hf. J. Eiochem. Vol. 24, No. 7, pp. 1051-1055, Printed in Great Britain. All rights reserved
1992 Copyright
0
0020-7 I I X/92 $5.00 + 0.00 1992 Pergamon Press Ltd
KINETIC EVIDENCE FOR THE EXISTENCE OF AN UNSTABLE INTERMEDIATE IN THE TRINITROPHENYLATION-INDUCED RHODANESE INACTIVATION REACTION EMMANUELT. RAKITZIS and THALIA B. MALLIOPOULOU Department of Biological Chemistry, University of Athens Medical School, Athens I15 27, Greece (Received
4 November
1991)
Abstract-l. Rhodanese inactivation by 2,4,6-trinitrobenzenesulphonate, in the presence of n-butylamine in the reaction medium, has been studied by a kinetic analysis of the data, based on the assumption that enzyme inactivation is brought about by direct reaction of this with the modifying agent. 2. Initial reaction rates for rhodanese activity loss were determined by a mathematical analysis of the first three recorded values of rhodanese residual activity. 3. It was found that fractional rhodanese activity values, at infinite reaction time with 2,4,6-trinitrobenzenesulphonate (end-point values), were significantly lower than the values calculated on the assumption of rhodanese inactivation being entirely due to direct trinitrophenylation of enzyme protein. 4. Also, initial enzyme inactivation values were higher in the presence, rather than in the absence, of n-butylamine. 5. These results indicate that 2,4,6-trinitrobenzenesulphonate-induced rhodanese inactivation, in the presence of n-butylamine in the reaction medium, is due to the generation of a highly reactive, unstable intermediate, probably a free radical species.
INTRODUCI’ION Rhodanese (thiosulphate sulphurtransferase, EC 2.8.1.1) is an ubiquitous enzyme catalysing the transfer of sulphur from sulphur donors such as thiosul-
phate, to a variety of nucleophilic acceptors (Westley, 1973; Weng et al., 1978; Sorbo, 1953). Rhodanese free amino groups trinitrophenylation results in the loss of enzyme catalytic function. By a comparison of enzyme inactivation and of protein modification rate constants, it was tentatively assumed that all rhodanese modifiable amino groups are essential for enzyme activity (Rakitzis and Malliopoulou, 1985; Malliopoulou and Rakitzis, 1988). In this communication studies of rhodanese inactivation by 2,4,6trinitrobenzenesulphonate, in the presence of n-butylamine in the reaction medium, are presented. Kinetic analysis of the results obtained indicates the existence of an unstable, highly reactive, intermediate in the primary amino groups trinitrophenylationinduced rhodanese inactivation reaction. A preliminary communication of these results has been published (Rakitzis and Malliopoulou, 1991). MATERIALS
AND METHODS
Rhodanese, purified from bovine liver, and 2,4,6-trinitrobenzenesulphonic acid were obtained from Sigma Chemical Co., St Louis, MO., U.S.A. n-Butylamine was a product of Merck, Schuchardt, Fed. Rep. Germany. 2,4,6-Trinitrophenol was obtained from Sclavo Diagnostics, Italy. Rho-
danese activity was determined by the method of Sorb0 (1953), with thiosulphate and cyanide as substrates. Thiocyanate production was found to be linearly dependent on rhodanese concentration, at least up to an absorbance reading of 12OOcm-’ at 46Onm, under the experimental conditions used (Rakitzis and Malliopoulou, 1985). Trinitrophenylation-induced rhodanese activity loss was determined by preparing an enzyme/modifying agent mixture, the final composition of which was: 0.1 M sodium phosphate buffer; 0.01 M sodium thiosulphate, 9.12 PM rhodanese, 0.25 mM 2,4,6-trinitrobenzenesulphonic acid, and n-butylamine, as required. The sodium phosphate buffer/n-butylamine mixture was, for each n-butylamine concentration used, adjusted to pH 8.0 as follows: two solutions containing 0.1 M sodium phosphate buffer, 0.01 M thiosulphate, and n-butylamine as required, were prepared in each instance. The pH values of these solutions were approx 7.5 and 8.5. Finally, a pH 8.0 (determined by means of Radiometer pH meter) mixture of these two solutions was prepared. Inclusion of one of the substrates, namely thiosulphate, in the reaction medium, was considered necessary for the enzyme to be maintained in the sulphur-rhodanese form; in the absence of thiosulphate the persulphide form of rhodanese is easily oxidized, and the enzyme is thereby rendered inactive (Wang and Volini, 1968). After incubation of the enzyme/modifying agent mixture in a water bath at 25°C. for the time periods required, aliquots were withdrawn and used for the determination of rhodanese activity. The extent of the reaction of 2,4,6_trinitrobenzenesulphonate with rhodanese, or with the rhodaneseln-butylamine mixture was monitored by taking absorbance readings at 345 nm in a Gilford 240 spectrophotometer (Goldfarb, 1966).
1051
1052
EMMANUEL T. RAKITZISand THALIAB. MALLIOPOULOU
Results of rhodanese inactivation experiments in the absence of n-butylamine from the reaction medium, were studied by plotting the log of fractional enzyme activity vs the reaction time used. Since the modifying agent concentration was in large excess over the enzyme concentration used, reaction kinetics are expected to be first-order with regard to enzyme concentration (Rakitzis, 1984). To rule out the possibility of n-butylamine itself being a reversible, or an irreversible inhibitor of rhodanese, a reaction mixture was prepared containing 0.1 M sodium phosphate buffer. sodium thiosulphate 0.01 M, ~-butyiamine 0.12 M and rhodanese 9.12 PM. At different times from the time of the addition of all reaction components, aliquots were taken and used for rhodanese activity determination. It was found that rhodanese activity, at zero time, as well as at different times, up to 60min after the time of addition of the reactants, was the same as that obtained in the absence of ~-butylamine from the reaction medium. It was accordingly concluded that n-butylamine is not a reversible, or an irreversible, inhibitor of rhodanese. Results of rhodanese inactivation experiments, in the presence of n-butylamine in the reaction medium, were studied in two different ways: (i) By a comparison of observed and of calculated values for the end-point of the rhodanese inactivation reaction. Calculation of the enzyme activityend point is based on the kinetics of disappearance of 2,4,6-trinitrobenzenesulphonate from the reaction medium (see below). (ii) By the determination of the initial velocity of the reaction of trinitrophenylation-induced rhodanese inactivation. This is carried out by fitting the enzyme inactivation data to the equation (Rakitzis, 1987): E,,,,,(r)=a,, + a, t
+ aZtZ+ ” + u,t’.
(1)
where L,, is inactive enzyme; an equation of the form of equation (1) can be shown to define any single-vaiued continuous function (Cornish-Bowden, 1976). Since a, = 0 at f = 0, the coefficients u,. a,, . . , a, can be determined by setting up j simultaneous equations inj unknowns (Freund, 1969). The E:,,,, (0) value (where the superscript ’ is used to indicate the first derivative with regard to reaction time), i.e. a,, was determined for the trinitrophenylation-induced rhodanese inactivation experiments by using the first three recorded values of fractional enzyme activity, from the start of the reaction, at different n-butylamine concentrations. Theoretical
The two most likely reaction schemes are: (i) Enzyme inactivation is only due to enzyme protein trinitrophenylation: k”X1
E+M-E* km&
M-t-A-m where E is enzyme, M is modifying agent, i.e. 2,4,6trinitrobenzenesufphonate, A is the nucleophile used to bring about the pseudofirst-order reaction disappearance of M, i.e. n-butylamine, E* is the covalently modified enzyme, m is trinitrophenylated n-butylamine, and k,,,,, and k&_ are the relevant secondorder reaction rate constants.
(ii) Enzyme inactivation is due to the effect of an unstable, reactive intermediate, said intermediate being generated by the reaction of 2,4,6_trinitrobenzenesulphonate with primary amino groups: I;
1992 Copyright
0
0020-7 I I X/92 $5.00 + 0.00 1992 Pergamon Press Ltd
KINETIC EVIDENCE FOR THE EXISTENCE OF AN UNSTABLE INTERMEDIATE IN THE TRINITROPHENYLATION-INDUCED RHODANESE INACTIVATION REACTION EMMANUELT. RAKITZIS and THALIA B. MALLIOPOULOU Department of Biological Chemistry, University of Athens Medical School, Athens I15 27, Greece (Received
4 November
1991)
Abstract-l. Rhodanese inactivation by 2,4,6-trinitrobenzenesulphonate, in the presence of n-butylamine in the reaction medium, has been studied by a kinetic analysis of the data, based on the assumption that enzyme inactivation is brought about by direct reaction of this with the modifying agent. 2. Initial reaction rates for rhodanese activity loss were determined by a mathematical analysis of the first three recorded values of rhodanese residual activity. 3. It was found that fractional rhodanese activity values, at infinite reaction time with 2,4,6-trinitrobenzenesulphonate (end-point values), were significantly lower than the values calculated on the assumption of rhodanese inactivation being entirely due to direct trinitrophenylation of enzyme protein. 4. Also, initial enzyme inactivation values were higher in the presence, rather than in the absence, of n-butylamine. 5. These results indicate that 2,4,6-trinitrobenzenesulphonate-induced rhodanese inactivation, in the presence of n-butylamine in the reaction medium, is due to the generation of a highly reactive, unstable intermediate, probably a free radical species.
INTRODUCI’ION Rhodanese (thiosulphate sulphurtransferase, EC 2.8.1.1) is an ubiquitous enzyme catalysing the transfer of sulphur from sulphur donors such as thiosul-
phate, to a variety of nucleophilic acceptors (Westley, 1973; Weng et al., 1978; Sorbo, 1953). Rhodanese free amino groups trinitrophenylation results in the loss of enzyme catalytic function. By a comparison of enzyme inactivation and of protein modification rate constants, it was tentatively assumed that all rhodanese modifiable amino groups are essential for enzyme activity (Rakitzis and Malliopoulou, 1985; Malliopoulou and Rakitzis, 1988). In this communication studies of rhodanese inactivation by 2,4,6trinitrobenzenesulphonate, in the presence of n-butylamine in the reaction medium, are presented. Kinetic analysis of the results obtained indicates the existence of an unstable, highly reactive, intermediate in the primary amino groups trinitrophenylationinduced rhodanese inactivation reaction. A preliminary communication of these results has been published (Rakitzis and Malliopoulou, 1991). MATERIALS
AND METHODS
Rhodanese, purified from bovine liver, and 2,4,6-trinitrobenzenesulphonic acid were obtained from Sigma Chemical Co., St Louis, MO., U.S.A. n-Butylamine was a product of Merck, Schuchardt, Fed. Rep. Germany. 2,4,6-Trinitrophenol was obtained from Sclavo Diagnostics, Italy. Rho-
danese activity was determined by the method of Sorb0 (1953), with thiosulphate and cyanide as substrates. Thiocyanate production was found to be linearly dependent on rhodanese concentration, at least up to an absorbance reading of 12OOcm-’ at 46Onm, under the experimental conditions used (Rakitzis and Malliopoulou, 1985). Trinitrophenylation-induced rhodanese activity loss was determined by preparing an enzyme/modifying agent mixture, the final composition of which was: 0.1 M sodium phosphate buffer; 0.01 M sodium thiosulphate, 9.12 PM rhodanese, 0.25 mM 2,4,6-trinitrobenzenesulphonic acid, and n-butylamine, as required. The sodium phosphate buffer/n-butylamine mixture was, for each n-butylamine concentration used, adjusted to pH 8.0 as follows: two solutions containing 0.1 M sodium phosphate buffer, 0.01 M thiosulphate, and n-butylamine as required, were prepared in each instance. The pH values of these solutions were approx 7.5 and 8.5. Finally, a pH 8.0 (determined by means of Radiometer pH meter) mixture of these two solutions was prepared. Inclusion of one of the substrates, namely thiosulphate, in the reaction medium, was considered necessary for the enzyme to be maintained in the sulphur-rhodanese form; in the absence of thiosulphate the persulphide form of rhodanese is easily oxidized, and the enzyme is thereby rendered inactive (Wang and Volini, 1968). After incubation of the enzyme/modifying agent mixture in a water bath at 25°C. for the time periods required, aliquots were withdrawn and used for the determination of rhodanese activity. The extent of the reaction of 2,4,6_trinitrobenzenesulphonate with rhodanese, or with the rhodaneseln-butylamine mixture was monitored by taking absorbance readings at 345 nm in a Gilford 240 spectrophotometer (Goldfarb, 1966).
1051
1052
EMMANUEL T. RAKITZISand THALIAB. MALLIOPOULOU
Results of rhodanese inactivation experiments in the absence of n-butylamine from the reaction medium, were studied by plotting the log of fractional enzyme activity vs the reaction time used. Since the modifying agent concentration was in large excess over the enzyme concentration used, reaction kinetics are expected to be first-order with regard to enzyme concentration (Rakitzis, 1984). To rule out the possibility of n-butylamine itself being a reversible, or an irreversible inhibitor of rhodanese, a reaction mixture was prepared containing 0.1 M sodium phosphate buffer. sodium thiosulphate 0.01 M, ~-butyiamine 0.12 M and rhodanese 9.12 PM. At different times from the time of the addition of all reaction components, aliquots were taken and used for rhodanese activity determination. It was found that rhodanese activity, at zero time, as well as at different times, up to 60min after the time of addition of the reactants, was the same as that obtained in the absence of ~-butylamine from the reaction medium. It was accordingly concluded that n-butylamine is not a reversible, or an irreversible, inhibitor of rhodanese. Results of rhodanese inactivation experiments, in the presence of n-butylamine in the reaction medium, were studied in two different ways: (i) By a comparison of observed and of calculated values for the end-point of the rhodanese inactivation reaction. Calculation of the enzyme activityend point is based on the kinetics of disappearance of 2,4,6-trinitrobenzenesulphonate from the reaction medium (see below). (ii) By the determination of the initial velocity of the reaction of trinitrophenylation-induced rhodanese inactivation. This is carried out by fitting the enzyme inactivation data to the equation (Rakitzis, 1987): E,,,,,(r)=a,, + a, t
+ aZtZ+ ” + u,t’.
(1)
where L,, is inactive enzyme; an equation of the form of equation (1) can be shown to define any single-vaiued continuous function (Cornish-Bowden, 1976). Since a, = 0 at f = 0, the coefficients u,. a,, . . , a, can be determined by setting up j simultaneous equations inj unknowns (Freund, 1969). The E:,,,, (0) value (where the superscript ’ is used to indicate the first derivative with regard to reaction time), i.e. a,, was determined for the trinitrophenylation-induced rhodanese inactivation experiments by using the first three recorded values of fractional enzyme activity, from the start of the reaction, at different n-butylamine concentrations. Theoretical
The two most likely reaction schemes are: (i) Enzyme inactivation is only due to enzyme protein trinitrophenylation: k”X1
E+M-E* km&
M-t-A-m where E is enzyme, M is modifying agent, i.e. 2,4,6trinitrobenzenesufphonate, A is the nucleophile used to bring about the pseudofirst-order reaction disappearance of M, i.e. n-butylamine, E* is the covalently modified enzyme, m is trinitrophenylated n-butylamine, and k,,,,, and k&_ are the relevant secondorder reaction rate constants.
(ii) Enzyme inactivation is due to the effect of an unstable, reactive intermediate, said intermediate being generated by the reaction of 2,4,6_trinitrobenzenesulphonate with primary amino groups: I;
