Biochemistry 1993, 32, 2845-2852
2845
Significance of Hydrophobic S 4 - P 4 Interactions in Subtilisin 309 from Bacillus lentus Lene M. Bech, Steen Bech Slarensen, and Klaus Breddam' Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark Received October 6, 1992; Revised Manuscript Received January 5, 1993
ABSTRACT: The subtilisins have an extended substrate binding cleft comprising at least 8 subsites. Two pockets at the SIand S4sites are particularly conspicuous, and the interactions between substrate and these two pockets are very important for the substrate specificity. Phe residues have mutationally been introduced at one of positions 102, 128, 130, and 132 of the subtilisin Savinase from Bacillus lentus to investigate the effects of introducing bulky groups along the rim of the S4 binding pocket. It is shown that the marked P4 preference of wild-type Savinase for aromatic groups is eliminated by the Gly102 Phe and Ser128 Phe mutations, indicating that bulky groups at positions 102 and 128 block the S4 binding site. In contrast, the activity toward hydrophilic P4 residues is not nearly as affected by these mutations, suggesting that the binding mode of the P4 side chain is dependent on its properties. Introduction of a bulky -CHIS-CH2-CH2-pyridyl group at position 128, by mutational incorporation of Cys followed by chemical Phe mutation hardly modification with 2-vinylpyridine, has essentially the same effect. The Serl30 affects the activity of the enzyme while the Ser Phe mutation at position 132 renders the preference for hydrophobic groups in P4 even more pronounced. This mutation furthermore affects the size of the S4 pocket. An analysis of double mutants at positions 132 and 104 suggests that the S4 region is flexible and is adjusted upon binding of substrates.
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The three-dimensional structures of subtilisins in complex with various proteinaceous inhibitors (Bode et al., 1987; McPhalen et al., 1985; McPhalen & James, 1988; Takeuchi et al., 1991a,b) suggest that two types of interactions between such enzymesand substrate are important: (a) hydrogen bonds between the main chains producing an antiparallel &sheet and (b) hydrophobic interactions between the side chains at the PI and P4 positions' and the correspondingbinding pockets at the SIand S4 subsites, respectively. The significance of the P 4 S 4interactions, remote from the catalytic event, for rapid hydrolysis (Gran et al., 1992; Gran & Breddam, 1992; Svendsen, 1977)is of particular interest,but the features within the S4 binding pocket influencing the catalytic efficiency are not well-established, The structures of the various enzymeinhibitor complexes suggest that the amino acid residues 107, 126, and 135: all located at the bottom of the pocket (Heinz et al., 1991; McPhalen & James, 1988), determine its hydrophobic nature, and the residue at position 104 appears to function as an adjustable lid on the pocket (Bech et al., 1992b; McPhalen et al., 1985). However, along the rim of the pocket a number of generally conserved, nonbulky amino acid residues are conspicuous (Figure 1) with residues Gly100Ser101-Gly102,Ser 125-Leu126-Glyl27, Serl30, and Ser 132 being invariant. Gly102 is involved in main chain to main chain interaction with the P4 amino acid residue (McPhalen & James 1988). The backbone of the peptide segment 128130 participates in formation of the S4 binding pocket (Bode et al., 1987; Hirono et al., 1984; McPhalen & James, 1988), and a study of a number of mutants produced by random mutagenesiswithin this region implied that position 128,when occupied by a hydrophobic residue, is fixed at the S4 binding pocket (Teplyakov et al., 1992). Residue 132 is also located I The binding site notation is that of Schechter and Berger (1967). Accordingly, P,denotes a substrate position, S, denotes the corresponding enzyme binding subsite. *The amino acid numbering is that of subtilisin BPN' (Markland & Smith, 1967).
0006-2960/93/0432-2845$04.00/0
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on the rim but in the most remote position relative to the P4 position of the substrate. However, this residue is of interest since it is suspected to change position upon binding of inhibitors (McPhalen & James, 1988) in concert with the movement of the side chain of Tyrl04 (Bech et al., 1992b; Kraut, 1977; Takeuchi et al., 1991a). The significance of these nonbulky amino acid residues on the rim of the P4 pocket is here investigated by mutational introduction of bulky side chains at positions 102, 128, 130, and 132 of the subtilisin 309, S a ~ i n a s efrom , ~ Bacillus lentus, followed by an extensive kinetic characterization of mutant enzymes. The mutants G102F and S128F which lack the marked P4 preference of wild-type Savinase for aromatic groups are used to elucidate to which extent the favorable S4-P.4 interactions are employed to stabilization of the transition state. Double mutants at positions 132 and 104 are constructed to investigate the interdependence of the effects of substitutions at these positions.
MATERIALS AND METHODS Materials. A Bacillus subtilis strain (Aapr, Anpr) was kindly provided by Sven Hastrup, Novo Nordisk AIS, Denmark. The Escherichia colilb. subtilis shuttle vector containingthe structural gene for Savinase (Bech et al., 1992b), theE. colistrain DH5a (Hanahan, 1985), and the M13mp18 subclone of the Savinase gene (Bech et al., 1992b) were from house collections. The synthetic oligonucleotides were synthesized on an Applied Biosystems 380 DNA synthesizer, and the oligonucleotide-directedin vitro mutagenesis system version 2 kit was from Amersham. Restriction endonucleases, ~~~~~~~~
Abbreviations: ABz, o-aminobenzoyl;-EP, ethylpyridine; pNA,paranitroanilide; Pyr, pyridine; Savinase, subtilisin 309 from B. lentus; SUC, succinyl; Tyr(NOz), 3-nitrotyrosine; Y', Tyr(N0-J; 4,the scissile bond; X102Z, Savinase where residue X at position 102 has been replaced by residue Z S128C-EP, Savinase where the mutationally introduced Cys at position 128 has been alkylated with 2-vinylpyridine.
0 1993 American Chemical Society
2846 Biochemistry, Vol. 32, No. 11, 1993
Bech et al.
t FIGURE1: Stereoview of the S4 binding site of Savinase (Betzel et al., 1992). The P4 amino acid residue is involved in main chain to main chain interactions with Gly102 (McPhalen & James, 1988) and the P4side chain protrudes into the S4binding pocket bounded by Ser128, Serl30, and Ser132. Val104 functions as an adjustable lid on the pocket. T4 polynucleotide kinase, and adenosine triphosphate were from Boehringer Mannheim, FRG. DNA sequencing was performed using the Taq DyeDeoxy terminator cycle sequencing kit and the Model 373A DNA sequencing system from Applied Biosystems. Prep-A-Gene was from Bio-Rad Laboratories. Mes, Hepes, Bicine, and bacitracin were from Sigma, and bacitracinSepharose was prepared as previously described (Stepanov & Rudenskaya, 1983). Sephadex G5O was from Pharmacia LKB Biotechnology,Sweden. Suc-AlaAla-Pro-Phe-JpNA was purchased from Bachem, Switzerland, and the fluorogenic substrates were synthesized as previously described (Grran et al., 1992; Meldal & Breddam, 1991). Production of Savinase Derivatives. In vitro mutagenesis was performed using a M 13mpl8 subclone, containing a 41 5bp EcoRI-PvuII fragment of the Savinase gene. High frequency of mutants was obtained using the oligonucleotidedirected in vitro mutagenesis system version 2 kit from Amersham, on the basis of the methods of Eckstein and coworkers (Nakamaye et al., 1986; Sayers et al., 1988; Taylor et al., 1988a,b), and the mutated sequences were reintroduced into an E. coli/B.subtilis shuttle vector as previouslydescribed (Bech et al., 1992b). Mutant enzymes were purified from a l-Lculturegrownat 37 OC for 2*/2dayswithvigorousshaking in LB-medium containing chloramphenicol (6 mg/L). The enzyme purifications were performed as described (Bech et al., 1992b) and followed by assay against 0.35 mM Suc-AlaAla-Pro-Phe-JpNA in 50 mM Bicine, 2 mM CaC12, 0.1 M KCI, 5% dimethylformamide, pH 8.5. The enzyme preparations were stored frozen in buffer at -1 8 OC. The enzymes were stable under these conditions. Reduction of S 128C with 100 mM j3-mercaptoethanoland a subsequent alkylation with 94 mM 2-vinylpyridine was performed in 0.05 M Hepes, 2 mM CaC12, pH 7.5, using an enzyme concentration of 46 pM. The reaction was followed with assays against Suc-Ala-Ala-Pro-Phe-4 pNA until the activity remained constant and addition of another aliquot (94 mM) of 2-vinylpyridinehad no further effect on activity. Characterization of Mutant Savinase. The purity of the mutant enzymes was ascertained by SDS-polyacrylamide gel electrophoresis on 20% homogeneous gels using the PhastSystem from Pharmacia LKB Biotechnology. The enzymes had previously been denatured by incubation in 0.1 M HCl for 30 min and then lyophilized in order to avoid autolysis on boiling in sample buffer containing SDS. The concentration of Savinase mutants was determined spectrophotometrically
using a €280 = 23 mM-' cm-l or 27 mM-l min-l (S128C-EP). These values were determined by amino acid analysis of a solution of enzyme with known absorbance, performed after acid hydrolysis for 24 h at 110 OC using a Pharmacia LKB CY plus amino acid analyzer. N-Terminal sequencing was performed on an Applied Biosystems Model 470 A gas-phase sequencer using the program provided by the company. The specific activity toward Suc-Ala-Ala-Pro-Phe-JpNa (0.35 mM) was determined by assay in 50 mM Bicine, 0.1 M KCI, 2 mM CaC12,5% dimethylformamide, pH 8.5, at 25 OC and 410 nm using a Perkin Elmer A7 spectrophotometer. The kinetic constants for hydrolysis of ABz-Asp-Phe-Arg-LeuPhe- 1Ala-Phe-Tyr (NO2)-AspOH3were determined using the nonlinear regression data analysis program Enzfitter (Leatherbarrow, 1987). Assays were performed in 50 mM Bicine, 2 mM CaC12, 0.1 M KCl, 2% dimethylformamide, pH 8.5, and hydrolysis rates were determined by monitoring the fluorescence emission at 420 nm after excitation at 320 nm using a Perkin Elmer LS50 luminescence spectrometer. The kcat/KM values for hydrolysis of the fluorogenicpeptide substrates were determined from initial rates using the following relation: kcat/KM= vo/SoEo which is valid at SO 520 min-I, K M > 10 pM. The standard deviations for the k,,/KM values are
