Eur. J. Biochem. 229, 107-112 (1995) 0 FEBS 1995

Purification of turnip mosaic potyvirus viral protein genome-linked proteinase expressed in Escherichia coli and development of a quantitative assay for proteolytic activity Robert MENARD', Henriette CHATEL', Robert DUPRAS ', CCline PLOUFFE' and Jean-Franqois LALIBERTI?

Biotechnology Research Institute, National Research Council Canada, Qutbec, Canada Centre de recherche en virologie, Institut Armand-Frappier, Qutbec, Canada

(Received 3 January 1995) - EJB 95 0006/4

The 49-kDa, nuclear inclusion a-like, viral protein genome-linked proteinase (VPg-Pro) of turnip mosaic potyvirus (TuMV) was expressed in Escherichia coli. The protein was produced in a soluble form at high levels and was active, as demonstrated by intermolecular cleavage of the polymerase capsid protein (Pol-CP) substrate. The VPg-Pro was purified by metal-chelation and ion-exchange chromato- graphies. Two forms of VPg-Pro, which differed in molecular masses, were obtained during isolation; their identities were confirmed by immunoblot analysis and N-terminal amino acid sequencing. Data indicated that cleavage took place at a site near the C-terminus of VPg-Pro and was the result of the proteolytic activity of the viral protein. The purified proteinase retained enzymic activity on its natural substrate (Pol-CP) and was also capable of hydrolysing the synthetic peptide acyl-Ala-Ala-Val-Tyr-His- Gln-Ala-Ala-NH,, derived from the consensus cleavage site for the TuMV polyprotein. Analysis by mass spectrometry of the two fragments resulting from this reaction indicated that cleavage took place between the Gln and Ala residues, as expected. A fluorogenic derivative of this peptide was hydrolysed by VPg- Pro, affording a convenient quantitative assay for intermolecular proteolytic activity, and was used to determine the pH-activity profile. The availability of large quantities of pure proteinase and of a rapid and sensitive assay will permit detailed kinetic and structural studies which are essential to obtain a better understanding of the mode of action of this and related viral proteinases, such as the 3C proteinase of picornaviruses.

Keywords. Potyvirus ; viral protein genome-linked ; proteinase; purification ; fluorogenic substrate.

Turnip mosaic virus (TuMV) belongs to the potyvirus group (Ward and Shukla, 1991). Potyviruses are quite similar in terms of their genomic structures and strategies of expression to the plant comoviruses and nepoviruses as well as to picornaviruses (Riechmann et al., 1992). The TuMV genome is made up of one single-stranded, positive-sense RNA molecule of 9830 nucleo- tides, polyadenylated at the 3' end and its 5' end is covalently linked to a viral-encoded protein (viral protein genome-linked, VPg; Nicolas and LalibertC, 1992). Upon infection, the viral RNA is translated into a large polyprotein with a calculated mo- lecular mass of 358 000 kDa, which is processed into at least nine polypeptides by three virus-encoded proteinases (Dou- gherty and Carrington, 1988; Riechmann et al., 1992). The small nuclear inclusion a protein (NIa protein) of tobacco etch virus (TEV) or the homologous 49-kDa protein found in other potyvi- ruses have been shown to possess proteolytic activity (Carring- ton and Dougherty, 1987; Garcia et al., 1989a; Ghabrial et al., 1990; Hellmann et al., 1988; Lalibertk et al., 1992). The 49-kDa

Correspondence to J.-F. LalibertC, Centre de recherche en virologie, Institut Armand-Frappier, 531 Boulevard des Prairies, Ville de Laval, QuCbec, Canada H7N 423

Fax: +l 514 686 5626. Abbreviations. NI, nuclear inclusion; NTA, nitrilotriacetic acid; PPV,

plum pox (poty)vims ; Pol-CP, polymerase capsid precursor protein; TEV, tobacco etch (p0ty)virus ; TuMV, turnip mosaic (poty)virUs ; VPg, viral protein genome-linked; VPg-Pro, viral protein genome-linked pro- teinase-precursor protein.

protein is a multifunctional protein, the N-terminal domain being the VPg which is likely to be involved in replication, and the C- terminal domain containing the proteolytic activity (Murphy et al., 1990; Dougherty and Parks, 1991).

Viral protein genome-linked proteinase-precursor protein (VPg-Pro) releases itself from the polyprotein through an intramolecular event and recognizes at least seven cleavage sites on the polyprotein (Ghabrial et al., 1990; Riechmann et al., 1992). In the case of TEV, the cleavage sites are defined by sequences of seven amino acids; the PI, P, and P, determinants are essential in defining the cleavage site while the P,, P4 and P5 residues are involved in regulating the rate of cleavage (Car- rington and Dougherty, 1988; Dougherty et al., 1988, 1989; the subscript number indicates the position of the amino acid pre- ceding the hydrolysed peptide bond, position 1 being the closest to the hydrolysed peptide bond). This implies that cleavage sites differ in their rate of hydrolysis and it has been suggested that their susceptibility to processing in cis (intramolecularly) or in trans (intermolecularly) can also vary (Carrington et al., 1988).

The proteinase domain of the VPg-Pro protein has many properties in common with the 3C proteinase of picornaviruses (Bazan and Fletterick, 1988; Dougherty et al., 1989). Purifica- tion of recombinant picornaviral 3C proteinases has been real- ized (Baum et al., 1991; Harris et al., 1992; Nicklin et al., 1988; Pallai et al., 1989) and the structures of two 3C proteinases have been obtained (Allaire et al., 1994; Matthews et al., 1994). How- ever, the studies carried out with the potyviral proteinases use

108 Mtnard et al. (Eux J. Biochem. 229)

VPg- P1 HC-PRO P3 CI 8KPro POI CP m L\\\\\\l tsl h\ssmim3

PET-PolCP

88 kDa 69 kD. 27 kDa

Fig. 1. Schematic representation of the ”UMV genome and cDNA recombinant molecules used in this study. The TuMV genome is depicted at the top of the figure and the individual coding regions are indicated by boxes. PCR-generated fragment3 were inserted into PET-lld to create PET-PolCP or into PET-21d to create PET-ProRQhis. The hatched and dotted boxes represent the polymerase-coding and capsid-protein-coding regions, respectively. Lines represent the location of coding region and the size of the expected precursor and processed proteins. l luck lines represent proteins which were immunodetected The seven amino acids making up the cleavage site between the polymerase and the capsid protein are shown by the single-letter amino acid code. The modified dipepude of the internal cleavage site of VPg-Pro is shown, the bold-typed letter representlng the His residue replacing Glu.

cell-free translation or E. coli expression systems acting on com- plex substrates and it has not been possible to determine the role and importance of individual amino acid residues of the enzyme or the role and importance of the cleavage sites on the mecha- nism and specificity of polyprotein processing. In this study, we report a simple and rapid procedure which resulted in the purifi- cation of a biologically active 49-kDa proteinase of TuMV when proteolysis was assayed using a natural substrate and against synthetic substrates.

MATERIALS AND METHODS

Plasmid construction. Plasmids PET-NIa, PET-NId24 and PET-NId26 were previously described (LalibertC et al., 1992). Plasmid pET-Pro/24his was generated as described above by the PCR reaction but the coding region was cloned into PET-21d (Novagen) using sense primer 5’-TCTGAACCCATGGCTCAT- GAA-3‘ and anti-sense primer 5’-TTTGTGCCTCGAGTGCC GTGCTAT-3’. Similarly, the coding region for the precursor polymerase-capsid protein (Pol-CP) was generated by PCR using the sense primer 5’-GTCTCCATGGAAACCCAGCA- GAAT-3’ and the anti-sense primer 5’-TATAGTCTACCAG- GATCCACTTCATAACCCCTGAAGGCC-3’, and cloned into the plasmid PET-lld, resulting in PET-PolCP (Fig. 1). The re- combinant plasmids were introduced into Escherichia coli strain BL21 (DE3; Studier et al., 1990).

Purification of recombinant VPg-Pro. E. coli BL21 (DE3) harboring pET-Pro/24his (500 ml) was grown in Luria-Bertani broth at 37°C to an A,oo of 0.3. At this cell density, isopropyl- 8-D-thiogalactopyranoside was added to the culture at a final concentration of 1.0 mM, and the bacteria were further incu- bated for 2 h at 30°C. Following the induction period, the bacte- rial suspension was centrifuged, washed and resuspended in 30 ml buffer A (50 mM NaPO,, pH 8.0, 300 mM NaCl), lysed in a French press and centrifuged at 100000 g. The supernatant was ajusted with 10 mM 2-mercaptoethanol and 0.1 % Tween- 20, mixed with 1 .O ml Ni” - nitrilotriacetic acid- Agarose (Qui- agen) equilibrated in the same buffer and incubated at 4°C on an orbital shaker. After 1 h, the suspension was loaded into a column and washed with buffer A containing 10% glycerol until the A,,, reached zero, after which time a linear gradient of 0- 100 mM imidazole in buffer A, adjusted at pH 6.0, was applied. The bound proteins eluted as a single peak at approximately 25 mM imidazole. The purified fraction was diluted with 2 vol. buffer B (50 mM NaPO,, pH 7.4, 10 mM NaC1) and dithiothrei- to1 at a final concentration of 2.0 mM was added. The protein

solution was applied to a Waters CM Protein Pak SP-5PW col- umn equilibrated with buffers A and B at a ratio of 1/2. The resin was extensively washed until the A,,, reached zero after which a 0.1-1.0-M NaCl gradient was applied. Fractions (0.5 ml) were collected and analyzed by SDSPAGE and irnmu- noblotting using an anti-TEV NI serum generously provided by W. G. Dougherty, Corvallis, Oregon, USA.

Transfer of the protein onto a poly(viny1idine difluoride) membrane was carried out according to the manufacturer’s in- structions (Bio-Rad). N-terminal sequences of proteins on poly- (vinylidene difluoride) membrane were obtained by automated Edman degradation performed on a model 470A gas-phase Se- quencer equipped with an on-line model 120A phenylthiohydan- toin analyzer (Applied Biosystem Inc.), employing the general protocol described in Hewick et al. (1981). Approximately 100 pmol blotted protein was loaded onto the sequencer with a trifluoroacetic-acid-treated glass-fiber filter. A standard program (03RPTH from Applied Biosystem Inc.) was employed for se- quencing.

Proteolytic assay on Pol-CP substrate. E. coli harboring PET-PolCP was induced as described above. The cells were centrifuged, resuspended in 0.1 vol. 50 mM Tris/HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl and lysed after multiple freeze/ thawing cycles, lysozyme treatments and ultra-sonic treatments. Lysates of BL21(DE3) containing various forms of the TuMV proteinase were identically prepared. Equal volumes of both ly- sates were mixed and incubated at 37°C for 2 h. After this peri- od, total proteins (10 pl) were separated by SDSPAGE and ana- lyzed by immunodetection using a monoclonal antibody raised against the TuMV capsid protein (generously provided by M. R. McDermott, McMaster University, Canada). For the assay using purified proteinase, 50 pl (0.4 g/l) of VPg-Pro was mixed with 100 pl crude lysate containing Pol-CP and treated as described above.

Proteolytic assay on small peptidyl substrates. Small syn- thetic peptides were synthesized and tested as substrates for VPg-Pro. These peptides were based on the observed sequences for VPg-Pro cleavage sites of TuMV (Nicolas and Lalibertk, 1992). As shown in Fig. 2, only Val at position P, is absolutely conserved while His and Gln are found in the majority of se- quences at positions P, and PI, respectively. The consensus se- quence for the cleavage sites is Xaa-Xaa-Val-Xaa-His-Gln-Yaa, where Xaa is a variable amino acid residue and Yaa is a small residue (Ala, Ser or Thr). Based on this sequence, the peptide Ala-Ala-Val-Tyr-His-Gln-Ala was chosen to form the template for designing two small peptidyl substrates. The first peptide prepared was acyl-Ala-Ala-Val-Tyr-His-Gln-Ala-Ala-NH,. This

MCnard et al. (Eul: J. Biochem. 229) 109

Lys - Glu - Val - Val - His - Gln - Ala Pro - Thr - Val - Tyr - His - Gln - Thr Glu - Ala - Val - Asn - His - Gln - Ser Glu - Pro - Val - Thr - His - Glu - Ala Val - Pro - Val - Asp - His - Glu - Ser Thr - Ala - Val - Tyr ~ Ala - Gln - Thr Ala - Cys - Val - Tyr - His - Gln - Ala

Consensus Sequence

Xaa - Xaa - Val - Xaa - His - Gln - Yaa Glu

Fig.2. Rational for the design of synthetic peptides. Amino acids found at the different cleavage sites recognized by VPg-Pro of TuMV are listed. Hydrolysis takes place between residues P, and P',. In the consensus sequence, Xaa and Yaa represent variable amino acid residues (Yaa = small residue, i.e. Ala, Ser, Thr). The subscript numbers indicate the position of the amino acid relative to the hydrolysed peptide bond, position 1 preceding the cleaved bond.

compound was synthesized by the solid-phase method on an Ap- plied Biosystem 430A peptide synthesizer, as described pre- viously (Szewczuk et al., 1992). A second substrate was pre- pared by first synthesizing the peptide O-aminobenzoyl-Ala- Ala-Val-Tyr-His-Gln-Ala by a manual solid-phase technique using a standard N-(9-fluorenylmethoxycarbonyl) synthetic strategy, (benzotriazol-l-yloxy)tris(dimethylamino)phosphoni- um hexafluorophosphate as coupling reagent and N-(9-fluor- enylmethoxycarbony1)-Ala-Sasrin (Bachem) as resin. After cleavage of the solid support, alanine-p-nitroanilide (Ala-NH- Np; Bachem) was attached to the protected peptide in solution using dicyclohexylcarbodiimidelhydroxybenzotriazole as cou- pling reagent. Protecting groups were removed by trifluoroacetic acid/EDTA/H,O (95 : 2.5 : 2.5) at room temperature to yield 0- aminobenzoyl-Ala-Ala-Val-Tyr-His-Gln-Ala-Ala-NH-Np. The peptides were purified and characterized as previously described (Szewczuk et al., 1992).

Assay of VPg-Pro against acyl-Ala-Ala-Val-Tyr-His-Gln- Ala-Ala-NH, was performed with 50 pl purified proteinase (0.4 g/l) and 0.46 mM peptide in a volume of 250 p1 with a final buffer concentration of 25 mM NaPO,, pH 7.0, 0.12 M NaCl, 1.6 mM EDTA, 1.6 mM dithiothreitol, 2 % glycerol and 4 mM imidazole. Product formation was measured by the absorbance at 214nm by reverse-phase HPLC on a Vydac C,, column (25 cmX0.46 cm) using a linear gradient (0-25% CH,CN for 25 min, all in 0.1 % trifluoroacetic acid) at a flow rate of 1 ml/ min. The identity of the cleavage site was confirmed by collect- ing the fractions from the HPLC and determining the molecular mass on an API I11 mass spectrometer (Sciex). With the sub- strate O-aminobenzoyl-Ala-Ala-Val-Tyr-His-Gln-Ala-Ala-NH- Np, the reactions were followed by measuring the increase in fluorescence resulting from cleavage of the Gln-Ala bond (exci- tation, 340nm; emission, 420nm) on a SPEX Fluorolog-2 spectrofluorometer. The pH dependence of activity was mea- sured under the following conditions: 50 mM buffer (sodium phosphate for pH 6.0-7.9, sodium borate for pH 7.9-9.4), 0.2 M NaC1, 5 mM EDTA, 2 mM dithiothreitol, 2.5% CH,CN, 2.0 pM substrate. The calibration factor to convert the fluores- cence into the product concentration was determined by measur- ing the fluorescence of intact and fully hydrolysed substrate at various concentrations. Variation of pH had no effect on the cali- bration factor. The pH-activity data was fitted by non-linear re- gression to a model where ionization of a single group leads to an increase in activity. All kinetic experiments were carried out at 25 "C.

Fig. 3. Intermolecular proteolytic activity of VPg-Pro in crude E. coli lysate. The assay was performed as described in Materials and Methods. The proteins were separated on a 10% polyacrylamide gel and release from PolCP was monitored by immunoblotting using an anti-(capsid protein) monoclonal antibody. Lane 1, PET-lld; lane 2, PET-PolCP; lane 3, PET-NId24; lane 4, PET-PolCP and PET-NIaf24; lane 5, PET- PolCP and pET-Pro/24his; lane 6, PET-PolCP and PET-NIa; lane 7, PET- PolCP and PET-NId26.

RESULTS AND DISCUSSION

Expression of the 49-kDa proteinase in E. coli. The TuMV 49-kDa VPg-Pro, modified by site-directed mutagenesis, has previously been described (LalibertC et al., 1992). By replacing the Glu residue with His at position P, of the internal cleavage site, the altered protein was unable to undergo proteolysis be- tween the VPg and Pro domains. In order to verify that it still retained enzymic activity, intermolecular proteolysis catalyzed by VPg-Pro was assayed by measuring the release of the capsid protein from the Pol-CP precursor protein. Previous studies have shown that this site is one of the most efficiently hydrolysed of all sites on the potyviral polyprotein (Garcia et al., 1989b, c). Fig. 3 , lane 4 shows that incubation of lysates containing both Pol-CP and VPg-Pro released a 27-kDa protein which reacted with a monoclonal antibody preparation directed against the cap- sid protein of TuMV and which had the expected size if hydroly- sis took place between the polymerase and the capsid protein. Similar results were obtained with a rabbit polyclonal antiserum raised against purified viral preparations of TuMV (results not shown). This protein species was not detected when either VPg- Pro or Pol-CP was omitted from the lysate (Fig. 3, lanes 2 and 3). Furthermore, incubation of the Pol-CP lysate with another lysate expressing an inactive proteinase (VPg-Pro/26 encoded by PET-NId26) did not result in the detection of the capsid- precursor-containing protein (Fig. 3, lane 7); in this case, the proteinase had been inactivated by site-directed mutagenesis which replaced the His of the catalytic triad by a Gln residue (LalibertC et al., 1992). Incubation of the Pol-CP with a lysate expressing the wild-type 49-kDa proteinase showed litle activity (Fig. 3, lane 6) which is interesting since only the 22-kDa VPg protein was detected by immunoblotting using an anti-NI serum, while protein intermediates containing the proteinase domain were undetected and presumed to be degraded in E. coli. Hence, a very low level of active proteinase-containing polypeptides must remain in the lysate. The precursor Pol-CP protein was not detected in our assay system and was a consequence of the in- ability of the monoclonal antibody to recognize it since this Pol- CP was detected on Coomassie-blue-stained polyacrylamide gels and was also detected using an antiserum directed toward CP (results not shown). The above experiments thus indicate that VPg-Pro, while unable to undergo self-processing, was fully active and hydrolysed Pol-CP precursor protein when supplied in trans.

Purification of VPg-Pro. Proteins containing several contigu- ous His residues are conveniently purified by metal-chelation

110 MCnard et al. (Eul: J. Biochem. 229)

Fig. 4. Purification of VPg-Pro by metal-chelation and ion-exchange chromatographies. Purification was performed as described in Materi- als and Methods. SDSPAGE analysis of: lane 1, total protein from E. coli harboring PET-1 Id; lane 2, total protein from E. coli harboring PET- Pro/24his induced at 37°C; lane 3, total protein from E. coli harboring pET-Pro/24his induced at 30°C ; lane 4, VPg-Pro-containing protein peak eluted from imidazole gradient during metal-chelation chromatog- raphy; lane 5 , VPg-Pro-containing protein peak eluted from NaCl gradi- ent during ion-exchange chromatography ; lane 6, inclusion bodies puri- fied from E. coli harboring PET-NId24his induced at 37 "C. The gel was stained with Coomassie blue.

chromatography (Hochuli et al., 1987). Consequently, the coding region for VPg-Pro was subcloned into pET-2ld, which resulted in the fusion of six His residues at the C-terminus of the protein- ase. The addition of these amino acids had apparently no effect on the proteolytic activity of VPg-Pro; the capsid protein was released from the precursor Pol-CP with the same efficiency as VPg-Pro without the His tail (results not shown).

Fig. 4 is a representative Coomassie-blue-stained SDS/ PAGE analysis of different fractions from the purification scheme described in Materials and Methods. Fig. 4, lane 2 shows the electrophoretic profile of VPg-Pro-containing E. coli proteins when the induction was performed at 37°C. At this tem- perature, VPg-Pro was synthesized to very high levels but was found in insoluble inclusion bodies (Fig. 4, lane 6). In contrast, when induction was carried out at 30"C, VPg-Pro was produced to a much lower extent but was soluble (Fig. 4, lane 3). Metal- chelation chromatography was performed, which proved to be a very efficient purification step (Fig. 4, lane 4), and contaminat- ing proteins were then eliminated by cation-exchange chroma- tography (lane 5) . The purified proteinase generally migrated as a doublet during SDSPAGE as judged by Coomassie-blue stain- ing and immunodetection with anti-TEV NI serum (results not shown) ; the size difference was estimated to be in the range 1 - 2 kDa. VPg-Pro was purified at a yield of approximately 1 mg/ 1 bacterial suspension.

Self-cleavage near the C-terminus of VPg-Pro. There are two possibilities for explaining the presence of two species of VPg- Pro on an SDS/polyacrylamide gel; either initiation took place at an internal AUG codon, or the lower band resulted from hy- drolysis of VPg-Pro. N-terminal amino acid sequencing indi- cated that the two species had identical sequences, thus eliminat- ing the internal initiation hypothesis and suggesting instead that hydrolysis had taken place at the C-terminus of the protein. In this case, hydrolysis could be the result of the proteolytic activ- ity of the recombinant protein itself or of E. coli proteins. In order to distinguish between these two possibilities, VPg-Pro and the inactive form of the protein (VPgPro/26) were produced in E. coli and analysed by SDSPAGE and immunoblotting. The inactive form of VPg-Pro migrated as a single species (results not shown) which supports the notion that VPg-Pro is responsi- ble for the hydrolysis. Interestingly, purification of the NIa pro-

8 E 5: 9

C

0 10 20 30

Retention Time (min)

Fig. 5. Proteolytic activity of purified VPg-Pro on the synthetic sub- strate acyl-Ala-Ala-Val-Tyr-His-Gln-Ala-Ala-NH,. Substrate and en- zyme were incubated for the periods of time indicated on the chromato- grams and product formation analyzed by HPLC. Absorbance was mea- sured at 214 nm. Conditions are given in Materials and Methods. The peak with a retention time of 21.8 min corresponds to acyl-Ala-Ala-Val- Tyr-His-Gln-Ala-Ala-NH, and the peak with retention time of 20.0 min to acyl-Ala-Ala-Val-Tyr-His-Gln.

teinase domain of TEV also resulted in the appearance of two protein bands (Dougherty and Parks, 1991). The exact location of this additional cleavage within the TuMV protein and its bio- logical significance remain to be experimentally determined.

Elution from Ni2+ -NTA-Agarose of the faster-migrating species, which should not have a His tail, is explained by its intrinsic affinity for the resin. Indeed, purification of VPg-Pro without a His tail was also achieved by metal-chelation chroma- tography; the recombinant protein bound to the resin and eluted in a similar manner to that of the faster-migrating species with an imidazole gradient and the two forms of VPg-Pro were also detected. However, the degree of purity obtained for the species without the His tail was less than that obtained for the species with the tag (data not shown) and, consequently, purification of His-tagged VPg-Pro was prefered.

Proteolytic activity of the purified VPg-Pro on synthetic substrates. The purified VPg-Pro was further characterized for its ability to cleave an eight-amino-acid peptide as substrate. It has previously been shown that a foreign protein, which har- bored an insertion of seven amino acids representing a cleavage site, can be hydrolysed by the 49-kDa TEV or PPV proteinases (Carrington and Dougherty, 1988; Garcia et al., 1989b, c) which suggested that no other structural features are necessary for cleavage to occur. The peptide acyl-Ala-Ala-Val-Tyr-His-Gln- Ala-Ala-NH,, derived from the consensus cleavage site on the polyprotein (Fig. 2), was incubated with purified VPg-Pro at room temperature and the reaction was followed by removing aliquots after various incubation times and analyzing the prod-

MCnard et al. ( E m J. Biochem. 229) 111

35

3 30 . ._ 3 5 25 e

:P 20 E

$ 15 rL m .e l o

a, - - .- -

5

0 1

6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5

PH

Fig. 6. pH-activity profile of VPg-Pro. Proteolytic activity was mea- sured using the fluorogenic peptide O-aminobenzoyl-Ala-Ala-Val-Tyr- His-Gln-Ala-Ala-NH-Np as described in Materials and Methods. The solid line represents the best fit of the data obtained by non-linear regres- sion to a model assuming that the activity is modulated by a single- ionizable group.

ucts by HPLC (Fig. 5). The peak for the intact peptide eluted with a retention time of 21.8 min. After adding the enzyme, the intensity of this peak decreased with time and a second peak appeared at a retention time of 20.0 min. Both of these peaks were collected and analyzed by mass spectrometry. Molecular masses of 871.5 Da (retention time = 21.8 min) and 730.0 Da (retention time = 20.0 min) were obtained, corresponding to the intact peptide and to the hydrolysed fragment acyl-Ala-Ala-Val- Tyr-His-Gln-COOH, respectively. This confirms that hydrolysis has taken place at the Gln-Ala bond, as expected. The other product (Ala-Ala-NH,) is most likely to be found in the void volume.

The HPLC procedure can be used to determine kinetic pa- rameters. However, this method is time consuming and rela- tively insensitive. The results obtained with the substrate acyl- Ala-Ala-Val-Tyr-His-Gln-Ala-Ala-NH, indicated that this pep- tide could form the basis for designing intramolecularly quenched substrates (Yaron et al., 1979). Hydrolysis of this type of substrate can be followed by continuously monitoring the changes in fluorescence and affords a more rapid and sensitive assay for proteinases (Bratovanova and Petkov, 1987). In the present case, O-aminobenzoyl-Ala-Ala-Val-Tyr-His-Gln-Ala- Ala-NH-Np has been chosen as a potential fluorogenic substrate. The fluorescence of the 0-aminobenzoyl group was partly quenched by the p-nitroanilide moiety in the intact substrate, but when hydrolysis of the Gln-Ala bond was catalysed by VPg- Pro, an increase in fluorescence was observed. Initial rate mea- surement at various substrate concentrations was performed and showed a linear relationship up to 10 pM substrate at pH 8.2 (data not shown), indicating that the K, is much higher than 10 pM. (Higher concentrations were not tested because of the insolubility and possible intermolecular quenching of the sub- strate at these concentrations.) Large Km values for peptide sub- strates are often observed for viral proteinases (Hopkins et al., 1991 ; Malcolm et al., 1992) and probably reflect greater confor- mational flexibility of small peptides compared to the natural cleavage sites of these enzymes (Hellen and Wimmer, 1992). To test the potential of this assay for detailed kinetic characteriza- tion, this substrate was used to determine the pH-activity profile of the VPg-Pro (Fig. 6). The measurement of pH-activity data was carried out over a range of pH sufficient to obtain a good

definition of the profile for the curve-fitting procedure (i.e. where the plateaus at low and high pH values are reached). Higher pH values were not used due to the possibility for en- zyme instability interfering with the measurement of activity. The activity increased with pH following ionization of a single group with a pK, of 7.47. However, due to the precision of the data, we cannot rule out the possibility that other ionizable groups could have minor effects on activity. A somewhat similar profile has been obtained recently with the 3C proteinase of the hepatitis A virus, a member of the picornaviridae family (Jewel1 et al., 1992). The pH dependency of activity for the VPg-Pro is very similar to that observed for the trypsin family of serine proteinases and differs markedly from the pH-activity profiles of the papain family of cysteine proteinases which typically dis- play bell-shaped profiles with maximal activity at near-neutral pH (Lowe, 1970). The pK, describing the pH-activity profile of the VPg-Pro is believed to reflect ionization of the His residue within the active site. The value of 7.47 obtained for VPg-Pro is slightly higher than the corresponding pK, of approximately 7.0 found for trypsin-like serine proteinases, which indicates that ionization of the His residue is not greatly affected by the substi- tution of Ser for Cys as the active-site nucleophile in the cata- lytic triad.

In conclusion, the availability of large quantities of pure pro- teinase will enable kinetic studies on substrate specificities to be carried out, as well as enabling identification of the role of cer- tain enzyme residues in substrate recognition and catalysis. Moreover, kinetic studies of the TuMV proteinase will also pro- vide information concerning the structure/function relationship for other viral proteinases, such as 3C of picornaviruses.

This work was supported by a grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada (JFL). We thank F. Shareck (IAF) and L. Trempe (IAF) for the oligonucleotides synthesis, G. McSween (IAF) for ion-exchange chromatography, Jean Lefebvre (BRI) for peptide synthesis and France Dumas (BRI) for amino acid sequencing. Issued as NRCC publication number 38530.

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