysteine was found
to be much lower compared cysteines in
proteins. Crystal structures of the mutant
and the inhibitor complexes explain the
polarizability and reactivity of the
cysteine. Since the pKa is lower than
the physiological pH, cysteine exists as
a thiolate anion and interacts with sub-
strate methionine by forming C-H$$$S
hydrogen bond. The importance of this
interaction is underscored from the fact
that MetAPs process only peptides with
methionine on the amino terminus.
This is the first example of such a
weak interaction playing critical role in
the conserved function of a family of
essential enzymes.3428-Pos Board B156
Monitoring Conformational Changes that Occur to Blood Coagulant
Prothrombin as it is Activated to Thrombin
Muriel Maurer, Marina Malovichko.
Chemistry, University of Louisville, Louisville, KY, USA.
The blood coagulation protein thrombin is originally expressed as the zymogen
prothrombin (ProT). Prothrombin contains the F1.2 (GlaþKringle 1þKringle
2) domain and the inactive serine protease prethrombin-2 (PT2). Kringle 2 of
F1.2 protects an immature anion binding exosite on ProT called pro-ABE II.
The prothrombinase complex converts ProT to thrombin. Cleavage of ProT
after R271 releases F1.2 from PT2. Cleavage of PT2 at R320 helps to generate
the active, two-chain species thrombin that contains an A and B chain linked by
a disulfide. Hydrogen-Deuterium Exchange coupled with MALDI-TOF mass
spectrometry was used to monitor the conformational dynamics of ProT,
F1.2 þ PT2, PT2, and thrombin. During the activation process, the A-chain
and the autolysis loop undergo changes in solvent accessibility that respond
to the individual R271 and R320 cleavages. The resultant conformational prop-
erties aid in creating a protease that can accept substrates at the active site.
Importantly, this coagulation protein system also uses anion binding exosites
to control its functions. F1.2 protects pro-ABE II on both ProT and PT2
from solvent exposure. Moreover, this binding event exhibits a long-range


Structural and Functional Basis for Substrate Specificity and Catalysis of Levan Fructotransferase 

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Structural and Functional Basis for Substrate Specificity and Catalysis of Levan Fructotransferase

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