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Inactivation of Aspartyl Proteinases by Butane-2,3-Dione Modffication Oftryptophan and Tyrosine Residues and Evidence Against Reaction of Arginine Residues

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Inactivation of Aspartyl Proteinases by Butane-2,3-Dione Modffication Oftryptophan and Tyrosine Residues and Evidence Against Reaction of Arginine Residues Biochem. J. (1981) 193, 55-65 55 Printed in Great Britain Inactivation of aspartyl proteinases by butane-2,3-dione Modffication oftryptophan and tyrosine residues and evidence against reaction of arginine residues Jean-Claude GRIPON* and Theo HOFMANNt Laboratoire de Biochimie et Technologie Laitieres, LN.R.A., 78350 Jouy-en-Josas, France, and tDepartment ofBiochemistry, University ofToronto, Toronto, Ont., Canada MSS IA8 (Received 22 April 1980/Accepted 7 September 1980) Butane-2,3-dione inactivates the aspartyl proteinases from Penicillium roqueforti and Penicillium caseicolum, as well as pig pepsin, penicillopepsin and Rhizopus pepsin, at pH 6.0 in the presence of light but not in the dark. The inactivation is due to a photosensitized modification of tryptophan and tyrosine residues. In the dark none of the amino acid residues, not even arginine residues, is modified even after several days. In the light one arginine residue in pig pepsin is lost at a rate that is comparable with the rate of inactivation; however, the loss of the single arginine residue in the aspartyl pro- teinase of P. roqueforti and the second arginine residue of pig pepsin is slower than the loss of activity; penicillopepsin is devoid of arginine. Loss of most of the activity is accompanied by the following amino acid losses: P. roqueforti aspartyl proteinase, about two tryptophan and six tyrosine residues; penicillopepsin, about two tryptophan and three tyrosine residues; pig pepsin, about four tryptophan and most of the tyrosine residues. Modification of histidine residues was too slow to contribute to inactivation. None of the other residues, including half-cystine and methionine residues (when present), was modified even after prolonged incubation. The inactivation ofP. roqueforti aspartyl proteinase and pig pepsin appears due to non-specific modification of several residues. With penicillopepsin, however, the reaction is more limited and initially affects only those tryptophan and tyrosine residues that lie in the active-site groove. In the presence ofpepstatin the rate of inactivation is considerably diminished. After prolonged reaction a general structural breakdown occurs. Some years ago Kitson & Knowles (1971) identified the modified arginine as that arginine reported that phenylglyoxal at pH 5.5 decreased the residue that is located 12 residues from the C- activity of pig pepsin to about 45%, as measured terminus (subsequently identified as arginine-3 16; with haemoglobin as substrate. This modification Tang et al., 1973). Kitson & Knowles (1971) had had a marked effect on the pH-dependence of the suggested that an arginine residue might function by enzyme. The inactivation was accompanied by interacting with an active-site aspartic acid residue, incorporation of one residue of phenylglyoxal per thereby accounting for the low pK of one of the molecule of pepsin. The site of modification was not catalytic groups. If that were the case, then other identified, but the authors suggested that one of the aspartyl proteinases would be expected to contain an two arginine residues of pepsin had been modified, equivalent basic residue, since the mechanisms of the because phenylglyoxal is an arginine-specific re- well-characterized aspartyl proteinases are very agent. At about the same time Huang & Tang (1972) similar (Hofmann, 1974; Fruton, 1976). It was there- studied the action of butane-2,3-dione, a reportedly fore interesting to find that in the amino acid sequence arginine-specific reagent, on human and pig pepsin, of penicillopepsin (Cunningham et al., 1976) one of and reported that the loss of one arginine residue the five lysine residues (penicillopepsin is devoid of was accompanied by a loss of activity. They arginine) occupied a position that corresponded to one of the two arginine residues of pig pepsin, but Abbreviations used: Ac, acetyl; peptide T-II, Ac- this was arginine-308 and not the apparently Ala-Ala-Lys(Nph)-Ala-Ala-NH2; Nph, p-nitrophenyl; important arginine-3 16 identified by Huang & Tang Ac-Trp-OEt, a-N-acetyltryptophan ethyl ester; Ac-Tyr- (1972). Position 308 is in a region of great sequence NH2, N-acetyltryosine amide. similarity, whereas position 316 is just outside this Vol. 193 0306-3275/81/010055-1 1$01.50/1 (© 1981 The Biochemical Society 56 J.-C. Gripon and T. Hofmann region. Modification of penicillopepsin with diethyl Penicillopepsin was prepared as described by pyrocarbonate led to inactivation and suggested that Hofmann (1976). Penicillium roqueforti aspartyl a lysine residue has an important function in the proteinase was prepared by the method of Zevaco et activity (Mains, 1973; Fenje, 1979). al. (1973); Rhizopus pepsin was from Miles This suggestion received strong support from the Laboratories, Elkhart, IN, U.S.A. X-ray analysis of penicillopepsin (Hsu et al., 1977), Penicillium caseicolum aspartyl proteinase was which showed the unusual feature of a partly buried purified as described by Lenoir et al. (1979). ion-pair between lysine-308 and aspartic acid-i1 Butane-2,3-dione monomer was obtained from BDH that is located at the end of the deep substrate- Chemicals, Poole, Dorset, U.K. Ac-Trp-OEt and binding cleft near the catalytic site. An important Ac-Tyr-NH2 were from Cyclo Chemical Corp., Los role for this ion-pair has been suggested (James et Angeles, CA, U.S.A. al., 1977). Position 316 on the other hand is on the outside of the molecule and a considerable distance Methods from the active site. Since the tertiary structure of Amino acid analyses. Protein samples were pig pepsin is very similar to that of penicillopepsin hydrolysed with glass-distilled 5.7M-HCI in vacuo (Andreeva et al., 1978), it is probable that arginine- for 20-24h. Additional samples were hydrolysed 308 rather than arginine-316 would form an ion-pair with 2-mercaptoethane-1-sulphonic acid for trypto- -with aspartic acid-il, a residue that is conserved phan as described by Penke et al. (1974). Amino in all nine aspartyl proteinases investigated so far acids were determined with a Beckman Spinco (Hofmann, 1974; Gripon et al., 1977; Faerch & model 121C automated amino acid analyser. In Foltmann, 1979). Position 308 is also highly some cases tryptophan was determined from the u.v. conserved and is occupied by arginine or lysine in all difference spectra, as described in the Results the proteinases studied (Hofmann, 1974; Faerch & section. Foltmann, 1979). All this evidence suggests that in U.v. spectra. Difference spectra were recorded pepsin arginine-308 and not arginine-316 might have with a Cary 118 spectrophotometer. Quartz cuvettes a role in the enzymic mechanism. of 2 x 0.437 cm (tandem cells) were used. The aspartyl proteinase of Penicillium roqueforti C.d. spectra. C.d. spectra were recorded on a is mechanistically related to pepsin and penicillo- Jasco J-41A spectropolarimeter at protein concen- pepsin (Gripon, 1976) and shows sequence homo- trations of about 1.2mg/ml (240-320nm) or logy with several aspartyl proteinases (Gripon et al., 0.15 mg/ml (200-250nm). 1977). It has only one arginine residue (Zevaco et Protein concentrations. Protein concentrations al., 1973), and it was therefore decided to test were determined by measuring the absorbance at whether this arginine residue could be modified in a 280nm. The specific absorption coefficients (for manner analogous to that of pig pepsin. When solutions of 1 mg/ml) were: pig pepsin, 1.4; penicillo- treatment with butane-2,3-dione inactivated the pepsin, 1.35; P. roqueforti aspartyl proteinase, 1.31. enzyme without concomitant loss of arginine, the possibility that the inactivation was due to the Enzyme activities reaction of residues other than arginine became very Aspartyl proteinases of P. roqueforti and P. strong. This is confirmed in the present study, which caseicolum and Rhizopus pepsin. The activities of shows that butane-2,3-dione inactivates several these enzymes were determined with haemoglobin as aspartyl proteinases, but that this inactivation substrate, as follows. Haemoglobin (4%, w/v; 2ml) proceeds only in the presence of light and not in the in 0.1 M-sodium acetate buffer, pH 3.5, was in- dark, although the arginine-specific reaction pro- cubated for 20min at 350C with 2ml of enzyme in ceeds readily in the dark (see Riordan, 1979). A the same buffer. The reaction was stopped by the study of the reaction of butane-2,3-dione with addition of 4 ml of trichloroacetic acid (5%, w/v). chymosin also led to the conclusion that inacti- After filtration, the absorbance at 280nm was vation is due to modification of residues other than determined. arginine when the reaction is performed in the Pig pepsin. The activity of pepsin was determined presence of light (A. A. Olsen & V. B. Pedersen, in the same way, except that haemoglobin and the personal communication). However, three of the five enzyme were dissolved in 0.1 M-glycine/HCl buffer, arginine residues of chymosin react in the dark with pH 2.0. about 20-30% reversible inactivation. Penicillopepsin. The activities of this enzyme were determined in three ways: (a) by the trypsinogen- Materials and methods activating assay as described by Hofmann (1976), (b) with bovine serum albumin as substrate (Hof- Materials mann, 1976) and (c) with peptide T-II (Hofmann et Pig pepsin (3x crystallized) was from Calbio- al., 1979). The assay with peptide T-II was chem, San Diego, CA, U.S.A. (lot no. 300001). performed in 20mM-sodium acetate buffer, pH 5.25, 1981 Reaction of aspartyl proteinases with butane-2,3-dione 57 at 200C at a substrate concentration of0.25 mm in a vation occurred (Fig. 1, controls). Treatment of pig recording spectrophotometer at 296nm. Initial rates pepsin with butane-2,3-dione gave the same result, were determined from the slopes. namely inactivation occurred only when the reaction Reaction with butane-2,3-dione.
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