ponents of the latex of Carica papaya (Baines & Brockle- hurst, 1982; Salih & Brocklehurst, 1983; Buttle & Barrett,. 1984; Brocklehurst et al., 1985a; Calam et al., ...
1226
BIOCHEMICAL SOCIETY TRANSACTIONS Table 1. Second-order rate constants for the reactions of the catalytic site thiol group of papain* with a number of substrate-derived two-protonic state reactivity probes in aqueous media at 25°C at I = 0.1 Probe
(Ut
CH, -C-
II
PH
NH -CH - C -NH(CH,), -S- S - 2 - Py
I II CH, 0 I
0
Ph
(11)
CH, -C
II
- NH -(CH,),
- S -S-
2 -Py
0
(111)
CH, - NH -C
II
- (CH,),
-S-S-
2 -Py
0
(IV)
CH, - C - 0 -
(CH,), - S-S - 2- Py
II
0
fV)S
CH, -S-S-2-Py
k(M-’S ’ )
4.0
2 400 000
6.5
4 600 000
9.0
2 700 000
3.5
I8 000
6.5
30 000
9.2
18 300
4.0
9 000
6.5
5 000
10.0
6000
4.0
15700
6.5
6 000
9.5
10000
4.0
95 000
6.5
22 000
10.0
32 000
*Whereas k is a maximum at pH6.5 for the reactions of probes I and I1 with papain, it is a minimum at this pH for the corresponding reactions with actinidin. tThe results for this probe. confirm those reported by Patel & Brocklehurst (1982). $A similar shape of pH-k profile is obtained for the reaction of papain with CH, -CH, CH, - S - S -2 - Py, although the values of k are lower.
We thank the Science and Engineering Research Council for project grants and for an Earmarked Studentship in support of this work. Brocklehurst, K. (1982) Merhocis Enzymol. 87C, 427-469 Kamphuis, I. G., Drenth, J. & Baker, E. N. (1985) J. Mot. Bid. 182,
3 17-329 Lowe, G . (1976) Tetrahedron 32,291-302 Lowe, G. & Yuthavong, Y. (1971) Biochem. J . 124, 107-1 15 Patel, G. & Brocklehurst, K. (1982) Biochem. Soc. Trans. 10. 216 217 Received 11 June 1986
Identification and characterization of chymopapains A and B from dried and fresh latex of Carica papaya BALDEV S. BAINES,S* KEITH BROCKLEHURST,* RAYMOND McKEE,? MAIREAD O’DRISCOLL,* ERDJAN SALIH*$ and HARRY SMITH? *Department of Biochemistry, St. Bartholomew’s Hospital Medical College, University of London, Charterhouse Square, London E C I M 6BQ, U.K., and ?Department of Botany, University of Leicester, University Road, Leicester LFI 7 R H , U.K. Recent interest in the various cysteine proteinase components of the latex of Carica papaya (Baines & Brocklehurst, 1982; Salih & Brocklehurst, 1983; Buttle & Barrett, 1984; Brocklehurst et al., 1985a; Calam et al., 1985; Baines et al., 1986) relates particularly to (a) the opportunities provided by this group of enzymes for the study of effects of structural variation within a group of homologous enzymes on active centre chemistry and catalytic mechanism [see also $Present address: Biotechnology Department, Glaxo Group Research Ltd., Greenford, Middx.. UB6 OHE, U.K. 6Present address: Department of Chemistry, Bradeis University, Edison-Lecks, 21 I , Waltham, MA 02254, U.S.A.
Willenbrock & Brocklehurst ( 1 984, 1985a,b) for a discussion of cathepsins B and H in this context] and (b) the value of these enzymes in various industrial and medical applications (see Brocklehurst et al., 1981; Calam el al., 1985). Aqueous extracts of dried papaya latex contain, as a minor component, the well-characterized enzyme papain (EC 3.4.22.2) (the least basic cysteine proteinase component), papaya proteinase R (originally called papaya peptidase A; see Brocklehurst et al., 1985, the most basic cysteine proteinase component) and a complex mixture of cysteine proteinases of intermediate basicity that are usually referred to as the chymopapains (see Baines rt ul., 1986). There are two major types of chymopapain active centre as judged by the reactivity characteristics of their thiol groups. These are readily distinguished by the markedly different shapes of plots of second-order rate constant (k) versus pH for the reactions with 2,2’-dipyridyl disulphide (see Brocklehurst et al., 1985a; Baines et al., 1986). Thus the pH-k profile for one type of chymopapain active centre (type B) contains a striking bell-shaped component, with optimal reactivity (k approx. 1 x 104-2 x 1 0 4 ~ - ’ s - at ’ ) pH 3-4, whereas that for the other (type A) lacks this bell-shaped I986
1227
619th MEETING, CAMBRIDGE component and instead contains a plateau at pH values around 4, with k approx. 2 x I O 3 ~ - ’ s - ’ decreasing , towards zero at lower pH. Bell-shaped components of high reactivity have been found not only in pH-k profiles for the reactions of B-type chymopapains with 2,2’-dipyridyl disulphide but also in the corresponding profiles for the reactions of many other cysteine proteinases including those of papain and papaya proteinase SZ (see Baines et al., 1986). In this communication we report some of the main findings of a study of four different types of papaya latex involving fractionation by ion-exchange chromatography on SP-Sephadex-C5O and characterization by two-protonicstate reactivity probe kinetics (Brocklehurst, 1982) and by chromatography by thiol-disulphide interchange (Brocklehurst et al., 19856). The four types of latex are: ( I ) a refined, spray-dried latex from India supplied by Powell and Scholefield, Ltd., Liverpool, (2) Sigma Type I latex which is crude, minimally processed, latex from Africa (3), fresh non-fruit papaya latex collected from the stem, leaves and petioles of the growing plant in Leicester, and (4) fresh fruit latex obtained from large papaya fruit picked in Edinburgh and sent to London for latex isolation and study. In all cases, one portion of the latex was treated with a large excess of 2,2’-dipyridyl disulphide to block all thiol groups and thus inhibit (reversibly) all cysteine proteinase activity and another portion was treated with a large excess of cysteine to provide opportunity for cysteine proteinase action on latex constituents. A striking result of this study is the existence of highly resolved chymopapain components that contain both thiol groups with A-type reactivity and thiol groups with B-type reactivity. It has not proved possible to separate these two types of thiol group by covalent chromatography by thiol-disulphide interchange in preparations where one type (A or B type) has been selectively blocked by reaction with 2,2’-dipyridyl disulphide. This suggests that these two types of thiol group reside either in the same protein molecule or in a tightly associated enzyme cluster. Another striking result is that extensive treatment with cysteine both of the latex and of at least one individual component of an ion-exchange profile results in the production of new proteins with different basicities and of changes
in the character of some residual proteins of a given basicity. All chymopapains of the Indian latex are of the (A + B)type before cysteine treatment and at least one A-type is present after cysteine treatment. The most basic chymopapain [(A + B)-type] is not present in Sigma latex until the cysteine treatment has been carried out (see also Calam et al., 1985). The chymopapains of the fresh non-fruit latex are all A-type (Brocklehurst et al., 1985a) but on cysteine treatment one (A + B)-type is generated. Fresh fruit latex is noteworthy for the presence of one purely B-type chymopapain, the others being (A + B)-type. Presumably, at least some of the changes in characteristics observed after treatment with cysteine are due to limited proteolysis. Preliminary characterization of some of the chymopapain components by gel-filtration suggests that they may be considerably smaller than papain ( M , 23 350). Such proteolytic fragments with perturbed catalytic sites that retain catalytic activity should be of value in the study of enzyme structure and function including the effects of remote parts of the protein structure on catalytic function. We thank the Science and Engineering Research Council for financial support, and the Royal Botanic Garden, Edinburgh for generous supplies of papaya fruit. Baines, B. S. & Brocklehurst, K. (1982) J. Protein Chem. 1, 1 1 9 ~139 Baines, B. S., Brocklehurst, K., Carey, P. R., Jarvis, M., Salih, E. & Storer, A. C. (1986) Biochem. J . 233, 119-129 Brocklehurst, K. (1982) Methods Enzymol. 87C, 427 -469 Brocklehurst, K., Baines, B. S. & Kierstan, M. P. J. (1981) Top. Enzyme Ferment. Biotechnol. 5, 262-335 Brocklehurst, K., McKee, R., Salih, E. & Smith, H. (19850) J . Protein Chem. 4, 103-127 Brocklehurst, K., Carlsson, J. & Kierstan, M. P. J. (19850) Top. Enzyme Ferment. Biotechnol. 10, 146-188 Buttle, D. J. & Barrett, A. J. (1984) Biochem. J . 233, 81 88 Calam, D. H., Davidson, J. & Harris, R. (1985) J. Chromarogr 326, 103~III Sa!ih, E. & Brocklehurst, K. (1983) Biochem. J. 213, 713 718 Willenbrock, F. & Brocklehurst, K. (1984) Biochem. J. 222, 8 0 5 ~ 8 1 4 Willenbrock, F. & Brocklehurst, K. (19850) Biochem. J . 227, 51 I 519 Willenbrock, F. & Brockelhurst, K. (1985h) Biochem. J. 227, 521 528 Received 1 1 June 1986
Catalytic mechanism of chloramphenicol acetyltransferase investigated by site-directed mutagenesis IAlN A. MURRAY, ANN LEWENDON. COLIN KLEANTHOUS and WILLIAM V. SHAW Department of Biochemistry, University of Leicester, University Road, Leicester LEI 7 R H , U .K .
Bacterial resistance to chloramphenicol is commonly conferred by the presence of chloramphenicol acetyltransferase (CAT; EC 2.3. I .28) which catalyses inactivation of the antibiotic via acetyl transfer from acetyl-coenzyme A (Shaw, 1983). Recent studies have concentrated on the highly active enterobacterial type 111 variant which has been used to prepare crystals suitable for high-resolution X-ray diffraction analysis (Leslie et al., 1986). The results of kinetic studies favour a ternary complex reaction mechanism with random order addition of substrates (Kleanthous & Shaw, 1984). The affinity reagent 3-bromoacetyl chloramphenicol inactivates CAT by alkylation of His-195, a uniquely reacAbbreviation used: CAT, chloramphenicol acetyltransferase
Vol. 14
tive histidyl residue believed to function as a general base during catalysis. N-3-carboxymethylhistidine was identified as the only alkylated amino acid, suggesting that a single tautomeric form of the reactive imidazole ring exists with the lone electron pair residing solely at N-3. It has been postulated that the preferred tautomer of His-I95 may be stabilized, at least in part, by hydrogen bonding with an adjacent acidic amino acid. Such stabilization might be expected to function in catalysis by increasing the basicity of N-3, thereby promoting abstraction from the primary hydroxyl of chloramphenicol before nucleophilic attack at the acetyl-coenzyme A thioester bond (Kleanthous et al., 1985). To test the hypothesis that tautomeric stabilization occurs within the catalytic centre of CAT by the above mechanism, we decided to replace the two conserved aspartyl residues (Asp-40 and Asp-199) by asparagine. The gene encoding type 111 CAT (cat,,,)was inserted into bacteriophage M 13 mp18 and subjected to dual-primer oligonucleotide site-directed mutagenesis (Zoller & Smith, 1984). Mutants identified by hybridization against 32P-
