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 <C-NH hydrogen bond to the tetrahedral intermediate. In modeling the Leu-155 variant on a computer graphics H--- -Ser system, it became evident that Leu-155 may form unfavor- The transition state then collapses to the tetrahedral inter- able contacts with Thr-220. However, close contacts with the mediate, Thr-220 side chain could be relieved by rotation about CO-CY, without significant readjustments in Xi, from the preferred 0- value of -60°. -C-NH METHODS O-Ser Subtilisin from Bacillus amyloliquefaciens has been cloned previously and expressed from plasmid vectors in Bacillus The publication costs of this article were defrayed in part by page charge subtilis (17, 18). In order to introduce oligonucleotide- payment. This article must therefore be hereby marked "advertisement" directed changes, the subtilisin gene together with flanking in accordance with 18 U.S.C. §1734 solely to indicate this fact. DNA sequences was subcloned into M13 mp9 in order to 3743 Downloaded by guest on September 26, 2021 3744 Biochemistry: Bryan et al. Proc. Natl. Acad. Sci. USA 83 (1986) 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.