Arch Microbiol (1988) 149: 198-206 © Springcr-Vcrlag 1988 Bacterial metabolism of side chain fluorinated aromatics: cometabolism of 4-trifluoromethyl(TFM)-benzoate by 4-isopropylbenzoate grown Pseudomonas putida JT strains * Karl H. Engesser l, Miguel A_ Rubio 2, a nd Douglas W_ RibbonsJ I Institut fUr Mikrobiologie def UniversitiH Stuttgart, 0-7000 Stuttgart I, Fedcral Republic of Gcnnany 1 AbteilungfUr Gewasserreinigungstcchnik, Technisehe Universitat Hamburg-Harburg, D-2100 Hamburg9Q, Federal Rt publicofGcnnany 3 Centre for Biotechnology, Imperial College of Science and Technology, London SW72AZ, UK Abstract_ Enzymes of the p-cymene pathway in Pseudomonas pulida strains eometabolized the intennediate analogue 4-trifluoromethyl(TFM)bcnzoate_ Three products, 4-TFM- 2,3-dih ydro-2,3-dih ydroxybcnzoa te, 4-TFM -2,3-dihydroxy- benzoate and 2-hydroxy-6-oxo-7, 7, 7-trifluorohepla-2,4- dienoate (7-TFHOD) were identified chemically and by spectroscopic properties. Certain TFM-substituted analogue metabolites of the p-cymcne pathway were transfonned at drastically reduced rates. Hammett type analysis of ring cleavage reactions of 4-substituted 2,3-dihydroxybenzoates revealed the negative inductive and especially mesomeric effect of substituents to be rate detennining. Whcreas decarboxylation of3-carboxy- 7-TFHOD was not affected by fluorine substitution the subsequent hydrolysis of 7-TFHOD proceeded very slowly. The negative inductive effect of thc TFM-group probably inhibited heterolysis of the carbon bond between Cs and C6 of7-TFHOD. Key words: Cymene pathway - Side chain fluo rinated aromatics - Cometabolism - Pseudomonas putida JT - 2-Hydroxy-6-oxohepta-2,4-dienoatc hydrolase (2.hydroxy- mueonic semialdehyde hydrolase) The metabolism of fluorinated benzoates by bacteria has been thoroughly investigated (Harper and Blakley 1971 ; Smith et al. 1968; Ali ct al. 1962; Milne el al. 1968; Husain et al. 1980; Clarke ct al.1975 ; Goldman et al. 1967). Side chain fluorinated aromatics the commonest of which are trifluoromethyl-substitutcd, however, have been studied to a much lesser extent (DeFrank and Ribbons 1976). In the preceeding paper (Engesser et al. 1988) evidence was pre- Abbreviations: DIIB, 1,2-Dibydroxy-2-hydrobenzoatc; DHC, 2,3- Dihydro-2,3-rlibyrlroxybenzoatc. this compound was lenncQ DHC simply to distinguish it from the similar 1,2·dihydroxy-2-hydro· benzoate (DHB) as described in the precccding paper (Engesser et al. 1988); HMS, 2-Hydroxymuconic semialdcbyde; HOD, 2· Hydroxy-6-oxohepta-2,4-dienoate; 7-TFHOO, 2-Hydroxy-6-oxo- 7,7,7-lrifluorohepta-2,4--dienoatc ; TFM , TrinuoromethyJ • This work was supported, in part, by the Gcsellsehaft fUr StrahleD- und Umwcltforschung, NeuhcrbcrgfMiinchen, FRG Ojjprint requests 10: K. H. Engesser scnted that side chain fluorination of methylbcnzoate severely impeded its catabolism in Pseudomonas pUti(la mt·2 and Rhodococcus rubroperlinclus N657. Neithcr of the three isomeric trifluoromethyl (TFM) substitu ted benzoatescould serve as a growth subslrate for these strains. With the aim of broadening our knowledge concerning the bacterial ca- tabolism of side chain fluorinated aromatics it seemed therefore worthwhile to examine their utilization by p-cumate (4-isopropylbenzoate) degrading organisms. 4-Isopropylbcnzoate is dissimilated via a pathway (Fig. 1) in Pseudomonas pUlida JT strains involving 2,3-dihydroxyla- tion of the benzoate nucleus compared with 1,2-dioxygena- tion of 3-methylbenzoate coded by TOL-plasmid bearing strains (DeFrank and Ribbons 1977a). Both pathways, how- ever, converge at the stage of the hydrolase substrate (DeFrank and Ribbons 1977b). Thereforc it was of interest to examine the specificity of the p-cymene pathway cnzymes towards trifluoromethyl substituted substrate analogues, of which 4-trifluoromethyl- benzoate had becn shown to be metabolized to a 2,3-dihy- drodiol by a mutant of Pseudomonas putida (DeFrank and Ribbons 1976). Materials and methods Chemicals. The isomeric lrifluoromethylbcnzoates were obtained from Bayer AG, Leverkusen, FRO. 4- lsopropyl- benzoate (p-cumate) was produced from 4-isopropylben7.al- dehyde (Meyer 1883). 177 g 4-1sopropylbenzaldehyde and 200 g NaOH were mixed with 700 ml HzO. 175 g KMn04 in 4 I HIO were added stepwise. The solution was stirred at room tempera ture until the reaction was finished (monitored by HPLC). Excess of permanganate was destroyed with methanol. After filtration with a Buchner funnel the product was precipitated with sulfuric acid and recrystallized from water/ethano1. The melting point was 11 9"C (Lit. 118°C). The yield was 45% of starting material. All other benzoates were bought from Aldrich Chemical Company, Steinheim, FRO. 4-Isopropyl-2,3-dihydro-2,3- dihydroxybenzoate (4-isopropyl-DHC) was produced from 4-isopropylbenzoa te employing the mutant Pseudomonas putida JT 832 (DeFrank and Ribbons 1976, 1977a; Wigmore and Ribbons 1980). The isolated product still contained about 4% (by weight) 4-isopropyl-3-hydroxybenzoate. This latter compound was accessible in pure fonn after acid catalyzed rcaromatization of 4-isopropyl-DHC. Rrvalue 6 ---¢ "'I "- • Q ~r " 0' 1 "'" - I "", =0 =0 ~ R-COOH - - - "'0' ~ , ,~, ¢r: H.C-~HO H,C-CO-COOH Fig. I. Pathway of p-isopropylbem.o8te (cumate) and p-ethyl- benzoate (R = isopropyl and ethyl rsp_) degradat ion in PseudQrnQn(l.)-pUlido JTlO] and JT8t 1 with solvent system [ on preparative TLC plates was 0.69. There was no fluorescence at ). = 366 nm i.c. no simulta- neously produced 4-isopropyl-2-hydroxybenzoate. 4-Tri- fluo romcthyl-2,3-dihydro-2,3-dihydroxybcnzoalc was pro- duced with the mutanl Pseudomonas putida lrl07. The sodium salt of this compound was purified by repea led crystallization (water/propanol). The rearomatization prod- uct 4-trifluoromethyl-3-hydroxybcnzoate CRr-value on TLC plates with solvent system I was 0.72) was not detected in the sample. 4-Methyl-2,3-dihydroxybenzoate was prepared accord- ing to a procedure described for the preparation of re- soreinylic acid (Wcsscly et al. 1950) . On TLC plates a major impurity with a Rrvalue of 0.52 (solvent system [) was separated from the product (Rrvalue = 0.79). After further purification on TLC plates (solvent system trichloro- methane/methyl ethyl ketone/methanol ; 60/26/40 by vol.) tbe parental ion at m/e = 168 could be observed in a mass spectrum. For characterization purposes 4-methyl-2,3- dihydroxybenzoate had to be produced also biologically using the mutant Pseudomonas pUlida JT833 but the turn- over of 4-methylhenzoate by this strain was incomplete. TLC purification (trichloromethane/methanol/acetic acid ; 9/3/ 1 by vol.) and sublimation of the product yielded pure sample. In a mass spectrum a parent iOn of mle = 168 was observed with fu rther fragmentations being at m/e = 150 (loss of water) and mle = 122 (additional loss of CO). In a 100 MHz NM R spectrum resonances at b = 2.26 ppm (3 H, s) b = 6.7 (I H , d, J 8.4 H z) and 7.3 (I H, d, J 8.4 Hz) were observed consistent with a methyl group and two vicinal protons respectively. 4-l sopropyl-2,3-dihydroxybenzoate was produced bio- logically with the mutant Pseudomonas pUlido JT833. After complete turnover of 4-isopropylbenzoate, the product was sublimed immediately after extraction and evaporation of solvcnt . There were no impurities as judged by TLC and 199 Table I. Strains of Pseudomonas pulido used Strain Genotype Phenotype Rererence l T1 01 (for- Cym +,Cum + DeFrank and merly PL.W) Ribbons 1977a JTt04 Gym K-71 Cym- .Cum - Defrank and Ribbons 1971b lTI07 Gym 1·51 Cym - , Cum - DeFrank and Ribbons 1976, 1977a 1T811 Gym R-l Cym+, Wigmore and Cum', c Ribbons 1980 JT832 Cym R-I , Gym /-3 Cym-, Wigmore and Cum - , C Ribbons 1980 1T833 Cym R-J , Cym 1-4 Cym - , Wigmorcand Cum - , c Ribbons 1980 C Constitutive HPLC. In the mass spectrum a parental ion of m/e = 196 was observed with further fragmentation being at mJe = 178 (loss of H 20) and mle = 150 (additional loss of CO). The most abundant peak at mle = 163 results from loss of H20 and methyl radical. The NMR spectrum gave reso- nances at b = 1.25 ppm (6 H, d), b = 3.4 ppm (1 H, m), (j = 6.8 (1 H, d, J 8.5 Hz) and (j = 7.4 (1 H, d, J 8.5 Hz) which are consistent with an isopropyl substituent and two adjaccnI aromatic protons. For characterization and preparation of 3-trifluoromethyl(TFM)-catcchol, 2-hyd roxy-6-oxo-hepta- 2, 4-dienoa te, 2-h ydroxy-7 -meth yl-6-oxo-octa-2,4-dienoa te and 2-hydroxy-6-oxo-7, 7, 7-trifluoro-hepla-2,4-dienoate (7- TFHOD) sec preceeding paper (Engesser et al. 1988). Chemicals for media were of the highest grade com- merciallyavailable. Organisms. Strains descriptions arc given in Table 1. For description of growth conditions, chromalog- raphical and spectr oscopical methods sec precceding paper (Engesser ct al. 1988). Isola/ion and derivatizDtirm of metaboliles. Extraction pro- cedures were described previously (Hartmann et al. 1979). Methylesters were prepared using N-methyl-N-nitroso-p- tolucnesulfonam ide(DeBoer and Backer 1954). lfnecessary, the reaction was followed by analytical TLC. J:.nzyme assays. P-Cumate dioxygenase was assayed using whole cells (Reineke and Knackmuss 1978a). 2,3-Dihydro- 2,3-dihydroxybenzoate(DH C)-dehydrogcnase was mea- sured according to previously published procedures (Rei- neke and Knackmuss 1978b) with the exception that 50 mM phosphate butTer (PH 7.4) was used. The exact concentra- tions of dienediol salts were estima ted by calculating the amount of NaDH at J. = 366 nm produced after complete substrate turnover using a crude extract ofthe mutant JT833 dialyzed (15 h) against 50 mM phosphate butTer (PH 7.4). 2,3-Dihydroxybenzoate-3,4-dioxygenase was assayed ac- cording to literature (DeFrank and Ribbons 1977b) using the mutant strain JT I04, which prevents further metabolism of products. The extinction coefficient uSL-d was e = 21.600 fo r 3-carbox y-2 -h ydroxy-7 -methyl-6-oxo-ocla-2,4-dienoa teo Tbe corresponding )'mu value was 345 nm . 3 - Carboxy - 2 - hydroxy -7 -met h yl- 6-oxo -octa -2,4 -dicnoa te- decarboxylase 200 was assayed as described (DcFranc and Ribbons 1977b). For extinction coefficients sec the test for 2,3-dihydroxy- benzoate-3,4-dio)l;ygenase. 2-Hydroxy-6-oxo-hepta-2,4-di- enoate(HOD)-hydrolase was measured by following the de- crease of absorption at respective )'mu values of substituted HOD analogues (DeFrank and Ribbons 1977b). The crude extracts from JT1 01 and Jrl 04 were dialyzed against 50 mM phosphate buffer (PH 7.4). For extinction coefficients see also proceeding paper (Engesser e l al. 1988). Results Production and identification of analogue metabolites As in the case of Pseudomonas putida ml-2 (Engesser ct al. 1988) the trifluorometbyl(TFM)-benzoatcs did not serve as growth substrates for the p-cumate utilizing Pseudomonas pUlida JT strains. In contrast to Pseudomonas putida mt-2, however, induction of the alkylbenzoatc degrading enzymes by 4-TFM-benzoatc could be observed (De Frank and Ribbons 1976, 1977a, b). Resting cells of p-cumate (4-isopropylbell2oate) grown Pseudomonas putjda JTI01 showed only low turnover rates for TFM-benzoates. Using exponentially growing cells, however, moderate transformation of 4-TFM-benzoate occurred, whereas the 2- and the 3-isomere were attacked only very slowly. The turnover of 4-TFM-benzoate had al- ready been demonstrated (De Frank and Ribbons 1976) with cells of Pseudomonas putjdaJTI01 growing with glucose and incubated with this side chain fluorinated analogue during exponential growth phase. When a cell suspension (cumate grown) of high optical density (OD~46 = 15) was divided and incubated with p-cumate (3.3 mM) as control and a mixture of p-cumate and 4-TFM-benzoatc (3.3 mM each), no influence of the latter substrate on turnover rate of p-cumate could be ob- served . This demonstrates the poor binding of the side chain fluorinated substrate 4-TFM-benzoate to the cumate dioxy- genase. Glucose-grown cells of Pseudomonas purida JTI01 induced for cumate degrading enzymes transformed 4-TFM- benzoate incompletely. TLC analysis (solvcnt systcm I) of thc culture supcrnatant revealed the presence ofthreemetab- olites. The most polar metabolite Jl had an Rrvalue of 0.29. A yellow metabolite 12 migrated with an Rr-value of 0.37 to 0.4, and metabolite n was located at Rrvalue of 0.73. The structure of metabolite 11 could be assigned most easily using a mutant of Pseudomonas pUlida JTI01 , strain JT107 which accumulates the dihydrodiols from 4-substituted benzoates (DeFrank and Ribbons 1976, 1977a). Using HPLC, metabolite 11 eluted with the biologically prepared 4 - trifluoromethyl- 2,3 -dihydro. 2,3 -dihydroxybenzoic acid (retention volumes with solvent system AI +25% Bll: 3.3 ml and 100% AI: 16.5 mI, see Materials and methods). Maximum absorption measured in situ was found for both compounds to be A. = 275 mm (phosphate buffer, 50 mM, pH 7.5). Metabolite 11 as well as the reference dihydrodiol from the mutant JTI07 showed the same chemical re- aromatization characteristics and could be hydrolyzed subsequently to hydroxyterepbtbalic acid. This can be ex- pected to happen with trinuoromethyl-substituted aromatic compounds bearing at least one ortho-hydroxyl group (Engesser et al. .1988). According to a previously made sug- gestion (DeFrank and Ribbons 1976) 3-hydroxy-4-TFM- benzoate was deduced to be the correct structural assign- OH HOOCCCOOH ~ c=o I CF, T co, OH t eooH ~ c=o I CF, Fig. 2a, b. Possible species of TFM-pieolinie acids produced by eyclization of 2-hydroxY-6-oxo-7,7, 7-trifluoro-hepta -2,3-dienoic acid (b) or its 3-carhoxy derivative (a). For conditions of eyelization in aqueous solutions of ammonia see Materials and methods ment instead of the 2-hydroxy isomere. This was based on the failure to detect any fluorescence of rcaromatization products of 4-TFM -2,3-dihydro-2,3-dihydroxybenzoates. Spectroscopic characteristics in acidic and basic solution suggested metabolite 12 to have a mueonic acid semial- dehyde chromophore. According to Fig. 1 and Fig. 2 two alternative structures had to be considered for the com- pound J2. Aftcr eyelization in the presence of ammonia, J2 was purified on TLC plates and methylated (see Materials and methods). One of the possible pyridine derivatives, 6-TFM-2,3-dicarboxypyridine would be likely to loose the carboxyl group attached to C-2 as was found for 2,3- dicarboxypyridine (Bialek 1962), yielding a nicotinic acid derivative. So even after decarboxylation of 6-TFM-2,3- dicarboxylic acid the resUlting 6-TFM-3-carboxypyridine would be clearly distinguishable from directly generated 6-TFM -2 -car box ypyridine. HPLC analysis using an authentic probe (Engesser ct al. 1988) clearly identified the derivatized metabolite 12 to be the methylcster of 6-TFM-2-carboxypyridine. (l",u mea- sured in situ at a solvent mixture of AI/40% BII were in each case 213 and 261 nm, l.m;~ was 236 nm). Metabolite 12 therefore is not attributable to a 3-carboxy-derivative of 2-hydroxy-6-oxo-7, 7, 7-trifluoro-hepta-2,4-dienoate (7- TFHOD) but to the decarboxylated intermediate 7-TFHOD itself. Analysis of product excretion patterns of mutants of the cymene pathway gave first hints concerning the structure of metabolite n. Firstly, a mutant defective in dihydrodiol dehydrogenase (PpJT 832) did not produce metabolite n during incubation with a mixture of glucose and 4-TFM- benzoate. Secondly, a mutant unable to metaboli7.e the ring fission product of 2,3-dihydroxybenzoates, strain JT104 accumulated metabolite n showing the same time depend- ence of excretion as the wild type. Finally, UV-absorption maxima were similar to 4-TFM-3-hydroxybenzoate (data not shown). These preliminary results strongly suggested metabolite n to have the structure 4-TFM-2,3-dihydroxy- benzoate. To obtain sufficient amounts of metabolite 13 for spec- troscopic characterization, a mutant was used (ppJT833) which is blocked in the step of ring cleavage of 2,3-dihy- droxybenzoates. it excreted metabolite J3 with intermediate formation of 4-TFM-2,3-dihydro-2,3-dihydroxybcnzoate. ,o~ " 8(} lQr: • 0 , co. ~ 6(} c , D • • 4(} ., ~ 1 0 • 20 .. .. 100 156 126 16' l 150 mi. o " ¢r: '" m/e-t84 204 200 M' 222 Fig. 3. Mass spectrum of 4-TFM-2,3-dihydroxybcnzoate, the metabolite J3 of cometabolism of 4-TFM-bcnzoatc by Pseudomf)nas putida JTIOI. For interpretation of the fragmentatio n pattern sec lext. Loss of water a nd hydrogen fluoride by migration of hydrogen via cyclic intermc The relative maximum lumover rales were detennincd a t saturating substrate t"QnccnLratlons (tOO ~M). They are ex:p resscd as percentages of that for 6.isopropyl-SA band 6-methyl-SA · taken as 100% in eaeh casc. K ... ·values were derived from Lilleweaver- Burk: plots. XI·values were calculated from replots of the slopes of lines representing d ifferent inhibi to r conccmrations against the corresponding inhibitor concentration o Concentration of 6-isopropyl-SA was varied d Concentration of 6-melhyl-S!\ was varied Discussion The detection of aromatics carrying the trifluoromcthyl (fFM) group as substituents in river wa ter (Lombardo 1979; Jungclaus ct al. 1978; Maier et al. 1978) promoted us to investigate the effects of side chain fluo rination on bac- terial metabolism 01" aromatics using toluate degrading bac- teria as model organisms (sec precceding paper, Engesser et ,1. (1988). Dehydrogenation of TFM substituted 1,2-dibydroxy-2- hydro·bell7.oatcs and hydrolysis of 2-hydroxy--6-oxo-7 ,7,7- trifl uoro·hepta-2,4-dienoate proved to be limiting reactions in PseudomontJ.f pUlido mt-2. Ortho-fission of 3-TFM- catechol was fac ile in the fonner strain but was critically restricted down in RhotWcoccu.s rubroperlinclUs N657. The innucncc of TFM groups on subst.rate binding of side chain nuorina led analogs vaned considerably dependi ng on the respective enzyme of the toluate pathway. In the present study similar observations were made using p·isopropylbcnzoate (p-cumate) aod p-ethylbcnzoatc dc- grading P.seudomonas pUlido JT strains as model orga nisms. With the exception of the decarboxylase (Fig. 1) all enzymes investiga ted showed decreased turnover ratcs with sub-- strates carrying a T FM·group instead of isopropyl. As in the case of Pseudomonas pulido mt-2, the initial enzyme, a non specific benzoate.2,3-dioxygenase shows some stenc constraints masking electronic influences of sub- stituents on reaction rates. Only 4-substitured substrates are dioxygenated with relative rates which are predicted by a Hammett·plot (Table 3). The low sensiti vity factor derived from the Hammett-plot indicates a modest dependence only of the dioxygenation rate from thc electron withdrawi ng character of the TFM·group. Accordi ngly, 4-TFM·benzoate 203 ~1:OHO_HE ~C:IOH HE ~ H ~ H R4 O~ eaOH R4 0'0 .3 '~O/ ·3 \ OH 0=-.) ~e:aH;y R. ~ ~r.¢VC:OH HE I H 3 I OH " R.. 0 R.. ~ R3 '0, c. , R3 0 ~ a o 4 5 -0.3 o Q3 0.6 substit uent constant S Fig. 6. Correlation betwccn 19 Vm ... -values of the 2,3-dihydroxy- bcnzoate-3,4-dioxygenasc reaction in Pseudomonas pu/ida lT104 with 4-substituted 2,3-dihydroxybenroates and the corresponding substituent constants. The V", •• -values are listed in Table 2, except of values for R = Hand R = OCH, which. are derived from literature (DeFrank and Ribbons 1977 b). The substituent constants have been published (Norman and Taylor \965); I 2,3- dihydroxybcnwate; 2 4-methyl-2,3-dihydroxybenzoate; 3 4-iso- propyl-2,3-dihydroxybenzoate; 4 4-methoxy-2,3-dihydroxy-bcn- 7J)ate; 5 4-trinuoromclhyl-2.3-dihydroxybcmwate (Bar tels et al . 1984). T he deactivating effect of electro- negative subslituents adjacent to the site of oxygen attak to catechols therefore seems to be a gcneral phenomenon for metapyrocatechases. The ring cleavage product of 4-TFM-2,3-dihydroxy- benzoate is subject to spontaneous decarboxylation yielding 2-hydroxy-6-oxo-7, 7, 7-trifluoro-hepta-2,4-dienoic acid (7- TFHOD). (This decarboxylation reaction seems to proceed fast enough even without dccarboxylase enzyme indicating a non cnzymatic reaction.) Obviously this non-enzymatic reaction is not hampered by substituents like fl uorine. The hydrolysis of 7-TFHOD would yield 2-hydroxy- pcnla-2,4-dienoate and trifluoroacetic acid. In Pseudomonas pUlida mt-2 (Engesser et al. 1988) as well as in Pseudomonas putida JT101 the 7-TFHOD shows a high affinity to HOD hydrolase (Table 4). T he turnover rates, however, are very low. To explain this phenomenon a reaction mechanism is prcsented (Fig. 5) which is bascd essentially on litera ture data (Shaw et a1. 1965; Duggieby and Williams 1986; Duggleby 1979). Accordingly an isomerization step is thought to precede a hydration reaction which is fo llowed by heterolysis of the bond between carbon atom 5 and 6. Similar electron shifts have been proposed for processing othcr mcta-ring cleavage products (Dagley 1978). The in- fluence of the TFM-group on the nucleophilic attack on the carbonyl can be estimated from thc well known tendency of carbonylic compounds, substituted wilh electronegative substituents, to form hydrates. Conversely electron-rich sub- stilucnts favour the non hydrated species. The equilibrium constants of the hydration reaction decrease in the series formic aldehyde, acetaldehyde and aeeton (K = 2 . 10+ 3 , 1.4 and 2 . 10 - 3 rsp.). Correspondingly tril1uoroaceta ldehyde- hydrate is so stable that it can not be dehydrated by a simple destillation (Braendlin and McBee 1963, Husted and Ahlbreeht 1952). It seems reasonable therefore, that the nucleophilic addition of XH (x = OH or an enzyme nucleophile) to the TFM group bearing carbon atom should be enhanced by the lattcr function. Conversely, however, the heterolysis of the carbon C~-C6 then may be severely re tarded (Fig. 5) by electronegative substituents. For Ihe HOD-hydrolase from PseudomonlL~ putida mt-2 the lurnover rates of 6-methyl-HMS are lOOO-fold greater than of 6-TFM-HMS even though the Km-value of the latter substra te is about 25-fo ld lower than that of the former, Since the proposed nucleophilic attack ofC6 in the 6-TFM- HMS should be facili tated, the dramatic decrease in turn- over rate may be due 10 inhibition of the heterolysis of the C~-C" bond by the electron withdr)lwing ~ll h~lill 1en l (Fig. S)_ Other subslituents e.g. hydrogen, ethyl, alkyl, n-propyl and isopropyl have similar Km- or Kj-values to 6-methyl-HMS, but they also show diminished turnover rates (Duggleby and Williams 1986; Table 5). This implies that the size of the 6-substituen t can also affect the heterolysis of the CS-C6 bond, This hypotheses are difficult to test, as substitution of the TFM-group as a probe for electronegative substituenlS by others like chlorine or fluorine directly attacked to the carbonyl-carbon would create acylhalides. 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