1Biotechnology Research Group of the Hungarian Academy of Sciences and 2Department of Biochemistry, Eötvös Loránd University, Pázmány sétány 1/C, 1117 Budapest, Hungary
3 To whom correspondence should be addressed. e-mail: graf{at}ludens.elte.hu
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Abstract |
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Keywords: autoactivation/serine protease/substrate specificity
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Introduction |
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To verify the structural basis of substrate specificity the amino acids at position 189 in trypsin and chymotrypsin were interchanged by site-directed mutagenesis. The D189S mutation failed to confer chymotrypsin-like activity to trypsin (Gráf et al., 1988). Apparently, interchanging of all different amino acids in the S1 regionsincluding two surface loops (L1, 185195 and L2, 217223)and also two further substitutions at sites 138 and 172 were needed for an almost complete conversion of trypsin to a chymotrypsin-like protease (Hedstrom et al., 1992
, 1994a
,b). This is in line with the view that the differential specificities of trypsin and chymotrypsin may be controlled by extended structural units (Gráf, 1995
; Perona et al., 1995
). To further test this hypothesis, reverse substitutions were introduced into chymotrypsin (Venekei et al., 1996b
). The S189D mutation greatly reduced the activity, while specificity remained basically chymotrypsin-like. Further substitutions in the S1 region resulted in non-specific enzymes with even lower activity. Substitutions at the same sites were introduced into trypsin in order to convert it to elastase as well, but the mutants had no measurable amidase activity (Hung and Hedstrom, 1998
). These later studies suggest that further sites might be involved in substrate discrimination, and a unique, even more extended structure determines each specificity. Such sites might be components of a co-evolving and mechanically coupled network in the trypsin family that are located outside the S1 region (Süel et al., 2003
).
The enzyme in the chymotrypsintrypsin conversion studies was chymotrypsin-B that contains an Ala at site 226. However, Gly is conserved at this site among trypsins that may indicate its important role in the determination of a trypsin-like substrate specificity. Indeed, the G226A mutation in trypsin reduces the activity on trypsin substrates by three to four orders of magnitude (Craik et al., 1985
). The crystal structure of the mutant revealed that Ala226 causes misalignment of both Arg and Lys substrates at the active site, and showed the lack of direct electrostatic interaction between the P1 Arg and Asp189 of trypsin (Wilke et al., 1991
). To investigate, if residue 226 is important for the chymotrypsin
trypsin specificity conversion as well, we made the A226G replacement in the S189D chymotrypsin-B mutant (S189D+A226G), and in one of our previously constructed chymotrypsin-B mutants with further substitutions in the S1 region (S1+A226G) (Figure 1).
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Materials and methods |
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Highly purified enterokinase was the product of Biozyme (EK-3). Bovine chymotrypsinogen, bovine trypsin, SBTI-Sepharose, MUGB, succinylAlaAlaProPheAMC, AMC and 7-methylumbelliferon were from Sigma Chemical Co. SuccinylAlaAlaProLysAMC and the oligopeptide substrate library were prepared as described (Gráf et al., 1988; Antal et al., 2001
).
Construction of mutants
Chymotrypsin-B mutants S189D+A226G and S1+A226G were constructed according to Kunkel (1985), from S189D and a multiple substituted S1 region mutant, respectively (Figure 1). The mutations were confirmed by DNA sequencing.
Similarly to the previous chymotrypsintrypsin mutants (Venekei et al., 1996a
,b) the new mutants were also expressed as a propeptide chimera in which the chymotrypsin propeptide was replaced by the trypsin propeptide and site Cys122 was mutated to Ser, so that they could be activated with enterokinase. This minimized the contamination of the enzyme preparations with trypsin and an accurate determination of even low tryptic activities of the mutant enzymes became possible. The enzyme kinetic parameters of the chimera do not differ from those of wild-type chymotrypsin under physiological conditions (Venekei et al., 1996a
). On the other hand, the wild-type propeptide increased the stability of the protein as studied under denaturing conditions (non-physiological pH, temperature and denaturing agents) (Kardos et al., 1999
). To examine if the disulfide-linked chymotrypsin propeptide would still affect the enzymatic properties of our new mutants, the chymotrypsin propeptide-containing forms of the mutants were also expressed and characterized. They did not, however, show any significant difference in their catalytic activities when compared with their chimeric counterparts.
Expression and purification of the mutants
The wild-type and the mutant chymotrypsinogen sequences were cloned into the pET-17b expression vector. These plasmids were transformed into the BL21 (DE3) pLysS Escherichia coli strain, the expression was conducted according to the manufacturers instructions (Novagen, 1997). After induction with IPTG, the cells were collected in a 1/10 volume TE buffer and frozen at 20°C. Then they were thawed and sonicated, and the inclusion body fraction was collected by centrifugation and washed three times with TE buffer. For renaturation of the expressed protein, the inclusion body fraction was solubilized with 6 M GuHCl, 0.1 M TrisHCl (pH 8.0), 100 mM DTT; the solution contained 10 mg/ml protein. The solubilized protein was diluted to 200-fold into the refolding buffer containing 1 M GuHCl, 5 mM cysteine, 1 mM cystine, 50 mM TrisHCl (pH 8.0), 5 mM EDTA. The renaturation process was conducted at 4°C, overnight. The renatured protein solutions were then dialyzed against 2 mM HCl, 10 mM CaCl2 and ultracentrifuged (45 000 r.p.m., 30 min, 4°C). Zymogens were activated by highly purified enterokinase at a 100:1 (w/w) zymogen/enterokinase ratio, and then the active forms were purified by affinity chromatography on an SBTI-Sepharose column. The purity of the preparations was analyzed by SDSPAGE. The enzyme concentration was determined by Bradford assay for the low-activity mutants, and by active site titration with MUGB and MUTMAC (Jameson et al., 1973
) for S189D+A226G mutant and wild-type chymotrypsin, respectively.
Enzyme assays
Amide hydrolysis was measured on succinylAlaAlaProPheAMC and succinylAlaAlaProLysAMC substrates in a 50 mM TrisHCl pH 8.0, 10 mM CaCl2, 0.1 M NaCl reaction buffer at 37°C using a Spex Fluoromax spectrofluorimeter. The data were analyzed with Enzfitter software.
Specificity profiling on a competing oligopeptide substrate library was also performed as described (Antal et al., 2001). Briefly, the oligopeptide library has seven members with a sequence HAAPXSADIQIDI, where X represents the different P1 residues, Lys, Arg, Tyr, Leu, Phe and Trp. X=Pro served as an internal standard. The individual peptide substrates compete for the proteinase during the enzymatic reaction. Enzyme concentrations were optimized to ensure comparable enzyme reaction rates: 0.6 µM for S189D and S189D+A226G mutants, 0.075 µM for chymotrypsin-B and 0.0012 µM for bovine trypsin. The concentration of each substrate mixture component was 40 µM. The reaction was monitored by RP-HPLC separation of the components.
Autoactivation experiments were conducted with incubation at room temperature in the reaction buffer above, and were monitored with SDSPAGE and activity measurement.
Chymotrypsinogen activation was carried out at 37°C in the reaction buffer above. Chymotrypsinogen (1 µM) was incubated with 16 µM S189D, 14 µM S189D+A226G and 5 nM bovine trypsin for 80 min. Chymotrypsin activity was measured every 10 min on succinylAlaAlaProPheAMC substrate and was expressed as a percentage of the total activity.
For the measurement of benzamidine inhibition of the S189D and S189D+A226G mutants, progress curves were recorded with enzyme concentrations of 80160 nM and with substrate concentrations of 50100 µM. Constants were determined by analyzing the progress curves by the DynaFit software (Kuzmic, 1996) according to the MichaelisMenten equation.
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Results and discussion |
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The A226G replacement exerted opposite effects on the mutants specificities (Table I). The S1 mutant changed to a more chymotrypsin-like protease, with two orders of magnitude higher activity on the chymotrypsin than on the trypsin substrate, while the basically chymotrypsin-like S189D mutant became trypsin-like by the single A226G substitution. The catalytic activity on the Lys-containing substrate was increased by two orders of magnitude and at the same time it was decreased by one order of magnitude on the chymotrypsin substrate. The trypsin-like activity of the mutant allowed the use of active site titration for the measurement of enzyme concentration. It is interesting to note that the increase in activities of both mutants, S189D+A226G and S1+A226G, resulted mainly from the elevation of the catalytic rate constants.
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The crystal structure of the S189D mutant (Szabó et al., 2003) shows that the S1 region contains severe deformations mostly in the loop segments 185195 and 217224, including the Cys191Cys220 disulfide bond. The Asp189 side chain at the bottom of the pocket is turned out to the solvent, presumably because, contrary to trypsin, the S1 region of chymotrypsin is not suitable for the stabilization of such a partially buried charge. These structural deformations explain the poor activity and the lack of trypsin-like specificity of the S189D mutant, but do not provide any useful information concerning the structural effects of the A226G substitution. From the increased trypsin-like features of the S189D+A226G mutant, however, we can conclude that the negative charge of Asp189 might somehow become more available in the S1 region. The wild-type level inhibition by benzamidine and the strong preference for the Arg substrate in the case of the oligopeptide mixture suggest that the P1 Arg and the Asp189 side chains can form even a direct charge interaction. To test this assumption, the three-dimensional structure of the S189D+A226G mutant would be needed, providing further insight into the structural basis of this unexpected specificity conversion.
The A226G substitution can be regarded as a conversion of S189D chymotrypsin-B to S189D chymotrypsin-A mutant. The chymotrypsin-B mutants were not appropriate counterparts of trypsinchymotrypsin mutants, since site 226 is a conserved Gly in trypsins. The fact that the G226A mutation in trypsin reduces the activity on trypsin substrates by three to four orders of magnitude (Craik et al., 1985
) also documents the importance of Gly replacing Ala at site 226. Therefore, the introduction of the S189D mutation into a chymotrypsin-A-like protease can be considered as the first successful attempt to convert chymotrypsin to a trypsin-like protease. Our unexpected finding is that the single mutation S189D in a chymotrypsin-A-like mutant converts the specificity profile of chymotrypsin to that of a trypsin-like protease with enough catalytic potential even for autoactivation. This seems to support the view (also suggested by Hung and Hedstrom, 1998
) that there may be different strategies and routes to convert trypsin to a chymotrypsin-like protease and vice versa and that the key residues involved in substrate discrimination and therefore to mutate to interchange the specificities of these proteases may not be identical in the two structures.
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Acknowledgements |
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References |
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Received September 4, 2003; revised October 31, 2003; accepted November 4, 2003 Edited by Valerie Daggett
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