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Enzyme-Induced Inactivation of Transaminases by Acetylenic and Vinyl Analogues of 4-Aminobutyrate by ROBERT A

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Enzyme-Induced Inactivation of Transaminases by Acetylenic and Vinyl Analogues of 4-Aminobutyrate by ROBERT A Biochem. J. (1979) 177,721-728 721 Printed in Great Britain Enzyme-Induced Inactivation of Transaminases by Acetylenic and Vinyl Analogues of 4-Aminobutyrate By ROBERT A. JOHN,* EIRIAN D. JONES* and LESLIE J. FOWLERt *Department ofBiochemistry, University College, P.O. Box 78, Cardiff CF1 1XL, Wales, U.K., and tDepartment ofPharmacology, School ofPharmacy, Brunswick Square, London WC1N lAX, U.K. (Received 25 August 1978) The reactions of two analogues of 4-aminobutyrate, namely 4-aminohex-5-ynoate and 4-aminohex-5-enoate, with three transaminases were studied. Three pure enzymes were used, aminobutyrate transaminase (EC 2.6.1.19), ornithine transaminase (EC 2.6.1.13) and aspartate transaminase (EC 2.6.1.1), and the course of the reactions was studied by observing changes in the absorption spectrum of the bound coenzyme and by observing loss of activity. All of the enzymes were inactivated by either inhibitor, but amino- hexenoate showed a marked specificity for aminobutyrate transaminase. Aminohexynoate was most potent towards ornithine transaminase, and with this enzyme transamination of the inhibitor is an important factor in protecting the enzyme. Most of the reactions could be analysed as first order, with the observed rate constant showing a hyperbolic dependence on inhibitor concentration. Ideally compounds intended to act as drugs by the resulting aminoacrylic intermediate reacts with specific inhibition of a particular metabolic step lysine-258 and cysteine-390 of the enzyme (John et should show an absolute selectivity towards the al., 1973). However, in addition there is a slow trans- target enzyme. One means of approaching this ideal amination that protects the enzyme from further in- is to prepare a compound that not only bears a activation, because the pyridoxamine form of the strong structural resemblance to the substrate, but enzyme, although fully active in the normal trans- also becomes reactive as an irreversible inhibitor only amination reaction, cannot combine productively as a result of the operation of the enzyme's normal with amino acids. If inhibition is investigated without mechanism. The pharmacological potential of such regard to the possibility of a protecting transamina- enzyme-induced irreversible inhibition has been tion it could be wrongly concluded that a compound pointed out (Fasella & John, 1969; Rando, 1974a) does not inhibit a particular enzyme. Furthermore, and inhibitors of this type have been referred to as the goal of very high specificity, which the enzyme- 'k,2,. inhibitors' (Rando, 1974a) and 'suicide enzyme induced type of inhibition might be expected to inactivators' (Abeles & Maycock, 1976). approach, may be made less easily attainable because Several inhibitors ofthis type have been synthesized of the existence of families of enzymes that share the with the intention of selectively inhibiting 4-amino- same catalytic mechanism. butyrate transaminase (EC 2.6.1.19) (Fowler & John, Two compounds, namely 4-aminohex-5-ynoic acid 1972; Jung & Metcalf, 1975; Lippert et al., 1977). and 4-aminohex-5-enoic acid, which might be con- The inhibition ofthis enzyme by a naturally occurring sidered as acetylenic and vinyl derivatives of 4- compound, gabaculine (5-amino-1,3-cyclohexadienyl aminobutyrate, have been prepared as enzyme- carboxylic acid), is also by an enzyme-induced induced inhibitors of aminobutyrate transaminase mechanism (Rando & Bangerter, 1977). Amino- (Jung & Metcalf, 1975; Lippert et al., 1977). They butyrate transaminase has received attention because inactivate the enzyme, and the mechanism proposed its substrate is an inhibitory neurotransmitter, and is one suggested for the inactivation of aspartate therefore its selective inhibition is pharmacologically transaminase by 2-aminobut-3-enoic acid (Rando, interesting. 1974b), in which the enzyme induces reactivity by The reactions of pyridoxal phospha-e-dependent introducing conjugation of double bonds. The enzymes with substrate analogues of this kind may inhibitors were also shown, by measuring rates of loss be complex, as is shown by the reaction of the gluta- of activity, to be relatively inert towards impure mate analogue, serine sulphate, with aspartate trans- preparations of some other pyridoxal enzymes. aminase (John & Fasella, 1969). In this case the The present paper is an account of investigations enzyme catalyses the elimination of sulphate from into the reactions of these compounds with some the f4-position, and there is evidence indicating that transaminases. The enzymes used were all purified Vol. 177 722 R. A. JOHN, E. D. JONES AND L. J. FOWLER to homogeneity and the study depends heavily on Determination ofkinetic constants observation of changes in the absorption spectrum When, the observed first-order rate coenzyme. As well as aminobutyrate graphically, of the bound to aspartate transaminase (EC 2.6.1.1) constants for inhibition (kobs.) were found vary transaminase, hyperbolically with inhibitor concentration, the and ornithine transaminase (EC 2.6.1.13) were of studied, the last particularly because, like amino- results were taken to indicate the initial formation transaminase, it catalyses transamination of significant amounts of a rapidly reversible complex butyrate The dissociation constant, K, for complex formation, groups on carbon atoms that bear no carboxy amino and the first-order rate constant, k, for irreversible group. inhibition were estimated using a weighted least- squares linear-regression analysis. Simple errors in kObs. were assumed and weights of ko2bs.I[1]2 were Experimental used. The method used was that described by Enzymes Wilkinson (1961) except that kobs. was substituted for v, k for Vmax., [I] for [S] and K for Km. The data Experiments with aspartate transaminase were were handled by a Hewlett-Packard 9820 A pro- carried out with the pure x-subform of the pig heart grammable calculator. cytoplasmic enzyme prepared by the method of Martinez-Carrion et al. (1967). Ornithine trans- Results and Discussion aminase was prepared from rat liver by the method of Peraino et al. (1969) and 4-aminobutyrate trans- The results of the experiments on all three enzymes aminase from rabbit brain by the method of John & and with both substrates will be interpreted in terms of Fowler (1976). Enzyme concentrations are expressed Schemes 1(a) and l(b) which are based on the classi- as concentration of bound pyridoxal phosphate and cal Snell-Braunstein mechanism for transamination were determined by using E280=7x 104 M-l-cm-l for (Braunstein, 1964; Guirard & Snell, 1964). Inactiva- aspartate transaminase (Birchmeier et al., 1973), tion will be presumed to occur as a result of reaction 6412= 5.2 x 103 M-1I cm-' for ornithine transaminase of a nucleophile in the enzyme with either or perhaps (G. M. Bridge & R. A. John, unpublished work) and both of the reactive intermediates El' and EKI'. 6415=1.2 x 104 M-1 cm-l for aminobutyrate trans- Several plausible routes are possible all leading aminase (L. J. Fowler & R. A. John, unpublished effectively to the same result, namely covalent binding work). The concentration of the last enzyme was also of the enzyme-induced inhibitor to the enzyme determined by titration with amino-oxyacetate (John protein. et al., 1978). Reactions ofaminobutyrate transaminase Chemicals The changes in coenzyme absorption spectrum that are seen when aminobutyrate transaminase The inhibitors 4-aminohex-5-ynoic acid and 4- reacts with aminohexynoate occur in two phases and aminohex-5-enoic acid were a gift from Merrell the rates of each phase are dependent on inhibitor International Research Centre, Strasbourg, France. concentration. Fig. 1 shows the changes that occur Tris (Puriss grade) and 2-oxoglutaric acid were from when the enzyme is treated with 0.63 mM-amino- Koch-Light Laboratories, Colnbrook, Bucks., U.K. hexynoate. Distinct changes are seen at three wave- Aspartic acid, ornithine and 4-aminobutyrate were lengths. Initially a rapid decrease at 412nm is from Sigma (London) Chemical Co., London SW6, accompanied by rapid increases at 330 and 550nm. U.K. Other chemicals used were supplied by BDH Thereafter a slow decrease at both 412 and 550nm is Chemicals, Poole, Dorset, U.K. accompanied by a further rise at 330nm. The course of these reactions followed at 412 and 550nm is Enzyme activity assays shown in Fig. 2. The slow process is first order, with the same rate constant that is observed when loss of Aspartate transaminase was assayed by the method enzyme activity is followed. This rate constant, of Karmen (1955), ornithine transaminase by the measured by activity loss, showed a hyperbolic method of Peraino & Pitot (1963) and aminobutyrate dependence on inhibitor concentration. The constants transaminase by the method of Salvador & Albers were estimated by measuring loss of activity (at 37°C (1959). in Tris/acetate pH 8.3, [acetate]=50mM) and are given in Table 1. Absorption spectra Both the rate constant (kF) and the amplitude of the fast change increased with inhibitor concentra- Absorption spectra were determined on a Beckman tion. One determination only was made at each of model 25 recording spectrophotometer. three inhibitor concentrations. The values obtained 1979 ENZYME-INDUCED INACTIVATION 723 CH CH "CH CH III III III III C C CH Lys C C 11 III III H N + H N-C-R H N=C-R H C H2N-C-R \ C H C H C H-C-N CH2NH2 +/C-R /1 0 -H+ + H20 -H' N N N N H+ N''H+ H H+ H+ EL ELI El' EKI' EM +H] [-H+ CH2 11 C11 (a) H N-C-R C NI- H+ EII CH2 7CH2 CH2 11 11 CH2 CH2 LCYS CH CH CH CH 11 H N + H2N-C-R H N-C-R H N=C-R SC-RK CH \t \ / a C H C H C H-C-N CH2NH2 + C-R +H+ +H20 II -H+ t -H t _ NZN' H+ H+ H H+ H+ EL ELI El' EKI' EM +H+] -H+ CH3 CH 11 H N-C-R (b) N' Ell Scheme 1.
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