THOMAS'. and KEITH BROCKLEHURST'. *Department of Biochemistry, Queen Mary & Westfield. College, University of London, Mile End Road, London, El.
Biochemical SocietyTransactions ( 1 994) 22
Existence of the Cys-His ion-pair state of cysteine proteinases may be an insufficient condition for catalytic competence. SURAPONG PINITGLANG', MANU PATEL'*, EMRYS W. THOMAS'. and KEITH BROCKLEHURST'. *Department of Biochemistry, Queen Mary & Westfield College, University of London, Mile End Road, London, E l 4NS, U.K. ?Department of Biological Sciences, University of Salford, Salford, M5 4JW, U.K. The dependence of rate constants on pH in experiments that are properly designed and interpreted (see [l] and references therein for discussion of some of the pitfalls) is a powerful tool in the investigation of mechanism. The proton is the least .sterically-demandinggeneral perturbant of protein structure and analysis of pH-dependent kinetics can provide information about kinetically-influentialionizations (macroscopicpK, values and values of pH-independent rate constants k, characteristic of individual ionization states). Analysis of complex pH-k profiles comprising a multiplicity of reactive states is facilitated by using the multitasking application program (SKETCHER) written in ANSI C running under RISCOS on an Acorn Archimedes microcomputer [2,3]. pH-dependent kinetic study of members of the cysteine proteinase family of enzymes (reviewed in [4]) continues to provide mechanistic insights (see e.g. [5]). Activity and catalytic site reactivity of these enzymes is determiried to various extents by a multiplicity of ionizations in the enzyme molecule. We recently introduced 4,4'dipyrimidyl disulphide @Ka 0.91) as a thiol-specific timedependent inhibitor and showed that by using both this compound and 2-pyridyl disulphides, such as 2,2 -dipyridyl disulphide @KO2.45), as reactivity probes it was possible to dissect the complex pH-dependent behaviour of papain (EC 3.4.22.2) and distinguish unequivocally the ionizations of the catalytic site Cys-25/His-159 interactive system from other kinetically-influential ionizations of the enzyme molecule [6,7]. Thus pK, values of 3.4 and 8.3 can be assigned to the former and pK, values of 4.0, 5.0 and 10.0 to the latter. A major conclusion is that, whereas in reactions of simple alkylating agents and of 2,2' -dipyridyl disulphide full nucleophilic character of (Cys-25)-S-/(His-I59)-Im+H is provided by protonic dissociation with pK, 3.3-3.4, in catalysis relatively little catalytic competence is produced consequent upon ion-pair formation. Substantial catalytic competence requires further protonic dissociation with pK, cu 4, and for cationic substrates further enhancement is produced by protonic dissociation with pK, cu 5. In the present communication the results of analogous experiments on another cysteine proteinase, ficin (EC 3.4.22.3) are presented as part of a study designed to ascertain the extent to which the existence of the ubiquitous (Cys)-S-/(His)-Im+H ion-pair of cysteine proteinase catalytic sites is a sufficient condition for catalytic competence as has been supposed hitherto. Ficin is intermediate between papain and actinidin (EC 3.4.22.14) in its P,-Sz stereochemical selectivity [8] and in its binding site-catalytic site signalling characteristics [9]. Studies on ficin, therefore, should contribute to the developing picture of structure-function relationships in the cysteine proteinase family. The pH-k profile for the reaction of ficin with 4,4'-dipyrimidyl Present address: $Department of Biochemical & Clinical Pharmacology, St. Jude Children's Research Hospital, 332, North Lauderdale, P.O. Box 318, Memphis, Tennessee 381010318, U.S.A.
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disulphide is of triple sigmoid form with k increasing with increase in pH across pK, values 2.5, 4.2 and 8.25. The small sigmoid wave with pK, 4.2 was not observed in the pHdependence of k for the analogous reaction of papain @K,s 3.4 and 8.3). The simplest interpretation of the data for the ficin reaction is that the pK, value of 2.5, associated with the major component of the pH-k profile in acidic media, is characteristic of the formation of the (Cys)-S-/(His)-Im+H ion-pair state of ficin. This pK, value is much lower than those around 4 previously assigned to this feature of the ficin molecule. A new fit to the pH-k data for the reaction of ficin with 2,2'dipyridyl disulphide [ 101 reveals kinetically influential ionizations with pK, values 3.3, 4.2 and 9.5 additional to the now known values (2.5 and 8.25) of the interactive Cys/His catalytic site system. The pH-dependence of k,,,/K, for the at ficin-catalysed hydrolysis of N-Ac-L-Phe-Gly-4-nitroanilide 25°C and I 0.1M is approximately bell-shaped with k,,,/K, maximal at pH cu 6 and characterized by the following values of the parameters (where gl, with units of M - k ' , represents the pH-independent value of k,,/K,,, for the ionization state developed by raising the pH across pK, and &, pK, etc. are similarly defined): pK, = 2.5, k, 5 1.0; pK, = 3.3, k2 = 40; pK, = 4.2, & = 215; pKrv = 8.3; L, = 20; pKv = 10, k5 = 0. As is the case for papain, very little catalytic activity develops consequent upon formation of the (Cys)-S/(His)-Im+H ion-pair state (Ll 5 1M-Is-lcompared with l2= 40 M%' and = 215 M-k'). Generation of the catalytic site (Cys)-S-/(His)-Im+H ion-pair, which supplies nucleophilic and acid and/or base catalysis at various stages of the catalytic process, is an insufficient requirement for catalytic competence. Proton loss from one or more additional sites in the enzyme molecule appears to be essential for development of substantial catalytic activity. We thank the University of Thai Chamber of Commerce for a Research Studentship for S.P. and the Science and Engineering Research Council for an Earmarked Research Studentship for M.P. 1. Brocklehurst, K & Topham, C. (1993) Biochem. J. 295, 898-899. 2. Brocklehurst, S.M., Topham, C.M. & Brocklehurst, K. (1990) Biochem. Soc. Trans. 18, 598-599. 3. Topham, C.M., Salih, E., Frazao, C., Kowlessur, D., Overington, J.P., Thomas, M., Brocklehurst, S.M., Patel, M., Thomas, E.W. & Brocklehurst (1991) Biochem. J. 280, 79-92. 4. Brocklehurst, K., Willenbrock, F. & Salih, E. (1987) Hydrolytic Enzymes (A. Neuberger & K. Brocklehurst, eds) New Comprehensive Biochemistry 16, 39-158. 5. Kowlessur, D., Topham, C.M., Thomas, E.W., O'D~scoll, M., Templeton, W. & Brocklehurst K. (1989) Biochem. J. 258, 755-764. 6. Mellor, G.W., Thomas, E.W., Topham, C.M. & Brocklehurst, K. (1993) Biochem. J. 290, 289-296. 7. Mellor, G.W., Patel, M., Thomas, E.W. & Brocklehurst, K. (1993) 294, 201-210. 8. Patel, M., Saleem, I., Mellor, G.W., Sreedharan, S., Templeton, W., Thomas,. E.W., Thomas, M. & Brocklehurst, K. (1992) Biochem. J. 281, 553-559. 9. Patel, M., Thomas, M.P., Kayani, IS., Mellor, G.W., Thomas, E.W. & Brocklehurst, K. (1993) Biochem. Soc. Trans. 21, 216s. lO.Malthouse, J.P.G. & Brocklehurst, K. (1976) Biochem. J. 159, 221-234.
