INT . J . RADIAT . BIOL .,
1975,
VOL .
28,
NO .
5, 4 8 5-488
CORRESPONDENCE Some implications from the inactivation of esterase activity in carboxypeptidase A by Br 2 - and other radicals
Int J Radiat Biol Downloaded from informahealthcare.com by Ohio State University Libraries on 12/03/14 For personal use only.
P . B . ROBERTSf and R . D . BUGDEN Division of Physics, Institute of Cancer Research, Sutton, Surrey SM2 5PX, U.K . (Received
19
September
1975 ;
accepted 6 October
1975)
Carboxypeptidase A (CPA) catalyses the hydrolysis of both peptide and ester substrates with certain C-terminal configurations (Snoke and Neurath 1949) . Extensive chemical modification and X-ray diffraction evidence (reviewed by Vallee and Riordan 1968, Quiocho and Lipscomb 1971) has led to the conclusion that peptidase activity involves at least one tyrosine residue . Tyrosine 248 has been assigned the role of proton donor during catalysis, and tyrosine 198 is believed to be involved in substrate binding, although this may be at an unproductive site . Auld and Holmquist (1974) demonstrated clearly that the mechanism of action of CPA is different for its two specificities, confirming what had been suspected previously from differences between the two activities in Michaelis-Menten parameters (Whitaker, Menger and Bender 1966), pH-activity profiles (Riordan and Vallee 1963) and other considerations . Of particular interest here is the effect of tyrosine acetylation, which eliminates petidase activity completely but does not impair the esterase function (Riordan and Vallee 1963) . Roberts (1973) showed that as the pH was raised around 9 . 4, the loss of peptidase activity induced by Br2- and CNS2 radicals increased sharply, as did both the rate of the reaction of these secondary radicals with CPA and the intensity of an absorption at 400 nm due to reaction products . The latter two parameters are known to increase around the pK of the aromatic hydroxyl group of tyrosine in free solution (Adams, Redpath, Bisby and Cundall 1973), and it was inferred that secondary radicals could detect essential tyrosines within an enzyme framework . Data obtained by Roberts (1973) on the rate of the Br2 reaction with CPA and the product spectra can be applied to considerations of either activity . It was of obvious interest, therefore, to study the effect of Br2 radicals on the esterase activity of CPA . Experimental methods have been published earlier (Roberts 1973), except that in this work esterase activity was assayed by the rate of cleavage of hippurylL-phenyllactate, observed at 254 nm . Inactivation due to Br2 was calculated by taking the yield of inactivation observed in solutions containing N 2 0 and 10 mM KBr (when H atoms and Br 2 - are the radicals produced in solution capable of reaction with CPA) and subtracting the relatively small yield of inactivation found in solutions containing N2O and 50 mM t-butanol (when H j' Present address : Institute of Nuclear Sciences, D .S .I .R., Private Bag, Lower Hutt, New Zealand . R .B .
Z L
Int J Radiat Biol Downloaded from informahealthcare.com by Ohio State University Libraries on 12/03/14 For personal use only.
486
Correspondence
atoms are the only reactive radicals produced in solution) . Throughout the pH range tested, the latter yield was essentially constant with a G value of 0 . 15 ± 0 . 03 . In the figure, the G value for the loss of both peptidase and esterase activity brought about by Br 2 - in the pH range 7 . 8-11 is indicated . The sharp increase in G (peptidase loss) around pH 9 . 4 reported by Roberts (1973) is clear . Above pH 10 . 5, G (peptidase loss) decreases due to a conformational change exposing reactive, but non-essential residues . The figure shows also that G (esterase loss) increases around pH 9 . 4, although the increase is only about one-half as great . Thus, a correlation exists between enhanced G (activity loss) and increased Br2 - attack upon a tyrosine residue(s) for both activities . For peptidase function, radiation inactivation through destruction of a catalytically-essential tyrosine agrees completely with the chemical modification and X-ray diffraction evidence .
2.0
18
16
1. 0
08
06 8
9
10
pH
The effect of pH on the loss of enzyme activity brought about by Br 2 - radicals, expressed as a G value . --0--, peptidase activity ;-*-, esterase activity . Points for the peptidase activity curve obtained from the data of Roberts (1973) . However, this is not so for the esterase activity, in which no direct role for tyrosine(s) is indicated . The smaller rise in G (esterase loss) may be a reflection of this relative unimportance of tyrosines . Several possibilities arise, including the ability of Br2 radicals to react with an essential tyrosine that is not accessible to normal chemical agents . The active sites for both forms of activity are a'most certainly situated in the same dead-end groove in the molecule, however, and the only tyrosines found to be close to bound substrates, tyrosines 248 and 198, are both susceptible to chemical modification (Quiocho and Lipscomb 1971) . The most probable explanation is that destruction of the integrity of
Int J Radiat Biol Downloaded from informahealthcare.com by Ohio State University Libraries on 12/03/14 For personal use only.
Correspondence
48 7
tyrosines 248 and 198, which must be close to the catalytic and substrate binding residues, breaks down the required conformation of the residues which carry out a mechanistic function . Thus tyrosines are essential to the esterase activity of CPA in a far looser sense than is the case in the peptidase action . Whatever the explanation, it is clear that care should be taken in imputing a function in the catalytic mechanism to residues shown to be important solely from data on the inactivation of the native enzyme by secondary radicals . Some simple parallel tests may determine if the implicated residues are biochemically essential or merely in a position to influence active site geometry . Measurement of Michaelis-Menten parameters after partial inactivation could indicate whether catalysis or substrate binding had been affected . Further useful information might arise from the irradiation of modified and inhibited or activated enzyme preparations . This approach was taken in a study of superoxide dismutase (Roberts, Fielden, Rotilio, Calabrese, Bannister and Bannister 1974) . As enzymes which are less well understood than those examined hitherto are studied by the secondary radical technique, such parallel studies may become increasingly important . During this work, data were obtained which enabled a comparison to be made between the sensitivities of the two activities towards the primary radicals e a,q- , OH and H atoms . The averages for several determinations of G (esterase loss) in N2 0, N2 and N2 0+t-butanol at pH 7 . 8 were 0 . 25, 0 . 17 and 0 . 12, respectively . Corresponding values for G (peptidase loss) were 0 . 44, 0 . 27 and 0 . 11 . When the contributions towards inactivation from the individual primary radical species are determined from these data (for details of the calculations see Roberts 1973), we find that the solvated electron is completely without effect on either function. H atoms are about equally effective at destroying either activity (about five radicals are required for one inactivating event) . Inactivation by OH radicals is less than one-half as effective in the case of esterase loss (44 radicals per inactivating event) compared with peptidase inactivation (17 radicals per inactivating event) . Once again the efficiency of inactivation by H atoms found with several enzymes (Mee, Adelstein and Stein 1972, Badiello, Quintiliani and Tamba 1974, Roberts et al . 1974) is highlighted . Amino-acid analysis of ribonuclease (Mee et al . 1972) and bovine superoxide dismutase (Barra, Bossa, Calabrese, Rotilio, Roberts and Fielden 1975) exposed to H atoms has indicated that only a few amino acid types are destroyed . The data presented here lend further support to the idea that H atoms are unusually effective. This may be due to either a high degree of selectivity for reaction at a few critical residues or the augmenting of the initial damage by intra-enzyme chain reactions (Lichtin, Ogdan and Stein 1973) .
REFERENCES ADAMS, G . E ., REDPATH, J . L., BISBY, R . H ., and CUNDALL, R . B ., 1973, ,. chem . Soc., Faraday Trans . 1, 69, 1608 . AULD, D . S ., and HOLMQUIST, B ., 1974, Biochemistry, 13, 4355 . BADIELLO, R ., QUINTILIANI, M ., and TAMBA, M ., 1974, Int .„ . Radiat. Biol ., 26, 311 . BARRA, D ., BOSSA, F ., CALABRESE, L ., ROTILIO, G ., ROBERTS, P . B ., and FIELDEN, E . M ., 1975, Biochem. biophys . Res . Commun ., 64, 1303 . MEE, L . K ., ADELSTEIN, S . J ., and STEIN, G ., 1972, Radiat . Res ., 52, 588 .
488
Correspondence
QviocHo, F . A ., and LIPSCOMB, W . N ., 1971, Advances in Protein Chemistry, edited by C . B . Anfinsen, Jr ., J . T . Edsall and F. M . Richards (New York : Academic Press), p. 1 . LICHTIN, N . N ., OCDAN, J ., and STEIN, G ., 1973, Radiat . Res ., 55, 69 . RIORDAN, J . F ., and VALLEE, B . L ., 1963, Biochemistry, 2, 1460 . ROBERTS, P . B ., 1973, Int . Y . Radiat . Biol ., 24, 143 . ROBERTS, P . B ., FIELDEN, E . M ., ROTILIO, G ., CALABRESE, L ., BANNISTER, J . V ., and
Int J Radiat Biol Downloaded from informahealthcare.com by Ohio State University Libraries on 12/03/14 For personal use only.
BANNISTER, W . H ., 1974, Radiat . Res ., 60, 441 . SNOKE, J . E ., and NEIJRATH, H ., 1949, Y . biol . Chem., 181, 789 . VALLEE, B . L., and RIORDAN, J . F ., 1968, Brookhaven Symp . Biol ., 21, 91 . WHITAKER, J . R ., MENGER, F ., and BENDER, M . L ., 1966, Biochemistry, 5, 386 .
1975,
VOL .
28,
NO .
5, 4 8 5-488
CORRESPONDENCE Some implications from the inactivation of esterase activity in carboxypeptidase A by Br 2 - and other radicals
Int J Radiat Biol Downloaded from informahealthcare.com by Ohio State University Libraries on 12/03/14 For personal use only.
P . B . ROBERTSf and R . D . BUGDEN Division of Physics, Institute of Cancer Research, Sutton, Surrey SM2 5PX, U.K . (Received
19
September
1975 ;
accepted 6 October
1975)
Carboxypeptidase A (CPA) catalyses the hydrolysis of both peptide and ester substrates with certain C-terminal configurations (Snoke and Neurath 1949) . Extensive chemical modification and X-ray diffraction evidence (reviewed by Vallee and Riordan 1968, Quiocho and Lipscomb 1971) has led to the conclusion that peptidase activity involves at least one tyrosine residue . Tyrosine 248 has been assigned the role of proton donor during catalysis, and tyrosine 198 is believed to be involved in substrate binding, although this may be at an unproductive site . Auld and Holmquist (1974) demonstrated clearly that the mechanism of action of CPA is different for its two specificities, confirming what had been suspected previously from differences between the two activities in Michaelis-Menten parameters (Whitaker, Menger and Bender 1966), pH-activity profiles (Riordan and Vallee 1963) and other considerations . Of particular interest here is the effect of tyrosine acetylation, which eliminates petidase activity completely but does not impair the esterase function (Riordan and Vallee 1963) . Roberts (1973) showed that as the pH was raised around 9 . 4, the loss of peptidase activity induced by Br2- and CNS2 radicals increased sharply, as did both the rate of the reaction of these secondary radicals with CPA and the intensity of an absorption at 400 nm due to reaction products . The latter two parameters are known to increase around the pK of the aromatic hydroxyl group of tyrosine in free solution (Adams, Redpath, Bisby and Cundall 1973), and it was inferred that secondary radicals could detect essential tyrosines within an enzyme framework . Data obtained by Roberts (1973) on the rate of the Br2 reaction with CPA and the product spectra can be applied to considerations of either activity . It was of obvious interest, therefore, to study the effect of Br2 radicals on the esterase activity of CPA . Experimental methods have been published earlier (Roberts 1973), except that in this work esterase activity was assayed by the rate of cleavage of hippurylL-phenyllactate, observed at 254 nm . Inactivation due to Br2 was calculated by taking the yield of inactivation observed in solutions containing N 2 0 and 10 mM KBr (when H atoms and Br 2 - are the radicals produced in solution capable of reaction with CPA) and subtracting the relatively small yield of inactivation found in solutions containing N2O and 50 mM t-butanol (when H j' Present address : Institute of Nuclear Sciences, D .S .I .R., Private Bag, Lower Hutt, New Zealand . R .B .
Z L
Int J Radiat Biol Downloaded from informahealthcare.com by Ohio State University Libraries on 12/03/14 For personal use only.
486
Correspondence
atoms are the only reactive radicals produced in solution) . Throughout the pH range tested, the latter yield was essentially constant with a G value of 0 . 15 ± 0 . 03 . In the figure, the G value for the loss of both peptidase and esterase activity brought about by Br 2 - in the pH range 7 . 8-11 is indicated . The sharp increase in G (peptidase loss) around pH 9 . 4 reported by Roberts (1973) is clear . Above pH 10 . 5, G (peptidase loss) decreases due to a conformational change exposing reactive, but non-essential residues . The figure shows also that G (esterase loss) increases around pH 9 . 4, although the increase is only about one-half as great . Thus, a correlation exists between enhanced G (activity loss) and increased Br2 - attack upon a tyrosine residue(s) for both activities . For peptidase function, radiation inactivation through destruction of a catalytically-essential tyrosine agrees completely with the chemical modification and X-ray diffraction evidence .
2.0
18
16
1. 0
08
06 8
9
10
pH
The effect of pH on the loss of enzyme activity brought about by Br 2 - radicals, expressed as a G value . --0--, peptidase activity ;-*-, esterase activity . Points for the peptidase activity curve obtained from the data of Roberts (1973) . However, this is not so for the esterase activity, in which no direct role for tyrosine(s) is indicated . The smaller rise in G (esterase loss) may be a reflection of this relative unimportance of tyrosines . Several possibilities arise, including the ability of Br2 radicals to react with an essential tyrosine that is not accessible to normal chemical agents . The active sites for both forms of activity are a'most certainly situated in the same dead-end groove in the molecule, however, and the only tyrosines found to be close to bound substrates, tyrosines 248 and 198, are both susceptible to chemical modification (Quiocho and Lipscomb 1971) . The most probable explanation is that destruction of the integrity of
Int J Radiat Biol Downloaded from informahealthcare.com by Ohio State University Libraries on 12/03/14 For personal use only.
Correspondence
48 7
tyrosines 248 and 198, which must be close to the catalytic and substrate binding residues, breaks down the required conformation of the residues which carry out a mechanistic function . Thus tyrosines are essential to the esterase activity of CPA in a far looser sense than is the case in the peptidase action . Whatever the explanation, it is clear that care should be taken in imputing a function in the catalytic mechanism to residues shown to be important solely from data on the inactivation of the native enzyme by secondary radicals . Some simple parallel tests may determine if the implicated residues are biochemically essential or merely in a position to influence active site geometry . Measurement of Michaelis-Menten parameters after partial inactivation could indicate whether catalysis or substrate binding had been affected . Further useful information might arise from the irradiation of modified and inhibited or activated enzyme preparations . This approach was taken in a study of superoxide dismutase (Roberts, Fielden, Rotilio, Calabrese, Bannister and Bannister 1974) . As enzymes which are less well understood than those examined hitherto are studied by the secondary radical technique, such parallel studies may become increasingly important . During this work, data were obtained which enabled a comparison to be made between the sensitivities of the two activities towards the primary radicals e a,q- , OH and H atoms . The averages for several determinations of G (esterase loss) in N2 0, N2 and N2 0+t-butanol at pH 7 . 8 were 0 . 25, 0 . 17 and 0 . 12, respectively . Corresponding values for G (peptidase loss) were 0 . 44, 0 . 27 and 0 . 11 . When the contributions towards inactivation from the individual primary radical species are determined from these data (for details of the calculations see Roberts 1973), we find that the solvated electron is completely without effect on either function. H atoms are about equally effective at destroying either activity (about five radicals are required for one inactivating event) . Inactivation by OH radicals is less than one-half as effective in the case of esterase loss (44 radicals per inactivating event) compared with peptidase inactivation (17 radicals per inactivating event) . Once again the efficiency of inactivation by H atoms found with several enzymes (Mee, Adelstein and Stein 1972, Badiello, Quintiliani and Tamba 1974, Roberts et al . 1974) is highlighted . Amino-acid analysis of ribonuclease (Mee et al . 1972) and bovine superoxide dismutase (Barra, Bossa, Calabrese, Rotilio, Roberts and Fielden 1975) exposed to H atoms has indicated that only a few amino acid types are destroyed . The data presented here lend further support to the idea that H atoms are unusually effective. This may be due to either a high degree of selectivity for reaction at a few critical residues or the augmenting of the initial damage by intra-enzyme chain reactions (Lichtin, Ogdan and Stein 1973) .
REFERENCES ADAMS, G . E ., REDPATH, J . L., BISBY, R . H ., and CUNDALL, R . B ., 1973, ,. chem . Soc., Faraday Trans . 1, 69, 1608 . AULD, D . S ., and HOLMQUIST, B ., 1974, Biochemistry, 13, 4355 . BADIELLO, R ., QUINTILIANI, M ., and TAMBA, M ., 1974, Int .„ . Radiat. Biol ., 26, 311 . BARRA, D ., BOSSA, F ., CALABRESE, L ., ROTILIO, G ., ROBERTS, P . B ., and FIELDEN, E . M ., 1975, Biochem. biophys . Res . Commun ., 64, 1303 . MEE, L . K ., ADELSTEIN, S . J ., and STEIN, G ., 1972, Radiat . Res ., 52, 588 .
488
Correspondence
QviocHo, F . A ., and LIPSCOMB, W . N ., 1971, Advances in Protein Chemistry, edited by C . B . Anfinsen, Jr ., J . T . Edsall and F. M . Richards (New York : Academic Press), p. 1 . LICHTIN, N . N ., OCDAN, J ., and STEIN, G ., 1973, Radiat . Res ., 55, 69 . RIORDAN, J . F ., and VALLEE, B . L ., 1963, Biochemistry, 2, 1460 . ROBERTS, P . B ., 1973, Int . Y . Radiat . Biol ., 24, 143 . ROBERTS, P . B ., FIELDEN, E . M ., ROTILIO, G ., CALABRESE, L ., BANNISTER, J . V ., and
Int J Radiat Biol Downloaded from informahealthcare.com by Ohio State University Libraries on 12/03/14 For personal use only.
BANNISTER, W . H ., 1974, Radiat . Res ., 60, 441 . SNOKE, J . E ., and NEIJRATH, H ., 1949, Y . biol . Chem., 181, 789 . VALLEE, B . L., and RIORDAN, J . F ., 1968, Brookhaven Symp . Biol ., 21, 91 . WHITAKER, J . R ., MENGER, F ., and BENDER, M . L ., 1966, Biochemistry, 5, 386 .
