James A. Endrizzi Klaus Breddam S.James Remington

Biochemistry 1994, 33,11106-11120

Background

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

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

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

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

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

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

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

Crystallographic Refinement:-The atomic model was refined with the TNT package.

- The model was subjected to10-30 cycles of TNT refinemet

Results

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

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

Data Collection: – Diffraction data sets were collected from single crystals of native

CDP-Y, three heavy atom derivatives and CDP-Y complexed with benzylsuccinic acid.

Results

Phase Calculation:– Heavy atom parameters and their reaction sites are given on the

table below

Results

Model building/refinement:– 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.

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 CPD-

WII in fig 2.

Results

Active Site:– Residues comprising the catalytic triad (Ser 146-Asp 338-His 397)

lie near the bottom of a large hydrophobic pit

Discussion

Comparison of CPD-Y and CPD-WII fold:– They are distantly related enzymes with only 26% of amino

acid sequence identity.– In solution, CPD-Y is a single chain monomer, while CPD-

WII is a homodimer.

Discussion

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

Disulfide Bridges:– Disulfide bonds occur

between residues 56-303,210-222, 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.

Insertions and Deletions:

– Relative to CPD-WII, CPD-Y has 8 insertions, totaling 3 amino acids and 9 deletions, totaling 31 amino acids.

Discussion

Residues involved in substrate recognition:– There are at least three binding subsites in serine

carboxypetidase: The s1’ subsite (which binds to p1’ side chain of substrate) The S1 binding site The carboxylate binding site

Discussion

PH dependence of Activity:– 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

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 CPD-WII suggests that this insertion domain has changed more rapidly during evolution than the rest of the molecule.

References

Biochemistry 1994, 33, 11106-11120

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