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Sequential Autolytic Processing Activates the Zymogen of Arg-gingipain

2003; Elsevier BV; Volume: 278; Issue: 12 Linguagem: Inglês

10.1074/jbc.m210564200

1083-351X

Jowita Mikolajczyk, Kelly M. Boatright, Henning R. Stennicke, Tamim Nazif, Jan Potempa, Matthew Bogyo, Guy S. Salvesen,

Biochemical and Structural Characterization

Most proteases are synthesized as inactive precursors to protect the synthetic machinery of the cell and allow timing of activation. The mechanisms used to render latency are varied but tend to be conserved within protease families. Proteases belonging to the caspase family have a unique mechanism mediated by transitions of two surface loops, and on the basis of conservation of mechanism one would expect this to be preserved by caspase relatives. We have been able to express the full-length precursor of the Arg-specific caspase relative from the bacterium Porphyromonas gingivalis, Arg-gingipain-B, and we show that it contains N- and C-terminal extensions that render a low amount of latency, meaning that the zymogen is substantially active. Three sequential autolytic processing steps at the N and C terminus are required for full activity, and the N-propeptide may serve as an intramolecular chaperone rather than an inhibitory peptide. Each step in activation requires the previous step, and an affinity probe reveals that incremental activity enhancements are achieved in a stepwise manner. Most proteases are synthesized as inactive precursors to protect the synthetic machinery of the cell and allow timing of activation. The mechanisms used to render latency are varied but tend to be conserved within protease families. Proteases belonging to the caspase family have a unique mechanism mediated by transitions of two surface loops, and on the basis of conservation of mechanism one would expect this to be preserved by caspase relatives. We have been able to express the full-length precursor of the Arg-specific caspase relative from the bacterium Porphyromonas gingivalis, Arg-gingipain-B, and we show that it contains N- and C-terminal extensions that render a low amount of latency, meaning that the zymogen is substantially active. Three sequential autolytic processing steps at the N and C terminus are required for full activity, and the N-propeptide may serve as an intramolecular chaperone rather than an inhibitory peptide. Each step in activation requires the previous step, and an affinity probe reveals that incremental activity enhancements are achieved in a stepwise manner. full-length wild-type 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol matrix-assisted laser desorption ionization time-of-flight Ac-biotinyl-Lys-Tyr-6-aminohexanoic-Arg-acyloxymethyl ketone t-butoxycarbonyl 7-amino-4-methylcoumarin synthetic complete Proteases of the gingipain family are virulence factors of the periodontal pathogenic bacterium Porphyromonas gingivalis(1Nakayama K. Kadowaki T. Okamoto K. Yamamoto K. J. Biol. Chem. 1995; 270: 23619-23626Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 2Curtis M.A. Aduse-Opoku J. Rangarajan M. Crit. Rev. Oral Biol. Med. 2001; 12: 192-216Crossref PubMed Scopus (121) Google Scholar, 3Genco C.A. Potempa J. Mikolajczyk-Pawlinska J. Travis J. Clin. Infect. Dis. 1999; 28: 456-465Crossref PubMed Scopus (86) Google Scholar). This group contains two genes that encode Arg-specific proteases (rgpA and rgpB) and one gene encoding a Lys-specific protease (kgp). Gene ablation studies have shown that RgpA and RgpB are required for the activation of Kgp, placing the Arg-specific proteases at the top of a proteolytic pathway required for bacterial growth (4Veith P.D. Talbo G.H. Slakeski N. Dashper S.G. Moore C. Paolini R.A. Reynolds E.C. Biochem. J. 2002; 363: 105-115Crossref PubMed Google Scholar, 5Kadowaki T. Nakayama K. Yoshimura F. Okamoto K. Abe N. Yamamoto K. J. Biol. Chem. 1998; 273: 29072-29076Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). The protein encoded byrgpB is predicted to consist of three distinct segments (see Fig. 1), but only two are found in the mature product isolated from bacterial cultures, the catalytic unit and an Ig domain. Consequently the 205-amino acid N-terminal segment may constitute an activation peptide that restrains the activity of the protease until it reaches its site of action. Structural analysis of the catalytic unit of RgpB (6Eichinger A. Beisel H.G. Jacob U. Huber R. Medrano F.J. Banbula A. Potempa J. Travis J. Bode W. EMBO J. 1999; 18: 5453-5462Crossref PubMed Scopus (155) Google Scholar) demonstrates that it shares its evolutionary origin with a common ancestor of caspases, proteases involved in apoptosis and cytokine activation (7Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6159) Google Scholar). Moreover, homology mapping suggests that the clan encompassing gingipains and caspases also may contain bacterial clostripain, plant and animal legumains (processing proteases) (8Chen J.M. Rawlings N.D. Stevens R.A. Barrett A.J. FEBS Lett. 1998; 441: 361-365Crossref PubMed Scopus (196) Google Scholar), and separase (required for sister chromatid separation during anaphase) (9Uhlmann F. Wernic D. Poupart M.A. Koonin E.V. Nasmyth M. Cell. 2000; 103: 375-386Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar). This clan is known as protease clan CD (10Barrett A.J. Rawlings N.D. Biol. Chem. 2001; 382: 727-733Crossref PubMed Google Scholar) or the caspase-hemoglobinase fold (11Aravind L. Koonin E.V. Proteins. 2002; 46: 355-367Crossref PubMed Scopus (137) Google Scholar). The majority of proteases are synthesized as zymogens that await activation at a suitable time to protect the biosynthetic machinery of the cell against activation and to act as a timing event in biological function (12Neurath H. Trends Biochem. Sci. 1989; 14: 268-271Abstract Full Text PDF PubMed Scopus (156) Google Scholar). Thus, one of the key events in any proteolytic pathway is the conversion of the zymogen to the active enzyme. Different protease clans utilize distinct strategies for zymogen maintenance and activation, but within clans there seems to be conservation of a particular strategy. On the basis of conservation of mechanism one would imagine that protease clan CD would embrace a similar activation pathway, meaning that the zymogens of caspases and RgpB should be stabilized by homologous molecular interactions. The molecular determinants of caspase activation have been elucidated (13Renatus M. Stennicke H.R. Scott F.L. Liddington R.C. Salvesen G.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14250-14255Crossref PubMed Scopus (370) Google Scholar, 14Chai J. Wu Q. Shiozaki E. Srinivasula S.M. Alnemri E.S. Shi Y. Cell. 2001; 107: 399-407Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 15Riedl S.J. Fuentes-Prior P. Renatus M. Kairies N. Krapp R. Huber R. Salvesen G.S. Bode W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14790-14795Crossref PubMed Scopus (194) Google Scholar), and this seems not to involve the removal of N-terminal segments common for other protease clans (12Neurath H. Trends Biochem. Sci. 1989; 14: 268-271Abstract Full Text PDF PubMed Scopus (156) Google Scholar). Consequently the understanding that the precursor of RgpB (pro-RgpB) may require truncation at its N terminus (or even C terminus) for its activation serves as a good model to test the conservation hypothesis for protease zymogen activation since these observations would seem to contrast with the caspase activation mechanism. This study presents a detailed investigation of the autocatalytic processing of recombinant pro-RgpB, including the characterization of intermediates on the activation pathway, to clarify the mechanism of pro-RgpB maturation. Saccharomyces cerevisiaestrain YG227 (Mata,Δalg6::HIS3,ade2–101, his3Δ200,ura3–52, lys2–801) was kindly provided by Markus Aebi (Institute of Microbiology, ETH Zentrum, Zurich).Escherichia coli DH5α was used as the host for construction and propagation of all plasmids. Standard and synthetic media were prepared and supplemented with nutrients appropriate for selection and maintenance of plasmids as described previously (16Johnston J.R. Molecular Genetics of Yeast: A Practical Approach. 141. IRL Press at Oxford University Press, Oxford1994: 123-134Google Scholar). Yeast cells were grown in 2% glucose as carbon source and 2% galactose and 1% raffinose to induce protein expression fromGAL1 promoter. The pRS316Gal(ΔAcc65I) expression vector was produced for internal laboratory purposes as follows, although the deletion of Acc65I was not specifically necessary for the cloning strategy used in this report. The pRS316(ΔAcc65I) was first generated by digestion of pRS316 (17Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) with Acc65I and blunt ending of the overhang by T4 polymerase and religation in the presence ofAcc65I. Clones lacking the KpnI/Acc65I site were identified by restriction enzyme digest and used to generate the expression vector. The sequence encoding the Gal1promoter was amplified from genomic yeast DNA using primers Gal1f (5-ttggagctcacatggcattaccaccatatacatatc-3) and Gal1r (5-agagcggccgccggtaccgttttttctccttgacgttaaagt-3). This fragment was introduced intro pRS316(ΔAcc65I) as aSacI-NotI insert resulting in pRS316Gal plasmid. The full-length pro form of RgpB was amplified from genomic DNA isolated from P. gingivalis strain HG66. The 24-residue signal peptide of gingipain was replaced by the signal peptide of yeast carboxypeptidase Y amplified from pRA21 plasmid (18Nielsen T.L. Holmberg S. Petersen J.G. Appl. Microbiol. Biotechnol. 1990; 33: 307-312Crossref PubMed Scopus (36) Google Scholar). The insert was then ligated into the yeast expression plasmid pRS316Gal, and insertion of a FLAG epitope sequence (DYKDDDDK) at the C terminus resulted in the pro-RgpB FL1-WT construct (Fig. 1). The XhoI restriction site encoding two amino acids (Leu and Glu) separates the FLAG tag sequence from the authentic C-terminal sequence of pro-RgpB. Specific mutants (Fig.1) were constructed using an overlap polymerase chain reaction technique and the pro-RgpB FL-WT constructs as a template. The double mutant 2The minus sign before the number indicates residues in the RgpB prodomain counting backward using the first residue of the mature protein as the origin. Arg−1→ Ala/Arg−103 → Ala was generated in the same way but with pro-RgpB FL Arg−103 → Ala as a template. All constructs were sequenced completely to confirm that no undesired mutations were present. Yeast cells were transformed by the lithium acetate method, and transformants were selected on SC plates without uracil (SC−uracil) for auxotrophic selection (19Gietz D. St. Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2895) Google Scholar). Large scale expression was performed as follows. Single colony transformants were used to inoculate 20 ml of SC−uracil medium, and cultures were grown at 30 °C for 24 h followed by inoculation to 1.0 liter of medium, and the culture was allowed to grow for 12 more h. Next cells were washed in water and inoculated into induction medium (SC−uracil, 2% galactose, 1% raffinose). Cells were harvested by centrifugation 6 h after induction, washed in water, and resuspended in 20 mmBis-Tris, 10 mm NaCl, 1 mm CaCl2, pH 6.5 containing the protease inhibitors 1 mmphenylmethanesulfonic acid and 100 μm3,4-dichloroisocoumarin (20Salvesen G. Nagase H. Beynon R.J. Bond J.S. Proteolytic Enzymes: A Practical Approach. IRL Press, Oxford1989: 83-104Google Scholar). A half-volume of glass beads was added, and the cells were mechanically broken in a bead beater. Cell debris were removed by centrifugation at 18,000 × g for 30 min followed by filtration, and the supernatant fluid was used for further purification. Yeast extract was applied to a Sepharose Q (HiTrapQ HP5, Amersham Biosciences) column equilibrated with 20 mm Bis-Tris, pH 6.5 and washed with 5 column volumes of the same buffer following which bound protein was eluted with a two-step gradient (0–300 mm NaCl, 20 column volumes; and 300–500 mm NaCl, 5 column volumes). Fractions were assayed for activity, and Western blot analysis was performed using an enhanced chemiluminescence detection system with anti-FLAG monoclonal antibodies (Sigma). Fractions containing recombinant protein were pooled and further purified by immunoaffinity chromatography by binding to the to M-2 anti-FLAG agarose gel (Sigma) slurry overnight at 4 °C. Next the gel was washed on the column with TBS buffer (20 mm Tris, 137 mm NaCl, pH 7.6). Bound protein was eluted with FLAG-peptide (100 μg/ml). 1-ml fractions were collected and assayed for active enzyme or by Western blot with anti-FLAG antibodies. Fractions containing the recombinant protein were pooled and store at −70 °C. Identification of the recombinant protein was achieved by using both N-terminal sequencing and mass spectrometry (MALDI-TOF). Zymogen processing was analyzed in two ways. First, purified pro-RgpB was subjected to self-processing by incubating the recombinant protein in assay buffer (200 mmTris, pH 7.6, 100 mm NaCl, 5 mmCaCl2, 10 mm cysteine) for 2 h at 37 °C. Part of the reaction mixture was used for affinity labeling and activity assay; the other part of the reaction was stopped by adding 100 μm leupeptin, and cleavage products were analyzed by Western blot with anti-FLAG antibodies or antiserum to the mature RgpB (21Potempa J. Pike R. Travis J. Infect. Immun. 1995; 63: 1176-1182Crossref PubMed Google Scholar). Second, purified pro-RgpB was tested for self-processing at different concentrations to test for inter-versus intramolecular activation. The RgpB antiserum was raised against a peptide corresponding to the N-terminal 35 residues of mature RgpB and preferentially recognizes denatured protein. It does not require a free N terminus for reactivity as demonstrated under "Results." All chemicals used in the synthesis of the acyloxymethyl ketone were purchased from Advanced Chemtech and Sigma. The biotinylated inhibitor BiRK was synthesized by a solid-phase method from an arginine chloromethyl ketone according to the procedure described in Ref. 22Lee A. Huang L. Ellman J.A. J. Am. Chem. Soc. 1999; 121: 9907-9914Crossref Scopus (65) Google Scholar with minor modifications. The chloromethyl ketone was synthesized essentially as described using Fmoc-Arg(Pbf)-OH where Fmoc is N-(9-fluorenyl)methoxycarbonyl and Pbf is 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (23Robinson A.J. Pauls H.W. Coles P.J. Smith R.A. Krantz A. Bioorg. Chem. 1992; 20: 42-54Crossref Scopus (18) Google Scholar). Following cleavage from the matrix and deprotection, BiRK was purified on a Waters C-18 reverse phase high pressure liquid chromatography column and verified by mass spectrometry. The association rate of BiRK was determined by titration against RgpB of known activity purified from P. gingivalis (24Potempa J. Mikolajczyk-Pawlinska J. Brassell D. Nelson D. Thogersen I.B. Enghild J.J. Travis J. J. Biol. Chem. 1998; 273: 21648-21657Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Enzyme was used at a concentration of 0.25 nm. Enzymatic activity was measured by cleavage of Boc-FPR-AMC as described above. Inhibition of RgpB with BiRK was determined by progress curve analysis as follows. Enzyme was pre-equilibrated in assay buffer for 10 min at 37 °C and then added to a pre-equilibrated reaction mix containing 0–10 nm BiRK and 2 mm Boc-FPR-AMC in assay buffer to a final volume of 100 μl. Kinetic constants were obtained by a nonlinear least-squares fit of the data to the equation:y = (vst − (vs − v 0)(1 − exp−kt)/k obs) −A. The slope of the plot of k obs versus inhibitor concentration was used to determine the second order rate constant ka = 3.5 × 105m−1s−1. Active site labeling was performed with a trace (non-saturated) and a high (saturated) biotinylated affinity probe concentration. The time and concentration required to obtain trace and saturation probe binding were estimated according to the equation t 12 = ln2/ka[I] where I is the concentration of inhibitor required for free enzyme to decrease by 50% (half-lifet 12). A 30-μl portion of purified recombinant proenzyme (126 ng) subjected to autoprocessing was activated in assay buffer for 10 min at 37 °C. BiRK was added to a final concentration of 10 nm for 5 min at room temperature (trace probe condition) and 5 μm for 45 min at room temperature (saturating probe condition). Active site labeling was terminated by adding leupeptin (100 μm final concentration) for 5 min followed by boiling samples in SDS-PAGE sample buffer. Routinely the activity of recombinant forms of gingipain was determined by recording the release of 7-amino-4-methylcoumarin (AMC) generated by cleavage of Boc-QGR-AMC (100 μm) at 37 °C by measuring the increase in fluorescence at excitation/emission 380/490 nm using an fmax fluorescence microplate reader (Amersham Biosciences) operating in the kinetic mode. Assays were performed in 100 μl of assay buffer. Specific activity was defined as the amount of AMC released/min/μg of purified recombinant protein used for assay. The catalytic parametersK m and k cat were calculated by a non-linear regression fit to the Michaelis-Menten equation using substrate at a concentration ranging from 2.3 to 300 μm. Active enzyme concentration of fully processed recombinant wild-type RgpB was based on active site titration with leupeptin. Because of the low concentration of double Arg−1 → Ala/Arg−103 → Ala pro-RgpB mutant and its relatively poor activity, we were not able to accurately evaluate the enzyme concentration by active site titration. Therefore we estimated the enzyme concentration based on protein concentration determined by a modified Bradford assay (Pierce) with adjustment for the active pro-RgpB using incorporation of BiRK. Heterologous expression of soluble and active full-length or mutant forms of RgpB inE. coli was unsuccessful. Therefore we adapted the constructs to a Saccharomyces expression system that is engineered to drive synthesis and secretion. We added a FLAG epitope tag at the C terminus of all constructs for ease of purification and identification. Recombinant proteins were purified by a two-step protocol using anion exchange (Q-Sepharose) followed by anti-FLAG immunoaffinity chromatography and analyzed initially by Coomassie protein staining (Fig. 2 A) and Western blot using a FLAG antiserum (Fig. 2 B). Yields were low and ranged from 10 to 20 μg of protein/liter of yeast culture. Overexpression of the full-length pro-RgpB resulted predominantly in the expected 82-kDa protein but also in partial processing demonstrated by bands of lower molecular mass (68 and 56 kDa). The N-terminal sequence could not be obtained for this 82-kDa protein, probably due to a blocked N terminus, but based on the molecular mass in SDS-PAGE and assignment of tryptic peptides by MALDI-TOF (80% sequence coverage), the 82-kDa form corresponds to full-length zymogen with signal peptide attached. This was unexpected since the expression system was designed for secretion with expected signal peptide removal. The observation may explain why the recombinant product was not released from the cells, necessitating the extraction of the protein from the cell pellet. N-terminal sequence analysis revealed that the 68-kDa protein began at Ala−104, while the 56-kDa protein began at Tyr1 (see Fig. 1 for a description of the numbering system). Since both of these residues follow Arg in the coding sequence, it is likely that they are generated by a self-processing mechanism. To clarify whether the conversion occurs autocatalytically or whether it is due to a yeast-encoded protease we expressed a catalytic mutant, Cys244 → Ala. Substitution of Ala for the catalytic Cys completely abolished the smaller products, confirming an autoprocessing mechanism (Fig.2, A and B). To assess the importance of autoprocessing we constructed individual Arg → Ala substitutions at the determined cleavage sites and a double mutant containing both substitutions. Both pro-RgpB Arg−103 → Ala and the double mutant were found to be unprocessed, whereas an intermediate 68-kDa form corresponding to pro-RgpB processed at Arg−103 was detected in the Arg−1 → Ala mutant (Fig. 2 B). This indicates that pro-RgpB undergoes sequential two-step processing in which cleavage at Arg−103 is required for subsequent processing at Arg−1. Native mature RgpB obtained from P. gingivalis is truncated at the C terminus (6Eichinger A. Beisel H.G. Jacob U. Huber R. Medrano F.J. Banbula A. Potempa J. Travis J. Bode W. EMBO J. 1999; 18: 5453-5462Crossref PubMed Scopus (155) Google Scholar), and consequently we were unable to purify this protein because the C-terminal FLAG tag would be removed. To obtain this derivative we generated the construct pro-RgpB-ΔC-WT that encodes the pro form lacking the 72-residue C-terminal sequence (Fig.1) Expression and purification of this protein gave a band pattern similar to that of a full-length protein, but as expected, each derivative was slightly smaller (Fig. 2 B). Recombinant pro-RgpB proteins were incubated at 37 °C for 2 h in assay buffer (see "Materials and Methods") to provide conditions for autolytic processing. After this time the full-length wild-type proenzyme was almost completely converted to the mature form (Fig.3, A and B). The fully mature recombinant enzyme appeared to be identical in mass to that of the native enzyme indicating cleavage at the same site. The possibility that a contaminating yeast proteinase was responsible for processing is unlikely because the general serine protease inhibitors phenylmethanesulfonic acid (2.5 mm) and 3,4-dichloroisocoumarin (0.1 mm), the metalloprotease inhibitor 1,10-phenanthroline (1.0 mm), and the aspartic protease inhibitor pepstatin (0.015 mm) had no effect on the self-processing (not shown). In contrast, the gingipain inhibitor leupeptin at 100 μm completely prevented proenzyme autoprocessing. Moreover, the catalytic mutant Cys244 → Ala incubated in the same conditions remained unprocessed (Fig.4 A). The double mutant (Arg−1 → Ala/Arg−103 → Ala) subjected to self-processing did not generate any bands recognized by antisera (Fig. 5, A andB). However, affinity labeling revealed additional bands that resulted from an aberrant cleavage (Fig. 5, C andD). The aberrant product did not accumulate during self-processing conditions, and we did not observe an increase in activity as determined by cleavage of Boc-QGR-AMC (Fig.6) or affinity labeling (Fig. 5,C and D).Figure 4Self-processing of pro-RgpB mutants.Pro-RgpB mutants were subjected to self-processing conditions and analyzed by Western blot using anti-FLAG and anti-N-terminal RgpB antiserum. A, full-length catalytic mutant; B, ΔC-WT mutant. In each panel lane 1 represents untreated protein, and lane 2 represents incubated gingipain.View Large Image Figure ViewerDownload (PPT)Figure 5Self-processing of pro-RgpB double mutant. Purified recombinant Arg−1 → Ala/Arg−103 → Ala mutant was incubated for 2 h at 37 °C, and the reaction products were visualized by Western blot or affinity labeling with BiRK. A, proteins probed with anti-FLAG; B, proteins probed with specific anti-N-terminal RgpB antiserum; C, proteins labeled with a trace concentration of BiRK; D, proteins labeled with a saturating amount of BiRK. In each panel lane 1 represents untreated protein, and lane 2 represents incubated protein. Aberrant products are highlighted by an asterisk.View Large Image Figure ViewerDownload (PPT)Figure 6Activation of pro-RgpB by self-processing. The indicated recombinant pro-RgpB forms were subjected to self-processing by incubating for 2 h at 37 °C. Activity was measured by Boc-QGR-AMC substrate hydrolysis. The untreated samples (white bars) were stored on ice for the same period of time as the treated ones (black bars) and incubated for 10 min at 37 °C in the assay buffer prior to assay.R-103A/R-1A, Arg−1 → Ala/Arg−103 → Ala.View Large Image Figure ViewerDownload (PPT) We determined the order of processing by using differential Western blot using antisera against the N-terminal and C-terminal (FLAG-tagged) regions. An intermediate with the N-terminal propeptide intact but lacking the C-terminal FLAG was not detected. This reveals that removal of the C-terminal region (residues 434–506) requires prior removal of the N-terminal pro segment. Surprisingly, conversion to the mature form was drastically retarded in the RgpB-ΔC-WT mutant zymogen exposed to autoprocessing conditions, indicating the importance of the C-terminal part of the protein in maturation (Fig.4 B). The RgpB-ΔC-WT must be relatively inactive because it does not autoprocess in vitro, although conditions in yeast must have been more favorable to produce the initial cleavage events. Our data demonstrate that mature gingipain can be formed in vitro by sequential autoprocessing with the ultimate product lacking both the N-terminal propeptide and the C-terminal extension exactly as seen in the active enzyme isolated from P. gingivalis. However, it is not clear whether the final product represents the most active species or what the relative activities of the zymogen and intermediate forms are. To address this issue and to test the relevance of the cleavages we used active site labeling of recombinant zymogen and its derivatives. This technique relies on the inherent ability of a single active site-directed affinity probe to react with kinetics that parallel the enzyme catalytic competence (25Yamin T.-T. Ayala J.M. Miller D.K. J. Biol. Chem. 1996; 271: 13273-13282Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). All intermediates in the processing of pro-RgpB have a potential active site and an intact catalytic apparatus as demonstrated by BiRK labeling (Fig. 3, C and D), but this does not mean that they have equivalent activities. If two species of an enzyme with different activities are present in an equimolar amount, they will be equally labeled only if inhibitor is in excess. Conversely, if the inhibitor is in deficit (enzyme is saturating) the relative labeling should reflect the activity of the forms: the most active form will be more heavily labeled. In other words, low BiRK concentrations reflect enzymatic activity, whereas high BiRK concentrations simply reflect total concentration. This useful property enables us to quantitate enzyme activity as a function of BiRK labeling. The probe we designed, BiRK, was intended for high reactivity with Arg-specific cysteine proteases but was broadly tolerant of extended subsite occupancy. Labeling was performed using a low probe concentration (10 nm) and short time of incubation (5 min) under conditions predetermined (see "Materials and Methods") not to saturate the potential active sites present (Fig. 3 C). To verify this procedure we also incubated the intermediates with probe under conditions calculated to completely saturate all available active sites (5 μm, 45 min) (Fig. 3 D). The intensity of the bands labeled at high probe concentration was comparable to the intensity of bands probed with antibodies, reflecting the total protein of each form (Fig. 3, B and D, comparelanes 1). In stark contrast, low probe concentrations revealed a completely different pattern with the fully processed enzyme, even though it is present in the lowest amount, displaying highest labeling (Fig. 3, B and C, comparelanes 1). Full-length zymogen, representing the highest concentration among the proteins, showed the lowest labeling under low probe conditions. These findings indicate that the enzyme gains activity with each of the three processing events. The same samples were also analyzed for enzymatic activity. When incubating with the fluorometric substrate Boc-QGR-AMC a 3-fold increase in substrate cleavage was detected in wild-type digest, and no increase in activity was observed for C-terminally truncated enzyme and double mutant (Fig. 6). Taken together, the data argue that pro-RgpB activation correlates with the enzyme processing and is accomplished by sequential cleavage of N-terminal propeptide followed by trimming of the C terminus. Zymogen processing could be either intramolecular (by the catalytic site within the precursor) or intermolecular (where a different catalytic site attacks the bonds in an adjacent molecule). These possibilities can be distinguished by determining whether the process is unimolecular or bimolecular (26Nagase H. Enghild J. Suzuki K. Salvesen G. Biochemistry. 1990; 29: 5783-5789Crossref PubMed Scopus (351) Google Scholar). The rate of autoprocessing was dependent on precursor concentration (Fig. 7), implying a second order process. Since this is consistent with a bimolecular mechanism, we conclude that processing is predominantly intermolecular, although we cannot rule out some intramolecular component. Probe labeling is consistent with an increase in activity during processing, and we attempted to confirm this by determining the kinetic parameters of full-length wild-type protein converted to the mature form by self-processing and full-length double mutant (Arg−1 → Ala/Arg−103 → Ala), which represents the zymogen form. Labeling with BiRK revealed the full-length zymogen and an aberrant product of about 45 kDa (Fig. 5, C and D). The latter probably corresponds to a cleavage C-terminal to Arg−1 since it is not recognized by the specific anti-RgpB antiserum raised to the first 35 residues of mature RgpB. The aberrant cleavage appears to be a result of mutating Arg−103 and Arg−1 and must be taken into account for quantitating activity due to the full-length zymogen. Quantitative image analysis revealed that the full-length zymogen corresponds to 66% of the total RgpB as determined under saturating BiRK conditions (Fi