The interactions between dibromophenyl. chlorodifluoroethyl, benithiaiolvl and tnchlorovinyl cysteine conjugates (DBPC, CDFC,. BTC and TCVC respectively) ...
Biochemical SocietyTransactions ( 1 996) 24 Rationalisation of the C-S lyase activity of aspartate amino transferase. PAIJI. H Tlil:Sl~ALI:-SPI~rTl.F.,HARRIETJ. ADCOCK, LA~JRENCE H PA'II'IXSONand LORRAINED. B~JCKBERRY School of Applied Sciences. De Montfort University, Leicester, LEI OBH. U K L-Cvsteine-S-conjugates are responsible for the mutagenic and c\ totoxic consequences which occur following exposure to certain xenobioticsl I I Following enLyme mediated C-S lysis of the cc steine conjugate. equimolar amounts of pyruvate, ammonia and a thiolate species are formed (Fig 1) It has now been established that one of the physiological rdles of C-S lyase enzymes is that of transamination Enrvmes exhibiting C-S lyase activity include glutamine transaminase K. hynurenine aminotransferase [2,3], aspartate aminotransferase and alanine aminotransferase 141 The substrate/activity profile for aspartate aminotransferase (ASAT) has been previously reported 141, however this profile has never been rationalised We have rationalised the structure activit). profile of ASAT for C-S lysis is based on (I) Van der Waals overlap between the cysteine conjugate and the en/\ me active site (11) Ease of elimination of the thiolate RS The interactions between dibromophenyl. chlorodifluoroethyl, benithiaiolvl and tnchlorovinyl cysteine conjugates (DBPC, CDFC, BTC and TCVC respectively) and the active site of ASAT were determined The crystal-structure coordinates of chicken heart ASAT complexed with L-aspartate pyndoxal-5'-phosphate 151 were utilised In each case, atom coordinates of the enzyme were fixed, and the cysteine conjugate, assimilated into the structure as the pyndohimine, was unconstrained The complex was energy minimised using the CVFF forcefield 161 until a denvative of 0 01 Lcal/mol/A was achieved using conjugate gradients algonthm [7] Van der Waals overlap of the conjugate R group with nearby residues (typically Senne 296) was measured and the maximal value is reported in table 1 Enthalpies of conversion of the parent conjugate into a thiolate ion and partial charges on the conjugated sulphur and thiolate sulphur atoms were determined using the AM1 semi-empincal Hamiltonian [El The values are reported in table 1 enthalpies are relative to TCVC Clearly, neither the partial charge on the sulphur of the conjugate or the thiolate correlate with ASAT C-S lyase activity, however the relative enthalpies of formation of the thiolate species follows the activity well The sole exception to this being DBPC which might, on this information alone, be expected to be a better substrate than CDFC The observed order can be rationalised, however, as one of the bromine atoms of DBPC exhibits a significant s t e m clash with the side chain of Senne 296 Fig 1 The mechanism of C-S lvsis
P
U I1
HI
Ahhreviations used AS AT (aspartate aminotransferase). BTC (S-henzthiazolylI=cy steine).CDFC(S-chlorodifluoroethyl-L-cysteine),DBPC(S-d~hromophenylI.-cy\teine), TCVC (S-trichlorovinyl-L-cysteine)
141 S
Table 1 . Summary of molecular modelling data DBPC
CDFC
BTC
TCVC
Lyase activity of ASAT (nmoliminlmg protein)
((30
33 3 9 i 58
40 56* I67
40 56i 161
Relative enthalpy of conversion of conjugate to thiolate (kcallmol)
998
13 I
162
ooo
Partial charge on conjugate sulphur
0277
0 150
0324
0309
Partial charge on thiolate sulphur
-0 585
-0 859
-0 449
-0 5 15
Largest Van der Waals overlao
30%
8%
6 5%
11%
In conclusion, the C-S lyase activity of ASAT can be rationalised through consideration of molecular fit of the cysteine conjugate pyndoximine complex in the enzyme active site, and the stability of the thiolate produced on lysis. These observations can be used in future to determine the propensity of a cysteine conjugate to undergo C-S lysis, and can therefore be utilised in prediction of the formation cytotoxic species following exposure to a cysteine conjugable xenobiotic. W e wish to thank the Wellcome Foundation for financial support (HJA). Computational results were obtained using software programmes from Biosym Technologies of San Diego - molecular mechanics calculations were done with Dlscover(R) and graphically displayed in Insight II@ Semi-empirical MO calculations were done in MOPAC, interfaced to Insight II.
Shaw, P.N. & Blagborough, I S (1989) in Sulphur Containig Drugs And Related Organic Compounds (Damani, L.A., ed). vol. 2B, pp. 135-155, Ellis Honvood Ltd, Chichester. Buckberry. L.D., Blagborough, I.S., Bycroft, B.W. & Shaw, P.N. (1990), Toxicol. Lett., 53, 253-255. Buckbeny, L.D., Blagborough, I.S., Bycroft, B W & Shaw, P.N. (1992), Toxicol L e t t , 60, 241- 246. Gaskin, P.J., Adcock, H.J., Buckbeny, L.D., Teesdale-Spittle, P.H. & Shaw, P.N. (1995) Human Exptl Toxicol, 14, 422-427. Malashkevich, V.N., Toney, M.D., & Jansonius, J.N (1994), Biochemistry, 32, 1345 1 . Dauber-Osguthorpe, P., Roberts, V.A., Osguthorpe, D.J., Wolff, J., Genest, M. & Hagler, A.T. (1988), Proteins: Structure, Functions and Genetics, 4, 82-87. Fletcher, R., (1980) Practical Methods of Optimisation, Vol. 1 , Unconstrained Optimisation, John Wiley and Sons, New York. Dewar, M.J.S (1985), J. Am. Chem. SOC.,107, 3902-3909.
