ese tthe mena Sei or Bobo and Masuda By Ts Structural and Functional Roles of Asparagine 175 in the Cysteine Protease Papain* (Received for publication, January 5, 1995, and in revised form, May 10, 1995) ‘Thierry Vernet, Daniel C. Tessier, Jean Chatellier!, Céline Plouffe, Tak Sing Lee, David ¥. Thomas, Andrew C. Storer, and Robert Ménard§ From the Biotechnology Research Institute, National Research Council of Canada, Montréal, Québec H4P 2R2, Canada ‘The role of the asparagine residue in the Cys-His-Asn “eatalytie triad” of cysteine proteases has been investi- gated by replacing Asn" in papain by alanine and glu- tamine using site-directed mutagenesis. The mutants were expressed in yeast and kinetic parameters deter- mined against the substrate carbobenzoxy-1-phenylala- nyl(7-amino-4-methyleoumarinyl)-L-arginine. At the op- timal pH of 6.5, the specificity constant (kea/Ky)"™ was reduced by factors of 2.4 and 150 for the Asn! — Gin and Asn! — Ala mutants, respectively. Most of this effect was the result of a decrease in ky as neither mutation significantly affected Ky. Substrate hydrolysis by these mutants is still much faster than the non-cata- lytic rate, and therefore Asn'”* cannot be considered as fan essential catalytic residue in the eysteine protease papain, Detailed analyses of the pH activity profiles for both mutants allow the evaluation of the role of the Asn" side chain on the stability of the active site ion pair and on the intrinsic activity of the enzyme. Alter- ation of the side chain at position 175 was also found to increase aggregation and proteolytic susceptibility of the proenzyme and to affect the thermal stability of the mature enzyme, reflecting a contribution of the aspara- gine residue to the structural integrity of papain. The strict conservation of Asn'’* in cysteine proteases might therefore result from a combination of functional and structural constraints. Cysteine proteases are a class of enzymes requiring the thiol group of a eysteine residue for their eatalytie activity (1), The ‘additional involvement of an histidine residue in the catalytic process was inferred on kinetic grounds (2), and evidence for the location of an histidine in proximity to the eatalytic thiol ‘group was provided initially by the use of a bifunctional irre- versible inhibitor of papain (3). The Cys®-His!™ arrangement in the catalytic center of papain was established when the ‘three-dimensional structure of the enzyme was solved (4~6). "The papain molecule is folded to form two interacting domains dolimiting a cleft at the surface ofthe enzyme, Cys" and His!®? are located at the interface of this cleft on opposite domains of ‘the molecule; Cys** is part of the L1 a-helix atthe surface ofthe + This is Publication $8535 ofthe National Research Counel of Can sud. The costs of publication of this article were defrayed in part by the ‘ayment of page changes. This atile must therefore be hereby marked Fodoersement” im accordance with 18 U.S.C. Section 1734 solely to indicate this fact, ‘Present address: Institut de Biologie moleculaire et cellulaire, Laboratoire dimmunochimie, 16 rue R. Descartes, 67084 Strasbourg Cedex, France. '9To whom correspondence should be addressed: Biotechnology ‘Research Institute, National Research Council of Canada, 6100 Ave, Royalmount, Montréal, Quebec HAP 2R2, Canada. Te: 514-196-6317 Fax 514-496-5143, left domain, while His" is in @ B-sheet at the surface of the right domain of the enzyme. With the availability of the three-dimensional structure, other residues were found in the vicinity ofthe active site that could possibly play important roles in the mechanism of the enzyme. In particular, an asparagine residue that is conserved im all eysteine protease sequences of the papain femily, Asn”, was found to be adjacent to the catalytic His®* residue. The amide oxygen of the Asn! side chain is hydrogen-bonded to N@H of His! creating a Cys-His-Asn triad, which can be considered as being analogous with the Ser-His-Asp triad of serine proteases (Fig. 1). The side chain of Asn'7* is buried in 1 hydrophobie region of the enzyme composed mainly of res dues Phe, Vall™, Tep'”, and Trp!" Residues 141, 177, and 181 are located near the Asn!”*-His!™ hydrogen bond and can shield it from the external solvent. An important feature of the ‘Asn'"-His!™ interaction is that the hydrogen bond is approx ‘imately colinear with the His!" C?-C” bond, allowing rotation, of the imidazole ring about the O?-C” bond without disruption of the Asn!.tis!% hydrogen bond. Comparison of results from erystallographic studies with various forms of papain cither free or alkylated at the Cys®® sulfur atom by chloro- methyl ketone inhibitors have demonstrated that the His'®® side chain can change its orientation by about 30° (7), There- fore, it has been suggested that the role of Asn'”® is to orient the His" side chain in the optimum positions for various steps of tho catalytic mechanism. In the resting state of the enzyme, the His side chain would be coplanar tothe Cys? residue while during aeylation, the protonated imidazole ring would rotate to act as a proton donor to the nitrogen atom of the leaving group of the substrate 8) ‘An important feature of papain and other cysteine proteases in general is the high nucleophiliity ofthe sulfur atom of the active site eysteine residue. This is due to the fact that at the PH values where the enzyme is active, the sulfur atom is present as a thiolate anion. It is now generally accepted that the side chains of Cys** and His! possess unusual pk, values ‘and that the active form of the enzyme consists of a thiolate- imidazolium ion pair at neutral pH (9-12). However, the na- ‘tre and significance of the factors that are responsible for the formation and maintenance of the ion pair within a wide range of pH for the most part remain unknown, and this aspect has ‘been the object of many theoretical studies over the years (see, e., Refs. 13-19), Since the side chains of Asn!” and His!™ interact directly via hydrogen bonding, one of the obvious roles of Asn!” could be to stabilize the thiolate-imidazolium form of papain. It has been suggested that the proximity of the active site eysteine and histidine residues eould be one of the most ‘important factors contributing to the formation of an ion pair ‘and that the proton affinities of Cys# and His! at the active site of papain are strongly sensitive to the geometry of these residues (17, 19). Consequently, Asn!" could stabilize the ion 16645,ys25 Asnt75 Fic. 1. Schematic representation of the active site of papain showing the catalytic triad residues Cys", fand Asn'™. ‘The representation i derived from the erytalstructsre of Drenth et al (D-In the erystal structure, the active sive cysteine te oxidized, and ‘therefore the precise relative orientations of the Cys and His! side chains in the nonoxidized enayine might differ from the Mlustrated orientations, 9 in a favorable pair by keeping the imidazole ring of His! orientation. ‘There has been no quantitative experimental study address- {ng the role of the asparagine residue in the catalytic triad of cysteine proteases. In a preliminary study using random mu- ‘agenesis and screening of mutants, we have shown that re- placement of Asn'”® by several amino acids results in a signif icant loss of activity (20). However, due to the relatively low sensitivity of the assay, this system can unambiguously detect only mutants with activity similar to wild-type papain. In addition, enzyme inactivation could oceur for mutant enzymes that have a decreased stability under the relatively drastic conditions used to activate the enzyme precursors (low pH and hhigh temperature). The screening system we used cannot readily distinguish between a decrease in catalytic activity and a decrease in protein stability. In this study, the role of Asn!” at the active site of cysteine proteases was investigated by a detailed kinetic and functional characterization of papain mu- tants, Mutation of Asn" to a glutamine was chosen due to our previous observation that this mutation generates an enzyme that retains some activity (20), indicating that the conservative substitution of Asn!” by Gn is tolerated in the active site of papain. Complete removal of the hydrogen bonding capability of the side chain of residue 175 was accomplished by an Asn?” > Ala change. EXPERIMENTAL PROCEDURES Bspression and Purification of Papain Mutants—Expression of wild- type papain and of the Asn!” -> Gin and Asn”? — Ala mutant proen zymes in Saccharomyoee cerevisiae has been reported recent (20). ‘Yeast cella from I iter ofeulture (8 % 10” eellanl) were collected by contrfugation and resuspended in 20 ml of 10 ma Tris-HCl, pH 7.5, 1 mma EDTA to yield a Gnal volume of sbout 85 ml. The cella were lysed "using a French press (20,000 pas.) and the cellular debris removed by ' 10-min, 15,000 * g centeifigaton. Propapain present in the super natant was sonverted ta mattre papain by lied proteolysia with ‘ubtiisin BPN’ (Sigma). The soluble extract was incubated for 2-3 h at 37°C im the prosence of 0.1 mgm subtilisin. The extract waa then changed to pli 6.0 with sodium acetate buffer (30 ma, pl 4.0) and incubated at 55°C for 16 min, ARer a 10-min centrifugation at 15,000 Xf, precipitated proteins were discarded and the supernatant was made 80% ammonium sulfate and kept at 4°C overnight. This suspen son was centrifuged at 22,000 % g for 20 min and the protein pellet ecuspended in 4ml of 100 mit eodium acetate, 1 mM EDTA, pH 5.3. ‘This preparation was used te determine the protein half-life (see below), Role of the Papain Active Site Asn!” ‘The enzymes used for the kinetic characterization were further purified by covalent chromatography using & Uniopropyl-Sepharose column (21), Kinetics of Ireversble Thermal Incctivation—The kinetics of ire versible thermal inactivation of papain variants was determined described previously (22). Partially purified papain preparations (see above) were adjusted to pH 60 with 100 mu phosphate buffer and HigCl, added to5 mu They were incubated at 82°C for 0-60 min, and the residual papain activity was measured. The ly value (the time at ‘which the enzyme has let half ofits activity) was determined frou the lope of the Knearizd form of the data (22) ‘Aggregate/ Soluble Precursor Partitioning and Susceptibility to Pro tease Degradation—Total yeast extracts (3 ml) were prepared from 75 mi of culture grown under the conditions defined above. Processing of Dropapain waa prevented with 0.1 mw of E64 (1l(-trans-eponssucie nyl}-leueyllamine}-4-guanidinojbutane)(23).The extract was deglco- Slated by incubation for 2h at 37 °C in the presence of 36 mt sadam tcetatebuffor, pH 5.5, 200 mat f-morcaptoethanol, and 50 miliunits'ml ndoglycosdase H (Boehringer Mannheim). An aliquot of the mixtare ‘was eentrifaged at 15,000 x g for Smin. The supernatant was recovered and the pellet was resuspended in 200 yl of phosphate buffer, pil 6. ‘An aliquot of the pellet and supernatant degiveoylated fractions was ‘analyzed by Western blot prior to or fllowing incubation with 0.1 ‘mg/l subtiisin for 2 at $7 °C. Quantitative Western blot analyses ‘were performed using two rounds of antigen detection after separation of the proteins in SDS-polyacrylamide gel electrophoresis, Mature and proenzyme forms of papain were detected with an ant-papain rabbit polyelonal antibody 24). Papain-antibody amplexes were labeled with P*8abeled protein A Amersham Corp.) and visualized by autoradiog raphy, The antigen was then stained ina second reaction using alkaline ‘Phasphatace-conjugatod goat anti-rabbit IgGs (Bio-Rad). This proce- ‘ure facilitates accurate eutting ofthe immunoreactive bands for ra: dicactivity measurements using en LKB 1282 Compugamma counter. Kinetic MeasurementsThe kinetic parameters were obtained as described previously (22). The concentration of active purified enzyme was determined by titration with B-64 (25) Carbobenzoxy-1-phenylala _ayl(7-amino-tmethyleoumariny))-Larginine(Che-Phe-Arg MCA) was ‘sed as a substrate. The reaction conditions consisted of 50 mu phos phate buffer, 0.2 t NaCI § mat EDTA, 10% CH,CN, pH 6.5. Far the Aetermination of pH activity profiles, 60 mt citrate oF 50 mM borate were alto used as buffers and the substrate concentration was Kept wel, below the X,, value. Kinetic parameters at optimum pl (6.5) were determined by linear regresion ofthe initial rate) data to plot of fo versus 8 Hanes plots). The pl! activity profes were analyzed according to the model of Reaction 1 by nonlinear regression of the data tothe corresponding equation (Equation 1. ~ = EH 4 thal Kul Reaction 1 ha Kl me ke RT In this equation, (b/s represents the experimentally determined value of the apeifcity constant and (ky/Kyo!™ ithe limiting value ‘determined from nonlinear regression, ‘Computer Modeling -Compater modeling was used with the Asn" Gin mutant to predict the orientation of the Gin” side chain. The imdel representing free papsin wae obtained using the coordinates from the crystal structure of Drenthe a (7. Inthe model, the oxygen ‘toms on the oxidized Cys? residue were removed, and AMER partial charges were assigned considering that the active site residues are ‘present in the thiolateimidazoium ion pair state, In an inital step, the Systematic Search module of Sybyl 6.0 (Tripos Associates, Inc.) was tased to carry out @ search for tarcally allowed conformations of the ‘Aen! > Gln mutant, The Asa’ residue was replaced by Gin and the fide chain angler %,, Xz, and x of Gln!™ were varied by 2-degree increments. Two “groupe? of structures (structure 1 and structure 2) were found, both containing an hydrogen bond between the oxigen ‘tom ofthe Gln! eide chain amide and N'°H of His™, The structures ha Bal = ea.) "The abbreviations used are: Cbe-Phe-AntMCA, cacbobenzoxy- phenvlalanyl-(T-amino-4-methyleoumariayi-targinine, WT, wild-type