J. Biochem. 110, 939-944 (1991) Rat Cathepsin H-Catalyzed Transacylation: Comparisons of the Mechanism and the Specificity with Papain-Superfamily Proteases1 Hironobu Koga,*>2 Nobuko Mori,* Hidenori Yamada," * Yukio Nishimura,* Kazuo Tokuda,* Keitaro Kato,' and Taiji Imoto° 'Faculty of Pharmaceutical Sciences, Kyushu University 62, Maidashi, Higashi-ku, Fukuoka, Fukuoka 812; and "Department of Bioengineering Science, Faculty of Engineering, Okayama University, 3-1-1 Tushimanaha, Okayama, Okayama 700 Received for publication, August 20, 1991 We found that rat cathepsin H showed strong transacylation activity under physiological Downloaded from https://academic.oup.com/jb/article/110/6/939/755804 by guest on 28 September 2021 conditions. It is a feature of cathepsin H to utilize amino acid amides not only as acyl- acceptors but also as acyl-donors in the reaction. The pH-dependence of the transacylation activity was distinct from those of other papain-superfamily proteases. The alkaline limb (P-KOPP=7.5) could be regarded as the p-K, of the a-amino group of the acyl-donor, which was also involved in the original amino-peptidase activity. The acidic limb (pKcpp=5.8) was suggested to be involved in the deacylation step, where amino acid amide attacked the acyl-intermediate as a nucleophile in place of water in the hydrolysis. Although the Na-deprotonated acyl-acceptor, which is supposed to govern the nucleophilic attack, has a small population in the acidic pH range (above pH 5), the transacylation was detectable even at the acidic pH-range because of the high SI -site binding ability and suitable nucleophilicity of the acyl-acceptor. In the transacylation between various amino acid amides, the SI and SI' site appeared to prefer hydrophobic residues without and regardless of a branch at /5-carbon, respectively. From these results and the sequence homology in the papain superfamily, we concluded that the reaction was governed by the acyl-donor having a protonated amino group, the acyl-acceptor having a deprotonated amino group and the remarkable hydrophobic character (especially favoring tryptophan amide) of the SI' site, presumably reflecting the good conservation of Trpl77 in papain-superfamily proteases. Cathepsin H is a lysosomal cysteine protease (1) and has cathepsin H, we examine the generality of the transacyla- distinct aminopeptidase activity which recognizes the tion proceeding via the acyl-enzyme intermediate in the intact a--amino group of substrates (2). The enzyme papain-superfamily. belongs to the cysteine protease family named the papain It has been reported that papain (5) and cathepsin B (6, superfamily because of its amino acid sequence homology 8) favor hydrophobic residues such as Leu and Trp at their with papain (3, 4). Si' subsites in transacylation and hydrolysis. In the case of It is reported that papain (5) and cathepsin B (6, 7), papain, it is suggested that Trp 177 of the enzyme is another papain-superfamily protease, show transacylation involved in this specificity (9). Since the residues corre- which proceeds through an acyl-enzyme intermediate in the sponding with Trpl77 are well conserved in papain-super- same manner as hydrolysis. That is, in the transacylation, family proteases, it was expected that cathepsin H would a nucleophile such as an amino acid amide behaves as an show similar specificity of the Si' site. From this point of acyl-acceptor in place of water in the hydrolysis. We found view, we also studied the specificity of the SI' site of that cathepsin H also showed strong transacylation activity cathepsin H in the transacylation. In addition, since cathep- under physiological conditions. Therefore, employing rat sin H is originally an aminopeptidase, amino acid amide can also behave as acyl-donor. Thus, we also investigated the ' This work was supported in part by a Grant-in-Aid for Scientific specificity of the Si site in the reaction. Research from the Ministry of Education, Science and Culture of The mechanism of the cathepsin H-catalyzed transacyla- Japan. tion is discussed in terms of the function-structure rela- 'Present address: Drug Design department, Medicinal Research Laboratories I, Central Research Laboratories, Yamanouchi Pharma- tionship among papain-superfamily proteases. ceutical Co., Ltd., 21 Miyukigaoka, Tsukuba, Ibaraki 305. 3 To whom correspondence should be addressed. EXPERIMENTAL PROCEDURES Abbreviations: GlyNHt, glycine amide; AlaNHi, alanine amide; ValNHj, valine amide; DeNH , isoleucine amide; LeuNHj, leucine 2 Materials—Amino acid amides (GlyNH2, AlaNH2, Val- amide; TrpNHj, tryptophane amide; PheNH,, phenylalanine amide; NH , EeNHj, LeuNH , TrpNH , PheNH , TyrNH , Met- TyrNHj, tyrosine amide; MetNHj, methionine amide; ThrNHj, 2 2 2 2 2 threonine amide; ArgNH,, arginine amide; MCA, 4-methylcou- NH2, ThrNH2) and ArgNH2) were products of Sigma. maryl-7-amide; AMC, 7-amino-4-methylcoumarin; PAGE, poly- Aminomethylcoumarin substrate, Arg-MCA and AMC acrylamide gel electrophoresis; FAB, fast atom bombardment; were purchased from Peptide Research Foundation, Osaka. RP-HPLC, reverse-phase liquid chromatography. Sephadex G-75, DEAE-Sephacel, Mono S (FPLC), and Vol. 110, No. 6, 1991 939 940 H. Koga et al TrpNH2 Trp-TrpNH2 Trp-Trp-TrpNH2 XNH2 X-TrpNH2 X-Trp-XNH2 (amino acid amide) Trp-XNH2 X-Trp-TrpNH2 (dipeptide amide) Trp-X-XNH2 Trp-Trp-XNH2 (tripeptide amide) TrpCOOH XCOOH lamino acid) Downloaded from https://academic.oup.com/jb/article/110/6/939/755804 by guest on 28 September 2021 w Transcacylation Hydro Lysis Scheme 1 A. Preference of the S1 site from transacylation were analyzed with a reversed-phase column (6X150 mm) of YMC-Pack A-312 ODS (120 A, 5 x NH2CHCO f/m, Yamamura Chemical Lab.) with a Hitachi HPLC I system. The column was eluted with a gradient of 20 ml of S 10% acetonitrile and 20 ml of 40% acetonitrile, both X R containing 0.1% concentrated HC1 at a flow rate of 1.2 ml/ NHrCH-CONH-CH-CONH, min. The elution of products was monitored by measuring NH2CHCONH, the absorbance at 280 nm due to Trp. The yields were n calculated from the peak height relative to that of Trp, NH,CHCO whose amount was measured with an amino acid analyzer. R R Determination of amino acids with a Hitachi 835 amino NHrCH-CONH-CH-CONH2 acid analyzer was performed as follows, i) Amino acid composition; samples were previously hydrolyzed in 6 N HC1 under vacuum at 110'C for 20 h, lyophilized and SI sr dissolved in 0.02 N HC1 or hydrolyzed in 4 M methanesul- fonic acid containing 0.2% 3-(2-aminoethyl)indole (Pierce) B. Preference of the S1' site under vacuum at 118'C for 22 h, and neutralized with 3.5 N R NaOH. ii) Free amino acids; samples were diluted with NH,CHCO 0.02 N HC1. After these treatments, samples were subject- 1 NH,CHC0NH2 ed to amino acid analysis. a. » X _1 NHj-CHCONH-CHCONH, N-tenninal amino acid analyses of amino acid amide oligomers were performed with an Applied Biosystems Model 470A gas-phase protein sequencer. S1 sr R FAB mass spectra of products derived from cathepsin H- NH^CHCONH, ; THTPTOPHAHE ABIDE catalyzed transacylation were measured with a JEOL JMS Scheme 2 DX-300. Transacylation Reaction—TrpNH2 or a mixture of TrpNH2 and XNHj in appropriate buffer, pH 3-9, contain- molecular weight standard markers for SDS-PAGE were ing 3.8 mM 2-mercaptoethanol and 1 mM EDTA was obtained from Pharmacia Fine Chemicals. Acrylamide, incubated at 40"C with rat liver cathepsin H following 30 min i\r,i\T'-methylene bisacrylamide, SDS, and Triton X-100 of pre-incubation. The conditions (concentration of amides were purchased from Katayama Chemical. All other chemi- or amline/milliunits of enzyme/buffer/total volume/reac- cals used were reagent grade materials from various tion time/reference) were as follows: 10 mM TrpNH2/1.9/ commercial sources. 0.025 M borate buffer pH 8.6/1 ml/60 min/Fig. 1A; 1, 2, Enzyme Purification and Assay—Cathepsin H from rat 5, and 10 mM TrpNH2/8/0.1 M phosphate buffer pH 6.8/1 liver lysosomal fraction was isolated to homogeneity essen- ml/2 h/Fig. 2; 10 mM TrpNH2/0.76/7 = 0.1 acetate buffer tially by the method of Kirschke et al(10). Enzyme assays pH 3-4, phosphate buffer pH 5-7, borate buffer pH 8-9/1 were performed with Arg-MCA at pH 6.8 according to the ml/2 h/Fig. 3; 0.5 mM TrpNH2 and 10 mM aniline/0.76/ method described previously (11). Released AMC was 7=0.1 phosphate buffer pH 5-7/1 ml/2 h/Fig. 4; 10 mM determined by measuring fluorescence with a Hitachi TrpNH2 and 10 mM X-NH2/1.9/0.025 M borate buffer pH F-2000 fluorescence spectrophotometer (excitation at 380 8.4/1 ml/20 min/Table I. All reactions were terminated by nm, emission at 460 run). One milliunit of the enzyme adding 2 mM (final concentration) iodoacetic acid. activity was defined as that quantity releasing 1 nmol of Analysis of Specificities of the SI and SI' Sites in aminomethyl-coumarin per min at pH 6.8 and 40*C. Cathepsin H- Catalyzed Transacylation Reaction—As de- Analytical Methods—Trp-containing products derived scribed under "RESULTS," amino acid amides could behave J. Biochem. Rat Cathepsin H- Catalyzed Transacylation 941 as both acyl-donors and acceptors, that is, they could be mass spectroscopy (data not shown). Papain did not pro- incorporated in the resulting dipeptidyl amides at both N- duce any detectable transacylation product under the same and C-tenninal positions. Then we analyzed the speci- conditions (data not shown). Thus, the transacylation ficities of the Si and SI' sites in the reaction employing a between amino acid amides, which results in the production combination of TrpNH2 and XNHj as substrates.