FISHERIES AND MARINE SERVICE. Translation Series No. 3868

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1 1ARCIIIVES FISHERIES AND MARINE SERVICE Translation Series No Structural analogues of pyruvate. or inhibitors of LDH Behaviour as substrates by C. Lluis and J. Bozal OrIginal title: Analogos estructurales del piruvato. Comportamiento como substratos o inhibidores de la LDH From: Rev. Esp. Fisiol. 32: 9-13, 1976i Translated by the Translation Bureau(DC) Multilingual Services Division Department of the Secretary of State of Canada Department of the Environment Fisheries and Marine Service Halifax Laboratory Halifax, N.S pages typescript

2 d P., I; DEPARTMENT OF THE SECRETARY OF STATE TRANSLATION BliREAU. MULTILINGUAL SERVICES DIVISION reitbi e er'a CANADA SECRÉTARIAT D'ÉTAT BUREAU DES TRADUCTIONS DIVISION DES SERVICES MULTILINGUES TRANSLATED FROM - TRADUCTION DE Spanish AUTHOR - AUTEUR C. Lluis and J. Bozal INTO - EN English TITLE IN ENGLISH - TITRE ANGLAIS Structural Analogues of Pyruvate. Behaviour as Substrates or Inhibitors of LDH. TITLE IN FOREIGN LANGUAGE (TRANSLITERATE FOREIGN CHARACTERS) TITRE EN LANGUE ÉTRANGÉRE (TRANSCRIRE EN CARACTÉRES ROMAINS) Anàlogos estructurales del piruvato. Comportamiento como substratos o inhibidores de la LDH. REFERENCE IN FOREIGN LANGUAGE (NAME OF BOOK OR PUBLICATION) IN FULL. TRANSLITERATE FOREIGN CHARACTERS. RÉFÉRENCE EN LANGUE ÉTRANGÉRE (NOM DU LIVRE OU PUBLICATION), AU COMPLET, TRANSCRIRE EN CARACTÉRES ROMAINS. Revista espahola de fisiologia REFERENCE IN ENGLISH - RÉFÉRENCE EN ANGLAIS Spanish Review of Physiology PUBLISHER - ÉDITEUR DATE OF PUBLICATION DATE DE PUBLICATION PAGE NUMBERS IN ORIGINAL NUMÉROS DES PAGES DANS L'ORIGINAL PLACE OF PUBLICATION LIEU DE PUBLICATION YEAR ANNÉE 1976 VOLUME 32 ISSUE NO. NUMÉRO 9 to 13 NUMBER OF TYPED PAGES NOMBRE DE PAGES DACTYLOGRAPHIÉES 9 REQUESTING DEPARTMENT MINISTRE-CLIENT Fisheries and Environment TRANSLATION BUREAU NO. NOTRE DOSSIER NO Fisheries and Marine - Scientific BRANCH OR DIVISION TRANSLATOR (INITIALS) D. Collard DIRECTION OU DIVISION Information and Publications TRADUCTEUR (INITIALES) Dr P. H. Odense, Halifax Laboratory PERSON REQUESTING (via John Camp, Administrator, Scientific Documentation) DEMANDÉ PAR YOUR NUMBER VOTRE DOSSIER N 0 DATE OF REQUEST DATE DE LA DEMANDE O ctober 19th, 1976 NOV M SOS 200.I 0.0 (REV. 2/88)

3 , DEPARTMENT OF THE SECRETARY OF STATE TRANSLATION BUREAU MULTILINGUAL SERVICES DIVISION SECRÉTARIAT D'ÉTAT BUREAU DES TRADUCTIONS DIVISION DES SERVICES MULTILINGUES f.i i z 3S big CLIENTS NO. DEPARTMENT DI VISION/BRANCH cm No DU CLIENT MINISTÉRE DIVISION/DIRECTION VILLE Fisheries & Environment Fisheries and Marine Scientific Information Ottawa BUREAU NO. LANGUAGE TRANSLATOR (INITIALS) N DU BUREAU LANGUE TRADUCTEUR (INITIALES) Spanish DC November 12th, ] 976 STRUCTURAL ANALOGUES OF PYRUVATE BEHAVIOUR AS SUBSTRATES OR INHIBITORS OF LDH C. nun and J. Bozal Department of Biochemistry Faculty of Chemistry University of Barcelona The action of molecules the structures of which retain analogies with those of pyruvate and L-lactate, normal substrates of lacticodehydrogenase (LDH) is described, for the purpose of establishing some of the structural features which permit them to bind to the active centre of the enzyme and to behave as substrates or inhibitors of that enzyme. It has been suggested that the substrate of LDH is the cetyl form of pyruvate (2,4). Tienhaara (8) established that the true substrate is the unhydrated cetyl form rather than the enolic or hydrated forms; he suggests that the last series may be responsible for the inhibition caused by the pyruvate. LDH-NADH binary complexes bind with hydroxy acids of which the hydroxyl group in position a must possess the configuration L (9); although the substituent is not essential for the formation of the active tertiary complex it SOS

4 2 prevents the affected region of the enzyme from being occupied by an hydroxyl of the opposite configuration. The association of isocitrate, mandelate, and a-hydroxyisovalerianate in the binary LDH-NADH complex suggests that the introduction of bulky groups in position e does not prevent the formation of tertiary complexes; this does not take place if an hydroxyl group is in that position, as occurs with mesotartrate, e-hydroxybutyrate, and possibly with citrate. It has been postulated (6) that compounds with the structure R--00--COOH, in which R varies widely (fluoropyruvate, a-oxobutyrate, and a-oxovalerianate), may be slightly preferential substrates or inhibitors of LDH, but if the carboxyl group disappears, they are neither inhibitors nor substrates of the enzyme. The values of Km for a-oxobutyrate and a-oxovalerianate with respect to the isoenzyme H are less than with M while the K values of a-hydroxybutyrate 4 4' m with respect to both isoenzymes are practically equal (7). Material and Methods The enzyme is LDH from chicken liver obtained as described by Lluis et a/ (5). The specific activity of the prepared substance is 555 U/mg of protein (1 unit = A D.O./min). Each gram of the purified substance contains 200 mg of protein. The following have been used as substrates or inhibitors: sodium phenylpyruvate (Sigma); acetophenone, ethyl pyruvate, monohydrate glyoxylic acid, hydroxphenylacetic acid, and a-oxobutyric acid from Merck; ethyl acetoacetate, e-oxoglutaric acid and 2-oxovalerianic acid from Fluke; and sodium phenylpyruvate (Sigma).

5 3 LDH activity was measured by reading the decreases in NADH absorbency at a=340 nm on a Beckman DBGT spectrophotometer equipped with a recorder, at 30 ± 0.1 C, in 3 ml,cuvettes, allowing a light passage of 1 cm and in a medium of 50 mm sodium phosphate buffer with a ph of 7.4. The reaction began with the addition of LDH (5 pg purified) up to a volume of 3 ml. The samples in incubation contained NADH (1 x 10-4 M), and pyruvate (3 x 10-4 M),- which were replaced by the structural analogue when their behaviour as a substrate was investigated. The apparent kinetic parametere; Am and Vmax, were calculated using the Lineweaver-Burk method (3). The binding site for the two copresent substrates was determined by the Dixon method (1) which may be used with non-equimolecular mixtures of both substrates; this value for the same active centre is expressed by the equation:, F Vrcu = V. ^ -- Vb kt, 11) -!- K. ^ K,, and when the substrates act on enzyme centres which are independent of each other, by: Results V, a + Vt. b ^II) K +a Kt,+b Effect of structural analogues of pyruvate that are substrates of LDH. We studied the behaviour, as substrates br inhibitors, of compounds of the type R-CO--COOH, the influence which leads to variations in chain length in the -R grdup, and the intervention of the carboxyl group in the binding of the pyruvate to LDH.

6 4 The LDH concentration was as indicated except in the experiments carried out with a-oxovalerianate which was present in the amount of 50 pg in the 3 ml being incubated. Through graphic representation of the initial velocities (3), the values of K and V for the various compounds were obtained (Table I). m max The results suggest that the existence of the free carboxyl group is not essential for the substrate to bind to the enzyme (ethyl pyruvate) and that the lengthening of the aliphatic chain up to 3 carbon atoms (a-oxovalvalerianate) or the suppression of that chain (glyoxylate) does not prevent it from acting as a substrate. Ethyl pyruvate (5 x 10-3 M), a-oxobutyrate (5 x 10-2 M), and glyoxylate (2.4 x 10-2M) induce inhibition of LDH due to an excess of substrate; the inhibition caused by the latter increases as the concentration of NADH diminishes. In the pyruvate system (5 x 10-5 M to 3 x 10-4 N ) -LDH (5 pg/3 ml inc.)-nadh (1 x10-4 M), a-oxovalerianate (1 x 10-2 M and 5 x 10-2 M) is an.ecompetitive inhibitor with respect to NADH (K = 1.75 x 10-2 M) and a non-competitive.one with respect with NAD (K1. x 10-2 M); while in the inverse system, ip (3 x y1), L-lactate (5 x 10-3M to 1.66 x 10-2 M) and LDH (16.6 pg purified substance /3 ml inc.) inhibits competitively with respect to L-lactate (K. = 3.8 x 10-3 M). The results suggest that, when a-oxobutyrate acts as an ip inhibitor, it binds at the centre where the L-lactate binds, and this is a different centre from that at while the pyruvate binds. Table I Analogue substrates of LDH from chicken liver gubstr7ate Concentration Km V max Mx 10 M W 10 3 (D.0./min) x C1-13-CO.006CCH4 CH;CH CO.Ce DO - CH3-Ch CH2:CO-COCY HOC-000 1,3 tes A-20 3,3 Oi 5.0 6

7 5 kinetics of non-equimolar mixtures of substrates. Pyruvate and its structural analogues compete for the same active centre of the LDH, as we have been able to show by the Dixon method (1) working with binary nonequimolar mixtures composed of pyruvate and ethyl pyruvate (Fig 1 (a)), a-oxobutyrate (Fig 1(b)), or glyoxylate (Fig 1(c)). Experiments to measure the initial velocities of non-equimolar mixtures of pyruvate and the other substrate were performed in the usual manner. Values for K and V for the pyruvate used are: 5.3 x 10-5 M and m max 5.6 x 10-2 AD.P./min; values for other substrates appear in Table I. Figure 1 Kinetics of non-equimolecular binary mixtures 0.5 I.1C A ,.-' I- f.' 1, l, ;, e://, f J. 0.3 C.3 1. lee. ----'''-'" _/0/' t 0.2 ' C.!' 1 ' if/ e ' 0.1 I. C. I 1 r I v EETI7AL PYRUVATg Ft-miturriTAT LYÔXYLATE) to ta) (b) (c) i.2 dee >14 = 1 x 10-4 M;,1)11 = 5 pg in 3 ml; 50 mm phosphate buffers with a ph of 7.4; Km (pyruvate) = 5.3 x ; V (pyruvate) = 5.6 x 10-2 AD.O./min; (o) theoretical velocity et (10 theoretical velocity VT]-; (A) experimental velocity. (a) pyruvate (3 x )-ethyl pyruvate. (b) pyruvate (3 x 10-5 M)-a-oxobutyrate. (c) pyruvate (2.5 x )-glyoxylate.

8 6 Structural analogues which do not affect or inhibit LDH. We have tried to establish which modifications in the general R--00--COOH structure cause compounds to cease to be substrates of LDH and dictate whether the activity of the enzyme remains unaltered or is inhibited. The behaviour of the different compounds was determined under the usual experimental conditions on the direct system or with NAD (3.3 x 10-3 M) on the inverse system (Table 2). It is apparent that the disappearance of the carboxyl group (acetophenone) prevents the binding of the compound to the LDH; the same occurs when the carbonyl group is in position 0 with respect to the carboxyl group, as we see with ethyl acetoacetate and in 0-oxog1utarate. The fact that a-oxoglutarate and the phenyl pyruvate inhibit LDH activity confirms the need for the carbonyl and carboxyl groups to be in contiguous position so that they may bind to the enzymatic molecule in a fashion similar to that observed with L-lactate. Table II Different effectors of LDH activity of LDH from chicken liver Structure of the Compound Concentration Inhibition N x 10 3 direct inverse system system 9H3-CO-C Hs.. to. NO NO _ CH -CO CW.COOCH CH 50 Nell NO _2. *'00O-Ci' -CH -CO OCC-CI-1 -CO-CH ' NO NO... C FirCH-c0.coo b 2.. C H -CHOI-I-COO - ;Id 52 '

9 7 Discussion In this paper, we describe the structural requirements in order for the LDH substrates to establish an effective union with the enzyme. For this purpose, we have investigated the effects on enzymatic activity of specific molecules which are structural analogues of pyruvate, and which retain the character of a substrate throughout, together with the effect produced by substances which, although they may not behave like substrates, nonetheless exhibit an ability to bind to the enzyme molecule. Ethyl pyruvate is a substrate of LDH, which suggests that the presence of the free carboxyl group is not essential in order for the substrate to bind to the enzyme or for the catalytic conversion to take place. The effect of variation in the length of the aliphatic chain of the pyruvate has been observed with a-oxybutyrate, a-oxovalerianate and glyoxylate; these are all substrates of LDH and, like ethyl pyruvate, have K values exceeding that of m pyruvate, and induce inhibition of enzymatic activity due to an excess of substrate. Ethyl pyruvate, a-oxobutyrate, and glyoxylate are converted in the same active centre of the LDH as pyruvate, as has been demonstrated with non-equimolar mixtures of pyruvate and of each of the aforementioned substrates. a-oxovalerianate, which is a substrate of LDH, also acts as a noncompetitive inhibitor with respect to pyruvate and an acompetitive inhibitor with respect to NADH; it is a competitive inhibitor with respect to L-lactate in the presence of NAD. These findings permit us to state that the elimination of the aliphatic chain of the pyruvate, or its lengthening up to 3 carbon atoms, does not prevent the binding of compounds to the LDH molecule, although it does reduce the affinity of the enzyme for the substrate.

10 8 Elimination of the carboxyl group from the pyruvate results in the analogue compound ceasing to be a substrate or an inhibitor, as occurs with acetophenone. Moreover, the displacement of the carbonyl group to the position 8 with respect to the carboxyl group prevents the binding of compounds to the LDH molecule and they no longer behave as substrates or inhibitors (ethyl acetoacetate and 8-oxog1utarate). The fact that a-oxovalerianate is a substrate and inhibitor of LDH and that a-oxoglutarate and phenylpyruvate inhibit the enzyme confirms that the presence of a carbonyl group in position a with respect to the carboxyl is necessary for the attachment of the compounds to the LDH molecule, and that whether they act as substrates or inhibitors of the enzyme depends on the nature of the --R group. Summary Experiments conducted on various compounds structurally related to pyruvate, for the purpose of determining whether they are substrates or inhibitors of LDH, reveal some of the special features necessary for them to bind to the active centre of the enzyme. Pyruvate binds to the LDH molecule through the carboxyl group, which may be either free or esterified. Indeed, ethyl pyruvate behaves as a substrate, while acetophenone, ethyl acetoacetate, and 8-oxoglutarate and L-mandelate inhibit LDH. It appears that the structure of compounds that bind to LDH must contain a carbonyl group in position a with respect to the carboxyl group. Glyoxylate, a-oxobutyrate, and a-oxovalerianate are worse substrates of LDH than pyruvate. This indicates that the elimination or lengthening of pyruvate's aliphatic chain causes a decrease in the affinity of the enzyme for these pyruvaterelated compounds.

11 9 BIBLIOGRAPHY 1. DIXON, M. y WEBB, C. E.: Enzymes (204 Longman's Green, London, 1967, ed),. v FRomm, 11. G.: Biochim. Biophys.,Wu, 99, 540, LINEWEAVER. M. y I3uR, D.: J. Ant. Chem. Soc., , Lomu.$, F. A., CHEN, T. T., VF.NNESI.AND, S.: J. Biol. Clem., 212, 787, Li.uis, C., GUISERT, S. y B0ZA1., J.: Rev. esp- Fisioi., Si, 223, Nr..0.ANDs. C. B.: J. Biol. Chem., 225, 1036, NISSELItAIIN1, G. S., PACKER. E. y Botwls- KY, O.: J. Biol. Chem., 239, 2830, TIENHAARA, R. y MEANY, G.: Biochemistry, 12, 2067, WINER, A. D. y SCHWERT, G. W.: J. Biol. Chem., 231,