Le proprietà cinetiche di stato-pre-stazionario e di stato-stazionario relative all'idrolisi di p-nitrofenil esteri di N-$\alpha$-carbo-benzossi (-L-) aminoacidi catalizzata dalla bromelaina da fusto (da Ananas sativus; EC 3.4.22.4), dalla ficina (da Ficus glabrata; EC 3.4.22.3) e dalla papaina (da Carica papaya; E C 3.4.22.2) sono state determinate fra pH 2,5 e 6,0 (I= 0,1 M) a 21 ± 0.5 °C. Il diverso comportamento cinetico di queste proteinasi a cisteina, in particular modo nella dipendenza dal pH dello stato di deacilazione, è stato correlato alla natura del residuo aminoacidico presente in posizione 158. Infatti, differenze nell'intensità del campo elettrostatico indotte dal residuo 158 possono influenzare l'equilibrio acido-base della diade catalitica Cys25-His159, e quindi l'attività enzimatica.
Referenze Bibliografiche
[1] P. ADUCCI, P. ASCENZI, A. BALLO and E. ANTONINI (1984) - Catalytic properties of plant proteinases. A comparison between serine and thiol enzymes: spinach leaf proteinase, papain, ficin and bromelains, «Symposia Biologica Hungarica», 25, 453-466.
[2] P. ASCENZI, P. ADUCCI, G. AMICONI and A. BALLIO (1987) - Active-site titration of papain by specific chromogenic substrates. Determination of homogeneity and concentration of the active enzyme, «Gazzetta Chimica Italiana», 117, 469-473.
[3] P. ASCENZI, P. ADUCCI, A. TORRONI, G. AMICONI, A. BALLIO, E. MENEGATTI and M. GUARNERI (1987) - The pH dependence of pre-steady and steady-state kinetics for the papain-catalyzed hydrolysis of N-$\alpha$-carbobenzoxyglycine p-nitrophenyl ester, «Biochimica et Biophysica Acta», 912, 203-210.
[4] G. LOWE (1976) - The cysteine proteinases, «Tetrahedron», 32, 291-302.
[5] K. BROCKLEHURST, B.S. BAINES and M.P.J. KIERSTAN (1981) - Papain and other constituents of Carica papaya L., «Topics in Enzyme Fermentation and Biotechnology», 5, 262-335.
[6] L. POLGAR and P. HALASZ (1982) - Current problems in mechanistic studies of serine and cysteine proteinases, «The Biochemical Journal», 207, 1-10.
[7] F. WILLENBROCK and K. BROCKLEHURST (1985) - A general framework of cysteine-proteinase mechanism deduced from studies on enzymes with structurally different analogous catalytic-site residues Asp-158 and -161 (papain and actinidin), Gly-196 (cathepsin-B) and Asn-165 (cathepsin H). Kinetic studies up to pH 8 of the hydrolysis of N-$\alpha$-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide catalyzed by cathepsin B and of L-arginine 2-naphthylamide catalyzed by cathepsin H, «The Biochemical Journal», 227, 521-528.
[8] T. STUCHBURY, M. SHIPTON, R. NORRIS, J.P.G. MALTHOUSE, K. BROCKLEHURST, J.A.L. HERBERT and H. SUSCHITZKY (1975) - A reporter group delivery system with both absolute and selective specificity for thiol groups and an improved fluorescent probe containing the 7-nitrobenzo-2-oxa-1, 3-diazole moiety, «The Biochemical Journal», 151, 417-432.
[9] M. SHIPTON, T. STUCHBURY and K. BROCKLEHURST (1976) - -4-Chloro-7-nitrobenzo-2-oxa-1, 3-diazole as a reactivity probe for the investigation of the thiol proteinases. Evidence that ficin and bromelain may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain, «The Biochemical Journal», 159, 235-244.
[10] J.P.G. MALTHOUSE and K. BROCKLEHURST (1976) - Preparation of fully active ficin from Ficus glabrata by covalent chromatography and characterization of its active center by using 2, 2'-dipyridyl disulphide as a reactivity probe, «The Biochemical Journal», 159, 221-234.
[11] M. SHIPTON and K. BROCKLEHURST (1977) - Benzofuroxan as a thiol-specific reactivity probe. Kinetics of its reactions with papain, ficin, bromelain and low-molecular-weight thiols, «The Biochemical Journal», 167, 779-810.
[12] K. BROCKLEHURST, T. STUCHBURY and J.P.G. MALTHOUSE (1979) - Reactivities of neutral and cationic forms of 2, 2'-dipyridyl disulphide towards thiolate anions. Detection of differences between the active centres of actinidin, papain and ficin by a three-protonic-state reactivity probe, «The Biochemical Journal», 183, 233-238.
[13] B.S. BAINES and K. BROCKLEHURST (1979) - A necessary modification to the preparation of papain from any high-quality latex of Carioca papaya and evidence for the structural integrity of the enzyme produced by traditional methods, «The Biochemical Journal», 177, 541-548.
[14] K. BROCKLEHURST and J.P.G. MALTHOUSE (1980) - Evidence for a two-state transition in papain that may have no close analogue in ficin. Differences in the disposition of cationic sites and hydrophobic binding areas in the active centres of papain and ficin, «The Biochemical Journal», 191, 707-718.
[15] K. BROCKLEHURST (1982) - Two protonic-state electrophiles as probes of enzyme mechanism, «Methods in Enzymology», 87, 427-469.
[16] K. BROCKLEHURST, S.M. MUSHIRI, G. PATEL and F. WILLENBROCK (1982) - Evidence for a close similarity in the catalytic sites of papain and ficin in near-neutral media despite differences in acidic and alkaline media, «The Biochemical Journal», 201, 101-104.
[17] K. BROCKLEHURST, S.M. MUSHIRI, G. PATEL and F. WILLENBROCK (1983) - A marked gradation in active-centre properties in the cysteine proteinases revealed by neutral and anionic reactivity probes. Reactivity characteristics of the thiol groups of actinidin, ficin, papain and papaya peptidase A towards 4, 4'-dipyridyl disulphide and 5, 5'-dithiobis-(2-nitrobenzoate) dianion, «The Biochemical Journal», 209, 873-879.
[18] F. WILLENBROCK and K. BROCKLEHURST (1984) - Natural structural variation in enzymes as tool in the study of mechanism exemplified by a comparison of the catalytic-site structure and characteristics of cathepsin B and papain. pH-dependent kinetics of the reactions of cathepsin B from bovine spleen and from rat liver with a thiol-specific two-protonic-state reactivity probe (2, 2'-dipyridyl disulphide) and with a specific synthetic substrate (N-$\alpha$-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide), «The Biochemical Journal», 222, 805-814.
[19] B.S. BAINES, K. BROCKLEHURST, P.R. CAREY, M. JARVTS, E. SALIH and A.C. STORER (1986) - Chymopapain A. Purification and investigation by covalent chromatography and characterization by two-protonic-state reactivity-probe kinetics, steady-state kinetics and resonance Raman spectroscopy of some dithioacyl derivatives, «The Biochemical Journal», 233, 119-129.
[20] F. WILLENBROCK and K. BROCKLEHURST (1986) - Chemical evidence for the pH-dependent control of ion-pair geometry in cathepsin B. Benzofuroxan as a reactivity probe sensitive to differences in the mutual disposition of the thiolate and imidazolium components of cysteine proteinase catalytic sites, «The Biochemical Journal», 238, 103-107.
[21] S.S. HUSAIN and G. LOWE (1968) - The amino-acid sequence around the active site cysteine and histidine residues of stem-bromelain, «Chemical Communications», 1387-1389.
[22] S.S. HUSAIN and G. LOWE (1970) - The amino-acid sequence around the active-site cysteine and histidine residues, and the buried cysteine residue in ficin, «The Biochemical Journal», 117, 333- 340.
[23] S.S. HUSAIN and G. LOWE (1970) - The amino-acid sequence around the active-site cysteine and histidine residues of stem bromelain, «The Biochemical Journal», 117, 341-346.
[24] B. FRIEDENSON and I.E. LIENER (1972) - The active site sequence of multiple forms of ficin, «Archives of Biochemistry and Biophysics», 149, 169-174.
[25] J. DRENTH, K.H. KALK and H.M. SWEN (1976) - Binding of chloromethyl ketone substrate analogues to crystalline papain, «Biochemistry», 15, 3731-3738.
[26] S.D. LEWIS, F.A. JOHNSON, A.K. OHNO and J.A. SHAFER (1978) - Dependence of the catalytic activity of papain on the ionization of two acidic groups, «The Journal of Biological Chemistry», 253, 5080-5086.
[27] P. SCHACK and N.C. KAARSHOLM (1984) - Absence in papaya peptidase A catalyzed hydrolyses of a $pK_{a} \sim 4$ present in papain-catalyzed hydrolyses, «Biochemistry», 23, 631-635.
[28] L. PELLER and R.A. ALBERTY (1959) - Multiple intermediates in steady state enzyme kinetics. I. The mechanism involving a single substrate and product, «Journal of the American Chemical Society», 81, 5907-5914.
[29] R.C. NEUMAN jr., D. OWEN and G.D. LOCKYER jr. (1976) - High-pressure studies. XX: Deacylation of acyl-$\alpha$-chymotrypsins, «Journal of the American Chemical Society», 98, 2982-2985.
[30] P. ASCENZI, E. MENEGATTI, M. BOLOGNESI, M. GUARNERI and G. AMICONI (1986) - Catalytic properties of bovine $\alpha$-thrombin: a comparative steady-state and pre-steady-state study, «Biochimica et Biophysica Acta», 871, 319-323.
[31] L.A. BLUMENFEL'D (1972) - Elementary act in enzymatic catalysis, «Biofizika», 17, 954-959.
Con il contributo del Ministero per i Beni e le Attività Culturali