THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 14, pp.

8969 8975, April 4, 2008 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Substrate Specificity of Platypus Venom L-to-D-Peptide Isomerase*

Received for publication, November 29, 2007 Published, JBC Papers in Press, December 24, 2007, DOI 10.1074/jbc.M709762200

Paramjit S. Bansal, Allan M. Torres, Ben Crossett, Karen K. Y. Wong, Jennifer M. S. Koh, Dominic P. Geraghty, Jamie I. Vandenberg , and Philip W. Kuchel1 From the School of Molecular and Microbial Biosciences, University of Sydney, New South Wales 2006, Nanoscale Organisation and Dynamics Group, College of Health and Science, University of Western Sydney, Penrith South DC, New South Wales 1797, School of Human Life Sciences, University of Tasmania, Launceston, Tasmania 7250, and Mark Cowley Lidwill Cardiac Electrophysiology and Biophysics Program, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales 2010, Australia
The L-to-D-peptide isomerase from the venom of the platypus (Ornithorhyncus anatinus) is the first such enzyme to be reported for a mammal. In delineating its catalytic mechanism and broader roles in the animal, its substrate specificity was explored. We used N-terminal segments of defensin-like peptides DLP-2 and DLP-4 and natriuretic peptide OvCNP from the venom as substrates. The DLP analogues IMFsrs and ImFsrs (srs is a solubilizing chain; lowercase letters denote D-amino acid) were effective substrates for the isomerase; it appears to recognize the N-terminal tripeptide sequence Ile-Xaa-Phe-. A suite of 26 mutants of these hexapeptides was synthesized by replacing the second residue (Met) with another amino acid, viz. Ala, -aminobutyric acid, Ile, Leu, Lys, norleucine, Phe, Tyr, and Val. It was shown that mutant peptides incorporating norleucine and Phe are substrates and exhibit L- or D-amino acid isomerization, but mutant peptides that contain residues with shorter, -branched or long side chains with polar terminal groups, viz. Ala, -aminobutyric acid, Ile, Val, Leu, Lys, and Tyr, respectively, are not substrates. It was demonstrated that at least three N-terminal amino acid residues are absolutely essential for L- to D-isomerization; furthermore, the third amino acid must be a Phe residue. None of the hexapeptides based on LLH, the first three residues of OvCNP, were substrates. A consistent 2-base mechanism is proposed for the isomerization; abstraction of a proton by 1 base is concomitant with delivery of a proton by the conjugate acid of a second base.

Bioactive peptides that contain D-amino acid residues occur in lower animals such as snails (1, 2), crustaceans (3), and spiders (4, 5) and among more the highly evolved amphibians, frogs (6, 7). In mammals, to date, they have only been reported for the Australian duck-bill platypus (Ornithorhyncus anatinus) (8). These peptides are invariably components of venoms. In all cases there is only one D-amino acid residue present, and

* This work was supported by an Australian Research Council Discovery Project grant awarded (to P. W. K. and J. I. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence should be addressed: School of Molecular and Microbial Biosciences, Bldg. G08, University of Sydney, New South Wales 2006, Australia. Tel.: 61-2-9351-3709; Fax: 61-2-9351-4726; E-mail: p.kuchel@mmb.usyd.edu.au.

it is near the N terminus in the case of the frog and platypus peptides, but it is near the C terminus in the case of spider venom peptides. In the latter, the third amino acid residue from the C terminus is in the D-form (4, 9). In all cases the D-amino acid residues are the result of post-translational modification of the original all-L-peptide (10, 11). The stereo-isomerization is catalyzed by a specific enzyme called peptidyl aminoacyl L/Disomerase or, as we use hereafter, peptide isomerase. The crude venom of Australian male platypus contains 50 peptides (12); among them are a C-type natriuretic peptide (OvCNP) and defensin-like peptides (DLPs).2 Both OvCNP and one of the DLPs exist in two isomeric forms; OvCNP exists in an a and a b form with OvCNPa consisting of all L-amino acids, whereas OvCNPb has a D-Leu in position 2 (12). Similarly, DLP-4 consists of all L-amino acids, whereas DLP-2 contains D-Met at position 2 (13). The presence of the L-to-D-amino acid residue isomerase has been reported in several cases where peptides that contain the corresponding D-amino acid have been isolated and characterized. We hypothesized the presence of a similar type of isomerase in platypus venom; thus we reported the partial purification of the peptide isomerase and an estimate of its molecular size, 50 60 kDa (12). Because the peptides that contain all L-residues and those with a D-residue elute at significantly different times (on the order of minutes) in reverse phase-high performance liquid chromatography (RP-HPLC), a convenient assay was developed for the peptide isomerase. Earlier we reported D-amino acid isomerization in small N-terminal peptides derived from DLP-2/DLP-4 (IMFFEMQ and ImFFEMQ; where the lowercase letter denotes the D-form of the amino acid residue) (12). In this study we showed that only the first three amino acid residues were absolutely required for peptide isomerase activity on potential substrates. To increase their solubility in aqueous buffers, we appended a short peptide consisting of three polar D-amino acid residues (srs). We chose these residues to be D-type on the supposition that they would be stable in the presence of L-specific carboxypeptidases.
2

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The abbreviations used are: DLP, defensin-like peptide; Abu, -aminobutyric acid; DMF, dimethylformamide; Fmoc, [N-(9-fluorenyl)methoxycarbonyl]; RP-HPLC, reverse phase high performance liquid chromatography; MS, mass spectrometry; Nle, L-norleucine; nle, D-norleucine; PMSF, phenylmethylsulfonyl fluoride; TIPS, triisopropylsilane; PBS, phosphate-buffered saline.

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We report here on our systematic study of the substrate specificity of platypus venom peptide isomerase using a set of 26 synthetic hexapeptides; and we also propose a plausible mechanism for L-to-D isomerization. Venom Gland ExtractsVenom glands were dissected from deceased animals after tying the ducts with ligatures. The glands were finely sliced and the ligatures removed, and the tissue was placed in 50 ml of ice-cold 1% (w/v) trehalose solution. The sample was homogenized with a Bamix cutter using short bursts of cutting ( 5 s) to ensure that the temperature remained low and avoided enzyme denaturation. The mixture was centrifuged at 3000 g for 10 min and at 5 C; solid material separated by the centrifugation was discarded, and the supernatant was decanted and passed through a 0.2- m Minisart filter (Sartorious, Gottingen, Germany) to avoid clogging the HPLC col umn. The filtrate was dispensed into 1.5-ml Eppendorf (North Ryde, New South Wales, Australia) centrifuge tubes and frozen and stored at 80 C for later use. Isomerase AssayThe platypus gland extract (previously frozen) containing all the peptides was fractionated into two fractions by centrifugal ultrafiltration using an Amicon (Millipore Corp., Bedford, MA) Ultrafree-MC device with 30-kDa nominal molecular weight cutoff. 100 l of gland extract was added to the filter cup followed by addition of 30 l of PBS and spun at 3000 g for 5 min. The filtrate was retained for further isolation of the proteins. The peptide isomerase-active retentate that contained components with nominal Mr 30,000 was washed twice by reconstituting it with 2 volumes of 50 mM phosphate containing 0.9% (w/v) NaCl (PBS) at pH 7.4, and then repeating the ultrafiltration. Finally, the retentate was reconstituted to the original volume with 50 mM PBS. In a typical experiment, 20 l of fractionated venom gland extract, 10 l of peptide solution (1 mg ml 1, 50% A and B), and 10 l of 100 mM EDTA were incubated at 37 C in a water bath. Aliquots for assaying peptide isomerase with our HPLC method were withdrawn at 0, 2, 4, and 20 h for each peptide solution. New AssayFor isomerization studies, 1 mg ml 1 solutions of each peptide was prepared in 50% v/v acetonitrile. A 20- l aliquot of this solution was mixed with 10 l of 100 mM EDTA and 10 l of the gland extract. The progress of the reaction was measured by HPLC at 0-, 2-, and 4-h intervals. The enzymatic reaction was recorded in each direction, L-to-D, and D-to-L; and when the reaction occurred, it appeared to reach equilibrium within 20 h.

MATERIALS AND METHODS Amino Acids and RP-HPLCAll Fmoc amino acids (L and D) with appropriate protection (Fmoc-Ile-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Nle-OH, Fmoc-Abu-OH, Fmoc-AlaOH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Trp(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu), and Fmoc-Arg(Pbf)-OH) and other reagents were purchased from Auspep Pty. Ltd. (Melbourne, Victoria, Australia). RP-HPLC was used to purify synthesized peptides with a semi-preparative C18 Vydac column (Deerfield, IL) on a GBC system (Invitrogen; flow rate 3 ml min 1) and using a linear gradient of 1% B min 1 (solvent B: 90% v/v acetonitrile containing 0.1% v/v trifluoroacetic acid) and solvent A (water containing 0.1% v/v trifluoroacetic acid). The purity of the fractions was monitored by analytical RP-HPLC using a C18 analytical column (Vydac; 4.5 mm 250 mm, flow rate 1 ml min 1; gradient 1% B min 1). Mass spectrometry (MS) was performed over the appropriate mass/charge (m/z) range on a QSTAR XL mass spectrometer equipped with a MALDI source (Applied Biosystems Inc., Foster City, CA). The m/z values of the [M H] are listed in Table 1. Peptide SynthesisFmoc solid phase peptide synthesis methodology was used for all peptides. Typically, 2-chloro(chlorotrityl) resin (0.1 mmol, 68 mg) was swollen in dry dimethylformamide (DMF) for 2 h. The solvent was drained, and a solution of the appropriate amino acid (2 ml, 1 mM) in DMF, containing 100 ml of N,N-diisopropylethylamine, was added to the resin. The suspension was mixed thoroughly and maintained at room temperature for 2 h with occasional manual shaking. The solvent was drained again, and the resin was washed with DMF, followed by removal of the Fmoc group by treating it twice (1 min each) with a 50% solution of piperidine in DMF. The next amino acid (1 mM) was activated with 0.5 M 2-(1H-benzotriazol1-yl)-1,1,3,3-tetramethylu-roniumhexafluorophosphate in DMF (800 l), and 75 l of N,N-diisopropylethylamine was then added. The solution was vortex-mixed and then added to the de-protected amino acid on the resin. The contents of the reaction vessel were stirred with a steady stream of bubbled dry nitrogen. After 10 min, a small quantity of the resin was removed and tested for coupling yield. This cycle was repeated until the required chain assembly was achieved. After completion of chain assembly, the resin was washed with DMF followed by dichloromethane and dried under nitrogen. A known amount of the resin was taken in a reaction vessel, and the peptide was cleaved from the resin with simultaneous removal of the side chain protecting groups of the amino acids using a mixture of trifluoroacetic acid, water, and triisopropylsilane in the ratio 95:2.5:2.5 as a cleavage reagent. After 2 h the suspension was filtered to remove the resin, and the cleavage reagent was evaporated with a steady stream of dry nitrogen. Ice-cold diethyl ether was added to precipitate the peptide. The peptide was collected and purified by HPLC and then characterized by MS (Table 1).

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RESULTS Short Natural and Designer SequencesIt was found in our earlier studies that two 12-residue peptides (IMFFEMQsrsrs) and (ImFFEMQsrsrs) derived from the N terminus of DLP-4 and DLP-2, respectively, were effective substrates of the isomerase (12). These peptides were designed by appending a solubilizing peptide incorporating an all-D-amino-acids peptide (srsrs) to the C terminus of the 7-residue N terminus segment (IMFFEMQ). The heptapeptide as such was insoluble in any solvent that was suitable for further studies of the peptide isomerase. The addition of srsrs also made the heptapeptide stable to the action of carboxypeptidases (data not shown). To establish the minimum number of amino acid residues required for peptide isomerase activity, and to investigate the range of specificity of the enzyme, we chemically engineered shorter peptides as substrates, including only three amino acid residues from DLP. Thus, IMFsrs and ImFsrs and 26 other mutant pepVOLUME 283 NUMBER 14 APRIL 4, 2008

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TABLE 1 Mass spectrometry and HPLC data for the peptides
Serial no. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
a b

Peptide IMFsrs ImFsrs IFFsrs IfFsrs INleFsrs InleFsrs IYFsrs IyFsrs ILFsrs IlFsrs IVFsrs IvFsrs IIleFsrs IileFsrs IAFsrs IaFsrs IMWsrs ImWsrs LLHsrs LlHsrs IIGsrs IiGsrs FVQsrs FvQsrs IAbuFsrs IWFsrs IMHsrs IKFsrs

Mr (M 740.27 740.29 756.80 756.80 722.60 722.37 772.80 772.80 722.37 722.36 708.41 708.39 722.40 722.36 680.35 680.33 779.38 779.39 712.37 712.37 632.35 632.45 723.80 723.80 694.37 795.38 730.34 737.38

H)

Rt (min)a (L/D) 10.60 12.63 11.50 14.07 11.15 13.78 10.71 11.85 10.98 13.33 9.98 12.52 10.48 13.43 9.20 10.05 10.67 12.24 7.82 9.21 8.32 10.02 7.51 9.87 9.89 11.68 10.66 9.35

Rt

(RtL RtD) (min)b 2.03 2.57 2.63 1.14 2.35 2.54 2.95 0.85 1.57 1.39 1.70

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2.36

Rt denotes retention time. Rt denotes difference in retention time of a pair of mutant peptides.

tides (combinatorial variants) were synthesized using Fmoc solid phase peptide synthesis methodology (see under Materials and Methods). Remarkably, these peptide pairs showed significant differences in their HPLC retention times (Table 1); thus they formed the basis of a subsequent HPLC method for monitoring the activity of the peptide isomerase. IMFsrs in the presence of the peptide isomerase under our standard assay conditions (see under Materials and Methods) showed readily detectable conversion to ImFsrs; this interpretation was based on the product having exactly the same retention time as synthetic ImFsrs (Fig. 1A). Similarly, ImFsrs was isomerized to IMFsrs (Fig. 1A), but notably the reaction in this reverse direction was relatively slow (Table 2). This is consistent with the mature venom having more of the D-containing DLP-2 than L-containing DLP-4 (12) and the notion that it is resistant to proteolytic cleavage in the venom. Substrate SpecificityTo explore the range of substrate specificity, synthetic mutants of DLP peptides were made by replacing the Met residue with either L-or D-amino acids; these were Abu, Ala, His, Ile, Leu, Nle, Phe, Trp, Tyr, and Val instead of Met. All mutants were purified with HPLC and characterized by MS (Table 1). The presence of D-amino acid residues (srs) at the C terminus prevented the carboxypeptidase activity. Specificity Mapping Based on DLP-4, Long Side ChainsThe importance of the length of the side chain of substrates on the isomerase activity was identified for the mutants in which Met was replaced by Nle or nle. For these two cases the isomerization reaction was relatively faster, for L-to-D than to D-to-L, under identical assay conditions (Fig. 1B). Moreover, the conversion rate of L-to-D was greater (Table 2, 41:59) over a period of 4 h than for the hexapeptide with a Met residue (Table 2, 53:47), or indeed for the whole native peptide, but for D-to
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FIGURE 1. RP-HPLCs of isomerization of N-terminal hexapeptide mutants IMFsrs and ImFsrs (A), INleFsrs and InleFsrs (B), and IFFsrs and IfFsrs (C) after incubation at 37 C at 0- and 4-h time intervals.

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TABLE 2 L-to-D and D-to-L isomerization of mutant peptides at 4 h of incubation (RP-HPLC assay)
Isomerization reaction IMFsrs 3 ImFsrs ImFsrs 3 IMFsrs INleFsrs 3 InleFsrs InleFsrs 3 INleFsrs IFFsrs 3 IfFsrs IfFsrs 3 IFFsrs IIFsrs 3 IiFsrs IiFsrs 3 IIFsrs ILFsrs 3 IlFsrs IlFsrs 3 ILFsrs IVFsrs 3 IvFsrs IvFsrs 3 IVFsrs
a

4-h incubation at 37 C, ratio of peak areas IMFsrs:ImFsrs ImFsrs:IMFsrs INleFsrs:InleFsrs InleFsrs:INleFsrs IFFsrs:IfFsrs IfFsrs:IFFsrs IIFsrs:IiFsrs IiFsrs:IIFsrs ILFsrs:IlFsrs IlFsrs:ILFsrs IVFsrs:IvFsrs IvFsrs:IVFsrs 53:47 82:18 41:59 87:13 59:41 87:13 100:0 100:0 100:0 100:0 100:0 100:0

Relative ratea 0.79 0.31 1.0 0.22 0.69 0.22 0 0 0 0 0 0

Rate is relative to the most rapid conversion.

L-isomerization there was little difference. In a similar manner,

peptide isomerase was assayed with mutant peptides that contained Phe in place of Met (Tables 1 and 2; Fig. 1C). In these cases as well, the isomerization from L-to-D was faster than from D-to-L (Table 2). In summary, both of the peptide pairs (InleFsrs and InleFsrs) and (IFFsrs and IfFsrs) were active substrates of the peptide isomerase with the conversion rate of INleFsrs to InleFsrs being the fastest of all the tested peptides (Table 1, right-hand column). Specificity Mapping Based on DLP-4, Long Side Chains That Are PolarLys and Tyr have long side chains that are similar in length to Met and Phe, but unlike the latter they carry polar groups on the terminal carbon. Although tyrosine is simply the 4-hydroxy-substituted benzene derivative of Phe the mutant peptides, IYFsrs and IyFsrs were not substrates for the peptide isomerase. After 4 h of incubation with the isomerase, no change was observed by the HPLC-based assay. Similarly, on incubating IKFsrs with peptide isomerase, under the standard assay conditions, no conversion to IkFsrs was observed after 4 h (HPLC). From these two examples we concluded that the active site of the peptide isomerase does not effectively interact with polar moieties and therefore the binding sub-site for the second amino acid residue may be hydrophobic. Specificity Mapping Based on DLP-4, -Branched Chains The -branched category of synthetic mutant peptides that we studied included ILFsrs, IIFsrs, and IVFsrs and their D-counterparts. In all three cases isomerization did not occur (Fig. 2, AC) under the standard assay conditions. Thus the peptide isomerase was not active with peptides incorporating -branched amino acid residues. Therefore, the steric characteristics of the second amino acid also play an important role in the isomerization reaction. Specificity Mapping Based on DLP-4, Short Straight Chains Peptides having short one- and two-carbon side chains at the -carbon were synthesized. Ala, containing a methyl group at the -carbon, and Abu, with a two-carbon chain, CH3CH2-, at the -carbon were incorporated into IAFsrs, and its D-variant IaFsrs, and I(Abu)Fsrs. Under the standard assay conditions with peptide isomerase, no isomerization was detected. Specificity Mapping Based on OvCNPaThe first three amino acid residues of OvCNPa are LLH. We noted that His shares aromatic character with the Phe of IMF, but it is also polar and basic. So peptides were synthesized that reflected the

Downloaded from www.jbc.org by guest, on August 17, 2012 FIGURE 2. RP-HPLCs showing the lack of isomerization of N-terminal hexapeptide mutants IIFsrs and IiFsrs (A), ILFsrs and IlFsrs (B), and IVFsrs and IvFsrs (C) after incubation at 37 C at 0- and 4-h time intervals.

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anionic character at position 3 and not the aromatic character. A sub-set of peptides based on LLH with the added solubilizing (srs) tripeptide was synthesized: the native LLHsrs and its D-counterpart LlHsrs. A two-substitution L-D pair was made corresponding to DLP-1, FVQsrs, and FvQsrs in which the branched chain leucine was replaced by the smaller branched chain valine, and the anionic imidazole of histidine was replaced by the neutral amino acid residue glutamine. None of these peptides showed any isomerization under standard assay conditions. The finding that isomerization of FVQsrs did not occur was consistent with the fact that only one isomer of DLP-1 has been observed in the platypus venom (15). Therefore, we concluded that these peptides are also nonsubstrates. Specificity Mapping Based on OvCNPa, Partial Chimera with DLP-2/4Full-length DLP-4 and DLP-2 were modified at their respective N termini, with LLH and LlH, as occur naturally at the N terminus of OvCNPa and OvCNPb, respectively. Under standard assay conditions, significant but very slow isomerization took place with both variants. Such slow isomerization has also been reported for the full-length OvCNPa and OvCNPb (12). Clearly these results indicate that for the second L in LLH to become isomerized to its D-form, a longer peptide sequence or a structurally different one than simply srs needs to be attached. Tryptophan-containing HexapeptidesTryptophan has a side chain with both aromatic and polar character. LLH of the OvCNPs and the IMF of DLP-2/4 have aromatic ring side chains in position 3, whereas one variant of the second class, which turned out to be a substrate, was IFFsrs. Therefore, to explore the range of characteristics accepted by the enzyme, two types of mutant peptides were made with Trp; one by replacing Met and other by replacing Phe of IMFsrs with Trp. Thus IMWsrs and its counterpart ImWsrs were synthesized; and under standard assay conditions no interconversion of the isomers occurred. Similarly, the peptides IWFsrs and IMHsrs were nonsubstrates. Although Trp is aromatic, it is bulkier than either His or Phe; it carries a fused pyrrole ring that renders it polar and basic and that projects the benzene ring laterally. Thus we concluded that the angular disposition of the benzene ring relative to the peptide backbone is important for positioning the peptide in the active site at position 2. However, in IMHsrs, the polar His next to the Met is not accommodated in the correct orientation as in IMFsrs; and note that LLHsrs was not a substrate but the full-length OvCNPa, which has LLH at its N terminus is. Therefore, another requirement for a hexapeptide to be a substrate is that the third amino acid must have a benzene ring at the -carbon. Frog Venom PeptideThe peptide isomerase isolated from skin secretions of the frog Bombina variegata converts L-Ile to D-Ile at position 2 in a heptapeptide IIGPVGC-amide that is derived from the natural substrate (10). The frog enzyme, however, does not act upon the heptapeptide (LLHDHPC) that is based on the N terminus of platypus OvCNPa (10). Conversely, we investigated the action of platypus peptide isomerase on the peptide from the frog secretion that has the N-terminal sequence IIG. Two peptides IIGsrs and IiGsrs were synthesized. In our standard HPLC-based assay there was no isomerization of either peptide. We conclude that the platypus and frog
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enzymes have different active site configurations that would be consistent with what appears to be quite different fold structures based on preliminary chromatographic isolation procedures (16). Differences were explored further by adding reagents that are at least partially selective for amino acid side chains that could be involved in the isomerization reaction. Treatment of venom gland extract with 2.5 mM hydroxylamine that covalently modifies pyridoxal phosphate groups in enzymes such as transaminases (14) had no effect on the platypus peptide isomerase activity. Thus it appears that this cofactor is unlikely to be present in or near the active site. Isomerase activity was not inhibited by phenylmethylsulfonyl fluoride or iodoacetamide. These findings appear to rule out the presence of a serine-protease-like reactive triad or the involvement of an SH group of Cys in the active site. However, the peptide isomerase was inhibited by diethyl pyrocarbonate that modifies histidine residues. This suggests the involvement of an imidazole moiety of at least one histidine in catalysis, as has been reported for the peptide isomerase from the frog (14).

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DISCUSSION The platypus venom peptides OvCNPb and DLP-2 both contain a D-amino acid residue at position 2, whereas their counterparts OvCNPa and DLP-4 have all L-amino acid residues (8, 12, 13). Definitive evidence for this isomerization was provided by unambiguous chemical synthesis of all peptides, including those with D-amino acids (8). Across all phyla, peptides synthesized on ribosomes contain in the first instance L-forms of amino acids, so the presence of a D-amino acid residue is most likely to be due to post-translational modification without residue excision and replacement. We proposed that as with the frog skin toxin the platypus peptides undergo post-translational isomerization via the peptide isomerase (8, 12, 13). The presence of D-amino acid residues in peptides isolated from different animal species is well documented in the literature (19). The peptides that are bioactive are normally toxins that are used in defense or for the capture of prey, the latter being most obvious with spiders. Biochemically, the presence of a D-amino acid residue at position 2 from the N terminus imparts stability against amino peptidases (8, 9, 16). This stability bias is exacerbated by the rate of isomerization of the D-form of the substrate peptide being slower, in all cases measured in the present work, than the L-form. Hence, in an HPLC analysis of platypus venom, the greater abundance of the D-form of the substrate pairs is not only because of the equilibrium of the isomerization reaction being biased toward the D-form, but the L-form is more likely to be hydrolyzed by aminopeptidases in the medium, depleting it further than would otherwise be the case. The medium in which the peptides are immersed may simply be the venom itself, or more importantly, we posit, in the tissues of the victim of envenomation; the D-form would be more stable in the victim thus prolonging its effects such as pain. Platypus venom peptide isomerase catalyzes the isomerization of the second amino acid in the native peptides DLP-4 and OvCNPa isolated from the venom. We showed that the heptapeptide IMFFEMQ derived from DLP-4 is a substrate (12). In
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requirements occurs via additional amino acid residues elsewhere in the tertiary structure. Our hypothesis that it is essential that the second amino acid residue needs to be a long hydrophobic chain and the third amino acid residue must be a benzene ring was further corroborated as follows. Mutants based on DLP-1 (FVQsrs FIGURE 3. Proposed 2-base isomerization mechanism of platypus venom peptide isomerase. Abstraction and FvQsrs) and frog peptide of a proton from one face of an activated -carbon atom is concomitant with delivery from the opposite face by mutants (IIGsrs and IiGsrs) were the conjugate acid of a 2nd base. nonsubstrates for the isomerase. this work we showed that the substrate specificity is vested in We argue that this is because these peptides do not contain the the first three amino acid residues, although the activity is mod- sterically favorable amino acids in positions 2 and 3 (Table 1). Mechanism of IsomerizationIodoacetamide and hydroxylulated by the presence of further residues in the case of the derivatives of OvCNPa. Twenty six mutant hexapeptides based amine did not inhibit the peptide isomerase but diethylpyrocaron the IMF peptide of DLP-4 were synthesized with various bonate did. This points to the presence of at least one histidine amino acids at position 2 (Table 1). Of these peptides three were residue in or near the active site of the enzyme. Our conclusion shown to be substrates; these were peptides in which the second is corroborated by the finding that the candidate isomerase amino acid residue is hydrophobically (long carbon chain or gene contains two histidine residues in its central portion (data aromatic ring) attached at the -carbon, viz. Met, Nle, and Phe not shown). On the bases of these studies and similar earlier (Fig. 1, AC). In Nle this effect can be attributed to the presence reports (9), a two-base mechanism is proposed for the isomerof a chain similar in length to methionine at the -carbon atom, ase with the proposal that two His residues are positioned on whereas in the case of Phe the benzene ring is also long and yet the opposite sides of the substrate during isomerization, and flat, the latter property seeming not to impinge on the peptide they act as a proton abstractor and a proton donor, respectively. fitting into the active site. However, amino acids with same During peptide isomerization one of the histidyl bases depstereochemical characteristics but with a polar group such as rotonates the -carbon atom of the second amino acid (Met) the OH at position 4 of Tyr, or -NH2 of Lys, were not sub- of the substrate and forms a planar intermediate. Concomistrates (Tables 1 and 2). These findings point to the presence of tantly, the other histidyl base in its conjugate acid form repa hydrophobic region of the active site that favorably interacts rotonates the intermediate at the opposite side of the -carwith the hydrophobic side chains of Met, Nle, and the benzene bon atom to form the other isomer (Fig. 3). ring of Phe. Furthermore, in our studies we observed that isomerizaThe substitution of -branched amino acids (Leu, Ile, and tion of the L-to-D-amino acid residues was faster than for the Val) at position 2 of the substrates also rendered the isomerase D-to-L reaction. This suggests asymmetry at the active site in ineffective (Fig. 2, AC, and Tables 1 and 2). This may be the which one isomer binds more favorably than the other. This result of steric hindrance posed by the bulky -substituents is not unexpected because the peptide substrates are diaste(methyl groups). This suggests that the active site has a narrow reomeric so the equilibrium constant most likely will not be hydrophobic cavity that cannot accommodate bulkier and 1.0. Thus in the 2-base mechanism, the L-isomer would be branched substituents at the -carbon atom. Also, the replace- more favorably positioned for proton abstraction than with ment of Met by Ala or Abu consisting of shorter chain amino the D-isomer (Fig. 3). In relation to the likely mechanism of acids did not exhibit any isomerization, i.e. they were not sub- the enzyme reactions there are two fundamental points: (i) strates (Tables 1 and 2). We conclude that when the amino acid identification of a target amino acid by platypus isomerase in in position 2 is flanked by two amino acids of longer carbon a peptide for isomerization; and (ii) deprotonation and repchain and a benzene ring in position 1 and 3, respectively, the rotonation of a weakly acidic proton (pKa values may be reactive groups in the active site do not gain sufficient access to higher than 20) from the -carbon atom of an amino acid the proton on the -carbon atom for effective catalysis. In other residue. Only peptides with a particular sequence will act as words, the second amino acid must have a substituent on its substrates. This means that the active site is very specific and restricted to accommodate only certain peptide sequences. -carbon atom that is at least four carbon atoms long. To explore further the importance of Phe at position 3, we The high energy process that involves the removal of a replaced it with other aromatic amino acids. Replacing Phe in weakly acidic proton may be explained by the involvement of IMFsrs with either of two other aromatic amino acids, Trp and a strong low barrier (high energy hydrogen bonds) that His, rendered the peptide a nonsubstrate. Thus we conclude results in the formation of tight binding between the enzyme that it is essential that the third residue be Phe, for isomerase and substrate to give a relatively stable reactive intermediate activity on a hexapeptide. This may appear paradoxical given (17, 18) (central complex in Fig. 3); this results in removal of that the first three residues of OvCNP are LLH having a histi- a weakly acidic proton (pKa values may even be higher than dine in position 3; but recall that the hexapeptide LLHsrs is not 20) with weakly basic catalysts (pKa values of conjugate acid a substrate, suggesting that modulation of the active site 7) (19). This outcome can be achieved through the forma-

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tion of a strong hydrogen bond with the carbonyl carbon of the amino acid thereby lowering the activation energy barrier for the formation of an enolic intermediate of the bound substrate; this will facilitate the removal of the proton. Alternatively, isomerization may be a concerted process where incipient carbanion formation, deprotonation, and reprotonation take place simultaneously in the transition state, similar to an Sn2-type mechanism that results in inversion of the configuration. A more accurate understanding of the mechanism should be possible when three-dimensional structural data on the enzyme-substrate complex become available. ConclusionsThe development of a series of artificial substrates for the platypus peptide isomerase paves the way to use them as probes of the enzyme in other tissues of this animal and, perhaps even more importantly, to search for the activity in other animals, including humans. If the peptide isomerase activity exists in higher animals and humans, the modification it makes to proteins and peptides will constitute another process in the growing list of post-translational modifications of proteins; and the findings could have implications for the etiology of diseases of protein turnover such as Alzheimer disease and amyloidosis. Introduction of D-amino acid residues into proteins and peptides can make them resistant to proteases and thus more stable in vivo. Our investigations, and related work reported elsewhere, could provide a means of making more stable therapeutic peptides by using platypus venom peptide isomerase.
AcknowledgmentsProfessor Paul Alewood, University of Queensland, is thanked for valuable discussions in the early stages of this work. Tim Larkin is thanked for helping in preparing Fig. 3. The mass spectrometric analyses were carried out at the Biomedical Node of the Australian Proteome Analysis Facility and was supported in part by grants from the Major National Facilities Program and the University of Sydney. REFERENCES
1. Kamatani, Y., Minakata, H., Kenny, P. T., Iwashita, T., Watanabe, K., Funase, K., Sun, X. P., Yongsiri, A., Kim, K. H., and Novales-Li, P. (1989) Biochem. Biophys. Res. Commun. 160, 10151020 2. Jacobson, R. B., Jimenez, E. C., dela Cruz, R. G. C., Gray, W. R., Cruz, L. J., and Olivera, B. M. (1999) J. Pept. Res. 54, 9399 3. Soyez, D., Van Herp, F., Rosier, J. P., Le Caer, J. P., Tensen, C. P., and Lafont, R. (1994) J. Biol. Chem. 269, 1829518298 4. Heck, S. D., Siok, C. J., Krapcho, K. J., Kelbaugh, P. R., Thadeio, P. F., Welch, M. J., Williams, R. D., Ganong, A. H., Kelly, M. E., and Lanzetti, A. J. (1994) Science 266, 10651068 5. Shikata, Y., Watanabe, T., Inoue, A., Kawakami, Y., Nishizawa, Y., Katayama, K., and Kuwada, M. (1995) J. Biol. Chem. 270, 16719 16723 6. Montecucchi, P. C., de Castiglione, R., Piani, S., Gozzini, L., and Erspamer, V. (1981) Int. J. Pept. Protein Res. 17, 275285 7. Richter, K., Egger, R., and Kreil, G. (1987) Science 238, 200 202 8. Torres, A. M., Menz, I., Alewood, P. F., Bansal, P., Lahnstein, J., Gallagher, C. H., and Kuchel, P. W. (2002) FEBS Lett. 524, 172176 9. Heck, S. D., Faraci, W. S., Kelbaugh, P. R., Saccomono, N. A., Thadeio, P. F., and Volkman, R. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 4036 4039 10. Jilek, A., Mollay, C., Tippelt, C., Grassi, J., Mignogna, G., Mullegger, J., Sander, V., Barra, D., and Kreil, G. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 4235 4239 11. Kreil, G. (1997) Annu. Rev. Biochem. 66, 337345 12. Torres, A. M., Tsampazi, M., Tsampazi, C., Kennett, E. C., Belov, K., Geraghty, P. C., Bansal, P., Alewood, P. F., and Kuchel, P. W. (2006) FEBS Lett. 580, 15871591 13. Torres, A. M., Tsampazi, C., Geraghty, P., Bansal, P. S., Alewood, P. F., and Kuchel, P. W. (2005) Biochem. J. 391, 215220 14. Volkmann, R. A., and Heck, S. D. (1998) D-Amino Acids in Sequence of Secreted Peptides of Multicellular Organisms (Jolles, P., ed) pp. 5771, Birkhauser Verlag, Basel, Switzerland 15. Torres, A. M., Tsampazi, M., Kennett, E. C., Belov, K., Geraghty, D. P., Bansal, P. S., Alewood, P. F., and Kuchel, P. W. (2007) Amino Acids (Vienna) 32, 63 68 16. Dooley, C. T., Chung, N. N., Wilkes, B. C., Schiller, P. W., Bidlack, J. M., Pastenak, G. W., and Houghten, R. A. (1994) Science 266, 2019 2022 17. Gerlt, J. A., and Gassman, P. G. (1993) Biochemistry 32, 1194311952 18. Cleland, W. W., and Hreevoy, M. M. (1994) Science 264, 18871890 19. Gerlt, J. A., Kozarich, J. W., Kenyon, G. L., and Gassman, P. G. (1991) J. Am. Chem. Soc. 113, 96679669

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