Transcript Slide 1

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James A. Endrizzi
Klaus Breddam
S.James Remington
Biochemistry 1994, 33,1110611120
Background
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Serine carboxypeptidases are exopeptidases
that remove C-terminal amino acids from
peptides.
Their optimum peptidase activity is at a pH of
4.5-5.5, depending on the enzyme
They are found in every eukaryote and are
divided into three general classes based on
substrate specificity
Background
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The less specific serine carboxypeptidases are grouped
into two classes : C and D
Carboxypeptidases C are subsets of the serine
carboxypeptidase family with high specificity for
hydrophobic residues at the P1’ position.
Carboxypeptidases D most efficiently hydrolyze basic
residues at P1’
Serine Carboxypeptidase from Saccharomyces
cerevisiae (CPD-Y) is a D carboxypetidase.
The atomic model of CPT-Y was determined by multiple
isomorphous replacement and refined at 2.8A resolution.
The model of CPT-Y was compared with that of wheat
serine carboxypeptidase II (CPD-WII)
Materials and Methods
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Deglycosylation/ Purification:
- CDP-Y was efficiently deglycosylated using endoglycosidase H.
- The reaction is 100 fold more efficient at pH 4.5 in acetate buffer as
opposed to pH 5.5 in citrate buffer.
- The preparative scale for deglycosylated reactions consisted of the
following:
*11ml of CPD-Y(18.8mg/ml of water),5ml of 0.5M sodium acetate
(pH 4.5), 27ml of water, and 0.1 unit of endo-H in 0.1ml of water. The
reaction was allowed to proceed 24 h at 35 C.
* CPD-Y was assayed spectroscopically at 340nm.
-The deglycosylated enzyme was repurified by affinity
chromatography.
-Sodium citrate (10mg, pH 5.3) was added per mg of CPD-Y, followed
by lyophilization.
Materials and Methods
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Crystallization:
- Crystals were grown by hanging drop vapor diffusion in tissue
culture plates.
- A total of 5ml of well solution 18-24% poly(ethylene glycol)
(PEG), Mr= 6000, 0.3M NaOAc, 0,05M NaCl, and 100 mM
imidazole/NaOH, pH 6-8, was added to 5ml of CPT-Y at
10mg/ml in 0.1M NaCl, 1mM DTT, and 20 mM citrate/ NaOH,
pH 6.5, on a salinized cover slip and inverted over wells
containing 1ml of precipitant.
- After several months, cubic crystals formed and stored in a
solution identical to the wells but containing 26-28% PEG-6000.
Materials and Methods
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Data Collection:
- Data were collected at room temp. on a San Diego Multiware
Systems area detector using graphite- monochromated Cu Kα
from a Rigaku RU200 rotating anode operated at 40kV and
150mA.
Materials and Methods
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Heavy Atom Derivatives:
- Potential heavy atom derivatives were screened by soaking
crystals in storage solution containing 1,10, and 100%
saturated compound for 1 day to 2 weeks, followed by precision
photography.
- These conditions were used:
*pCMB: 1mM, 3 days
* PtCl4: 10mM, overnight
* methylmercury iodine (MMI): saturated, 4 days
Materials and Methods
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Phase determination and Model Building:
-An electron density map was calculated at 3.5A resolution,
which immediately revealed helical segments of the correct
handedness. The α-carbon coordinates of CDP-WII were
positioned by hand using FRODO as a start point.l;
-In the initial model, 293 out of 421 amino acids were included
but did not include most residues 180-317.
Materials and Methods
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Crystallographic Refinement:
-The atomic model was refined with the TNT package.
- The model was subjected to10-30 cycles of TNT refinemet
Results
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Deglycosylation:
- CDP-Y contains four N-linked carbohydrate chains.
-Fully glycosylated CDP-Y was converted to products molecular
weight 57000 and 53000 within 1h indicating removal of
carbohydrate. After 24h, only 53000 molecular band remained
corresponding to a reduction in molecular mass of 13000Da.
-The final yield of Deglycosylated product was 70%.
Results
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Crystallization:
-Crystallized in two forms, Orthorhombic crystals and cubic
P213 crystals.
-The cubic crystals were chosen but further study because they
are less sensitive to X-rays and have higher symmetry. They
have a monomer in the asymmetric unit, with Vm=2.1A3/Da.
Results
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Data Collection:
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Diffraction data sets were collected from single crystals of native
CDP-Y, three heavy atom derivatives and CDP-Y complexed with
benzylsuccinic acid.
Results
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Phase Calculation:
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Heavy atom parameters and their reaction sites are given on the
table below
Results
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Model building/refinement:
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Electron density was verified for the following features:
single carbohydrate residues at Asn 87, Asn 168, Asn 368;
the catalytic triad and flanking residues, 5 disulfide bonds
and several model insertions in CPD-Y relative to CPD-WII.
Figure 1 illustrates the improvement in the quality of the
electron density between the 3.5A MIR map and the final
2.8 A 2Fo-Fc map for some turns not present in CPD-WII.
The final model consists of 3333 non-hydrogen atoms, all
421 amino acids, 3 of 4 N-linked carbohydrate residues and
38 water molecules. Has an R factor of 0.162 A for 10 909
independent reflections between 20 and 2.8A.
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The molecule has a central 11 stranded mixed β-sheet that
twists aprox. 180 degrees end to end. It has 14 α-helices
Disulfide bridges are found between residues 56-298,193-207,
217-240, 224-233, and 262-268. They are located surrounding
the active site.
Pro 54 and Pro 96 are in the cis config.
The secondary structure of CPD-Y is compared to that of CPDWII in fig 2.
Results
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Active Site:
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Residues comprising the catalytic triad (Ser 146-Asp 338-His 397)
lie near the bottom of a large hydrophobic pit
Discussion
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Comparison of CPD-Y and CPD-WII fold:
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They are distantly related enzymes with only 26% of amino
acid sequence identity.
In solution, CPD-Y is a single chain monomer, while CPDWII is a homodimer.
Discussion
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Insertion Domain and Hydrophobic Surface:
- Helix 230-251 contributes Val 230, Trp 231, Val 234, Pro 235, Ile 238, and
Tur 239 to the hydrophobic patch. This patch may be involved in substrate
recognition and possibly also substrate channeling. Specific hydrophobic
(CPD-Y ) to acidic /polar (CPD-WII) changes are important for determining
substrate.
Discussion
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Disulfide Bridges:
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Disulfide bonds occur
between residues 56303,210-222, and 246-268
of CPD-WII and residues
56-298, 193-207, 217-240,
224-233, and 262-268 of
CPD-Y. Disulfide 56-298 is
at the edge of the active
site, close to serine 146
nucleophile.
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Insertions and
Deletions:
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Relative to CPD-WII,
CPD-Y has 8 insertions,
totaling 3 amino acids
and 9 deletions, totaling
31 amino acids.
Discussion
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Residues involved in substrate recognition:
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There are at least three binding subsites in serine
carboxypetidase:
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The s1’ subsite (which binds to p1’ side chain of substrate)
The S1 binding site
The carboxylate binding site
Discussion
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PH dependence of Activity:
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Exhibit maximum activity between PH 4-5
depending on enzyme.
The lack of strong dependence of kcat on pH led
to the suggestion that the active site His has a
very low pKa (<3)
Unusual interaction between Glu 65 and Glu 145
(hydrogen bond)
Conclusion
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Serine carboxypeptidase also have large
insertion relative to the core topology
(residues 180-317) that may be functionally
important, in recognition of peptide
substrates.
The comparison between CPD-Y and CPDWII suggests that this insertion domain has
changed more rapidly during evolution than
the rest of the molecule.
References
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Biochemistry 1994, 33, 11106-11120
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