Proc. Natl. Acad. Sci. USA Vol. 83, pp. 3743-3745, June 1986 Biochemistry Site-directed mutagenesis and the role ofthe oxyanion hole in subtilisin (protein engineering/serine protease/oligonudeotide-directed mutagenesis) PHILIP BRYAN, MICHAEL W. PANTOLIANO, STEVEN G. QUILL, HUMG-YU HSIAO, AND THOMAS POULOS Genex Corporation, 16020 Industrial Drive, Gaithersburg, MD 20877 Communicated by David R. Davies, February 5, 1986

ABSTRACT Oligonucleotide-directed mutagenesis was The specific binding forces involved in stabilizing the tran- used to investigate the nature of transition state stabilization in sition state have been described crystallographically by using the catalytic mechanism ofthe serine protease, subtflisin BPN'. inhibitors that mimic the tetrahedral intermediate and have The gene for this extracellular enzyme from Bacillus amyloli- been extrapolated to true substrates by using model-building quefaciens has been cloned and expressed in Bacillus subtilis. In approaches (4-9). One of the most important hypotheses to the transition state complex, the carbonyl group of the peptide emerge from these investigations is that hydrogen bonding bond to be hydrolyzed is believed to adopt a tetrahedral between protein groups and the developing negative charge configuration rather than the ground-state planar configura- on the substrate carbonyl oxygen atom of the transition state tion. Crystallographic studies suggest that stabilization of this is a major contributing factor in lowering the free energy of activated complex is accomplished in part through the donation the activated complex. Indeed, a recent theoretical treatment ofa hydrogen bond from the amide side group of Asn-155 to the of trypsin supports this view (10). carbonyl oxygen of the peptide substrate. To specifically test In the mammalian serine proteases, trypsin and chymo- this hypothesis, leucine was introduced at position 155. Leucine trypsin, two peptide NH groups of the polypeptide backbone is isosteric with asparagine but is incapable of donating a form the so-called oxyanion hole by donating hydrogen bonds hydrogen bond to the tetrahedral intermediate. The Leu-155 to the negatively charged oxygen atom of the tetrahedral variant was found to have an unaltered K. but a greatly intermediate (3, 11, 12). The bacterial protease, subtilisin, is reduced catalytic rate constant, kcat, (factor of200-300 smaller) similar, yet here the side chain of Asn-155 is believed to when assayed with a peptide substrate. These kinetic results are provide one of the hydrogen-bonding groups in the oxyanion consistent with the Asn-155 mediating stabilization of the hole (4). Fig. 1 is a stereoscopic view of a hypothetical activated complex and lend further experimental support for complex formed between subtilisin and a peptide substrate in the transition-state stabilization hypothesis ofenzyme catalysis. the tetrahedral configuration. Unlike the active-site serine and histidine of serine Nearly 40 years ago, Pauling postulated that enzymic catal- proteases, whose function can be directly tested by a variety ysis is due to the preferential binding of substrates to the of chemical agents, it is not possible to utilize chemical- enzyme in a configuration that resembles the transition state modification methods to probe the function of the oxyanion complex (1, 2). By doing so, the enzyme lowers the activation hole because the groups involved, peptide NH and aspara- energy barrier of the reaction, resulting in an increase in gine, are essentially inert toward commonly used chemical reaction rate. Lowering of the activation energy barrier probes. However, oligonucleotide or site-directed mutagen- occurs because specific interactions between the enzyme and esis offers an alternate and much more specific method for the transition state conformation of the substrate forms an testing the role of any amino acid residue by selectively energetically more stable complex than the enzyme and altering DNA codon sequences using in vitro recombinant substrate in the ground-state configuration. This simple but DNA techniques. While the use of site-directed mutagenesis elegant idea continues to serve' as the single best explanation to study enzyme mechanisms is a field still in its infancy, it for enzyme catalysis and has received both experimental and is clear from those few examples in the literature (13-16) that theoretical support for various enzyme systems and espe- such an approach provides a powerful tool for probing the cially for serine proteases. In serine proteases, the carbonyl details of enzymic catalysis. Therefore, we were encouraged carbon atom of the susceptible bond adopts a tetrahedral to test the predicted role of the oxyanion hole in the configuration in the transition state as the serine-OH and subtilisin-catalyzed cleavage of a specific peptide substrate carbonyl carbon form a covalent bond (3). by changing Asn-155 to leucine. Leucine was chosen because leucine and asparagine are nearly isosteric and should result 08- in a minimum change in active site geometry with the exception, of course, that leucine is incapable of donating a

FIG. 1. Stereoscopic view of the subtilisin active site depicting the binding of a hypothetical tripeptide substrate in the tetrahedral transition state. provide single-stranded template DNA. The substitution in RESULTS AND DISCUSSION the subtilisin gene of the leucine codon for the codon for Asn-155 was accomplished by the procedure of Zoller and Vma, and Km values were determined by spectrophotometric Smith (19). The 27-base oligonucleotide used to direct the assay for the peptide substrate succinyl-L-Ala-L-Ala-L-Pro- mutation was homologous with the parental template gene L-Phe-p-nitroanilide as described by Delmar et al. (21). The with the exception that the codon for Asn-155, ACC, was results obtained from Lineweaver-Burk plots of the initial replaced with the leucine codon, CTC. Clones were screened rates of hydrolysis are summarized in Table 1. Two separate initially by hybridization and confirmed by sequencing ap- preparations of the Leu-155 variant yielded similar kinetic proximately 100 base pairs surrounding the site-directed constants. Our kinetic analysis demonstrates that conversion change. Two independently derived Leu-155 variants were of Asn-155 to Leu-155 lowers the catalytic constant, kcat, by cloned from mp9 into an Escherichia coli-B. subtilis plasmid a factor of about 200-300 with little effect on Km, indicating shuttle vector containing the high-copy-number pUB110 that Asn-155 plays an important role in the catalytic mech- origin of replication (20). In order to eliminate background anism but not in the initial binding of substrate. protease levels, a B. subtilis strain containing chromosomal That we observe no change in Km is interesting in the light deletions for both subtilisin and neutral proteases was used as of recent findings by McPhalen et al. (22) that the oxyanion the host for transformation. As expected in a chromosomal hole in subtilisin forms a hydrogen bond with the P1 carbonyl deletion strain, neither subtilisin activity nor protein could be oxygen atom in the enzyme-substrate complex (Fig. 1). detected, even in sensitive assays using specific antibody Therefore, one might have anticipated that the inability to against subtilisin. hydrogen bond with part of the oxyanion hole in the Leu-155 Wild-type subtilisin is expressed efficiently from this variant should have caused an increased Km. However, the vector in B. subtilis and secreted into the growth medium at refined 1.9-A subtilisin structure (B. C. Finzel, A. J. How- 100-200 gg/ml. Subtilisin is the major protein secreted in this ard, G. Gilliland, and T. L. P., unpublished data) shows that system and accounts for about 80% of the protein in the a water molecule occupies the oxyanion hole in the parent growth medium detectable by NaDodSO4/polyacrylamide structure; therefore, replacement ofthe water-oxyanion hole gel electrophoresis. The Leu-155 variant was expressed at hydrogen bond with the substrate carbonyl oxygen atom- considerably lower levels (25-50 ,g/ml). The lower mutant- oxyanion hole hydrogen bond probably contributes little to enzyme level is likely related to the mechanism ofprocessing the overall free energy ofbinding and thus to Km. Indeed, the the pro-form of subtilisin during secretion, which apparently present results provide evidence that the oxyanion hole does can involve an autocatalytic step. Neither of the two inde- not contribute to the free energy of binding in forming the pendently cloned Leu-155 variants exhibited any detectable initial enzyme-substrate complex. Alternatively, one of the activity in unpurified culture supernatants with hide powder referees pointed out that Leu-155 could adopt a conformation azure or tosyl-L-arginine methyl ester as substrates. To rule out the possibility that the loss in enzyme activity was due to 200 - undetected mutations that occurred during the mutagenesis 97.4 - procedure, one of the Leu-155 variants was reverted back to - Asn-155 by using oligonucleotide mutagenesis. As expected, 68.0 this revertant exhibited wild-type activity and secretion 43.0 - _ levels, demonstrating that the loss in activity resulted from the asparagine-to-leucine mutation. The following procedure was used to purify both parent 25.7 - and variant enzymes. Growth media from a 1.0-liter fermen- tation run was treated with saturating levels of ammonium sulfate to precipitate the subtilisin. The solubilized (10 mM phosphate buffer, pH 6.0) ammonium sulfate precipitate next 18.4 - 3 was treated batchwise with DEAE-cellulose, followed by fractionation of the filtrate with cold acetone. Subtilisin 14.3 - precipitates from filtrate containing between 50% and 65% acetone. The acetone precipitate was solubilized in 10 mM phosphate (pH 6.0) and chromatographed on a Whatman A B SE-53 column with a linear salt be- ion-exchange gradient FIG. 2. NaDodSO4/polyacrylamide gel electrophoresis patterns tween 0 and 0.4 M NaCl. After SE-53 chromatography, both for the purified Leu-155 variant (lane A) and wild-type (lane B) the wild-type and Leu-155 variant appeared as single major subtilisins. Molecular mass standards are at left with corresponding bands upon NaDodSO4/polyacrylamide gel electrophoresis kilodalton values. Approximately 25 ug of protein was applied to analysis (Fig. 2). each lane. Downloaded by guest on September 26, 2021 Biochemistry: Bryan et al. Proc. Natl. Acad. Sci. USA 83 (1986) 3745 Table 1. Kinetic parameters for the hydrolysis of ferent enzyme systems may reflect the overriding importance succinyl-Ala-Ala-Pro-Phe-p-nitroanilide of charge stabilization in enzyme-catalyzed reactions. In Enzyme Km, M s- kcat/Km, M-l s- conclusion, the results presented here confirm the important k,,t role ofthe oxyanion hole in serine proteases and supports the Wild type 2.07 x 10-4 44 2.2 X 105 role of electrostatic stabilization of the transition state in Asn-155 to Leu-155 enzyme-catalyzed reactions. Preparation 1 2.55 x 10-4 0.20 0.78 x 103 Preparation 2 1.88 x 10- 0.14 0.74 x 103 We thank Drs. N. Vasantha for providing the cloned subtilisin Assays were performed in 0.05 M Tris HCl/0.05 M KCl, pH 8.0 at gene, S. Fahnestock for the B. subtilis deletion strain, and J. Matthew for critically reviewing the manuscript. We also thank S. 250C. Young and S. Trattner for preparing the manuscript. We are grateful to one ofthe referees for drawing our attention to the potential steric enabling favorable hydrophobic contacts with the P1 residue, problem with threonine-220. thereby compensating for the loss of the substrate carbonyl oxygen-oxyanion hole hydrogen bond. That Km is not altered 1. Pauling, L. (1946) Chem. Eng. News 263, 294-297. also indicates that little if any significant disruption in 2. Pauling, L. (1984) Am. Sci. 36, 51-58. active-site geometry has occurred to the extent that substrate 3. Kraut, J. (1977) Annu. Rev. Biochem. 46, 331-358. binding is altered. 4. Robertus, J. D., Kraut, J., Alden, R. A. & Birktoft, J. J. The large decrease in kcat provides direct evidence that (1972) Biochemistry 11, 4293-4303. Asn-155 plays an important role in transition-state stabiliza- 5. Poulos, T. L., Alden, R. A., Freer, S. T., Birktoft, J. J. & tion. As predicted, our results indicate that a significant part Kraut, J. (1976) J. Biol. Chem. 251, 1097-1103. 6. Matthews, D. A., Alden, R. A., Birktoft, J. J., Freer, S. T. & of the energetic incentive in forming the tetrahedral interme- Kraut, J. (1975) J. Biol. Chem. 250, 7120-7126. diate derives from stabilization of the P1 oxyanion by 7. Robertus, J. D., Alden, R. A., Birktoft, J. J., Kraut, J., Pow- hydrogen bonding with Asn-155 and the peptide NH of ers, J. C. & Wilcox, P. E. (1972) Biochemistry 11, 2439-2449. Ser-221 (4). Nevertheless, Asn-155 appears not to be abso- 8. Segal, D. M., Powers, J. C., Cohen, G. H., Davies, D. R. & lutely essential because the mutant enzyme still retains some Wilcox, P. E. (1971) Biochemistry 10, 3728-3737. peptidase activity. This is not too surprising for two reasons. 9. James, M. N. G., Brayer, G. D., Delbaere, L. T. J. & First, the catalytic machinery involving Ser-221, His-64, and Sielecki, A. R. (1980) J. Mol. Biol. 139, 423-438. Asp-32 have not been altered nor have the specific binding 10. Nakagawa, S. & Umeyama, H. (1984) J. Mol. Biol. 179, forces that are responsible in forming the initial enzyme- 103-123. 11. Henderson, R. (1970) J. Mol. Biol. 54, 341-354. substrate complex been affected. Second, the P1 oxyanion 12. Henderson, R., Wright, C. S., Hess, G. & Blow, D. M. (1971) could still form a single hydrogen bond with the Ser-221 Cold Spring Harbor Symp. Quant. Biol. 36, 63-69. peptide NH group, which may provide sufficient stabilization 13. Villafranca, J. E., Howell, E. E., Voet, D. H., Strobel, M. S., ofthe oxyanion to generate the observed low level ofactivity. Ogden, R. C., Abelson, J. N. & Kraut, J. (1983) Science 222, Finally, we have used the method of Wilkinson et al. (23) 782-788. to estimate the change in transition-state stabilization in 14. Fersht, A. R., Shi, J.-P., Knill-Jones, J., Lowe, D. M., wild-type versus the site-directed variant. Using this method Wilkinson, A. J., Blow, D. M., Brick, P., Carter, P., Waye, and the data in Table 1, we estimate that the Asn- M. M. Y. & Winter, G. (1985) Nature (London) 314, 235-238. 155-oxyanion hydrogen bond contributes about -3.7 15. Estell, D. A., Graycar, T. P. & Wells, J. A. (1985) J. Biol. Chem. 260, 6518-6521. kcal/mol to the stabilization of the transition state. Using 16. Gardell, S. J., Craik, C. S., Hilvert, D., Urdea, M. S. & site-directed variants of tyrosyl-tRNA synthetase, Fersht et Rutter, W. J. (1985) Nature (London) 317, 551-555. al. (14) estimate a similar value (=4 kcal/mol) for hydrogen 17. Wells, J. A., Ferrari, E., Henner, D. J., Estell, D. A. & Chen, bonds between an active-site amino acid side chain and a E. Y. (1983) Nucleic Acids Res. 11, 7911-7925. charged substrate atom. It is particularly interesting that 18. Vasantha, N., Thompson, L., Rhodes, C., Banner, C., Nagle, Fersht et al. (14) find little contribution from protein-sub- J. & Filpula, D. (1984) J. Bacteriol. 159, 811-819. strate hydrogen bonds involving uncharged pairs but only in 19. Zoller, M. J. & Smith, M. (1983) Methods Enzymol. 100, uncharged-charged pairs. This correlates well with our 468-500. observation that Km i-s not altered significantly in the Leu-155 20. Jalanko, A., Palva, I. & Soderkund, A. (1981) Gene 14, variant where the enzyme-substrate complex does not in- 325-328. 21. Delmar, E. G., Largman, C., Brodrick, J. W. & Geokas, volve the interaction between charged atoms. However, once M. C. (1979) Anal. Biochem. 99, 316-320. a negative charge develops on the P1 carbonyl oxygen atom, 22. McPhalen, C. A., Svendsen, I., Jonassen, I. & James, hydrogen-bonding stabilization becomes a significant factor. M. N. G. (1985) Proc. Natl. Acad. Sci. USA 82, 7242-7246. Such similar stabilization energies for the analogous 23. Wilkinson, A. J., Fersht, A. R., Blow, D. M. & Winter, G. charged-uncharged hydrogen-bonded pairs in two very dif- (1983) Biochemistry 22, 3381-3386. Downloaded by guest on September 26, 2021