Some Properties of 3-Hydroxy-3-Methylglutaryl-Coenzyme a Synthase from Ox Liver

Biochem. J. (1978) 173, 925-928 925 Printed in Great Britain

Some Properties of 3-Hydroxy-3-methylglutaryl-Coenzyme A Synthase from Ox Liver

By M. ANN PAGE and PHILIP K. TUBBS Department ofBiochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1 Q W, U.K. (Received 16 January 1978)

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) was purified from ox liver, and obtained essentially free from 3-oxoacyl-CoA thiolases. The kinetic behaviour, like that'of the synthases from chicken liver and yeast, is compatible with a reaction pathway involving condensation of an acetyl-enzyme with acetoacetyl-CoA. The Km for acetoacetyl-CoA, less than 1 gm at pH 7.8, may possibly be low enough to permit rapid ketogenesis under physiological conditions without the need for a binary complex between the synthase and oxoacyl-CoA thiolase.

Although HMG-CoA synthase has often been Methods assumed to be the rate-limiting enzyme of keto- Enzyme purification genesis in mammalian liver very little is known about the enzyme from this tissue. Two distinct types of The synthase was usually prepared from frozen HMG-CoA synthase, mitochondrial and cytoplasmic, whole ox liver, but the same purification procedure have been purified from chicken liver (Clinkenbeard was also effective starting with frozen ftiitochondria. et al., 1975b; Reed et al., 1975); the former is sup- The liver was homogenized with 3 vol. of cold deion- posed to be involved in the synthesis of acetoacetate ized water, centrifuged at 15000g for 20min, and the and the latter in that of mevalonate (Clinkenbeard et supernatant fractionated at 0-5 C with (NH4)2SO4. al., 1975a). The HMG-CoA synthase of yeast has The protein that was precipitated between 225 and also been extensively studied (Middleton, 1972; 355 g of (NH4)2S04/litre was dissolved in 10mM- Middleton & Tubbs, 1972, 1974). potassium phosphate (pH 7.5) containing 0.5 mM- Kinetic and chemical experiments with the yeast dithiothreitol, and the solution was desalted by and chicken liver synthases have suggested that the passage through a large column of Sephadex G-25 formation of 3-hydroxy-3-methylglutaryl-CoA pro- equilibrated with this buffer. Glycerol was then added ceeds by the stepwise mechanism in Scheme 1. to 25% (v/v), and together with the dithiothreitol This scheme involves binding of acetyl-CoA (step was present in all potassium phosphate bufters used 1); acetylation (step 2) of the enzyme as a thioester in later steps, which were carried out at pH 7.5 and (Middleton & Tubbs, 1974; Miziorko et al., 1975); (except for the ultrafiltration) at room temperature. binding of acetoacetyl-CoA (step 3); reaction (step The solution was passed through a column of 4) of the acetyl-enzyme with acetoacetyl-CoA to DEAE-cellulose equilibrated with the above buffer yield a 1 ,5-dithioester of 3-hydroxy-3-methylglutarate containing 25 % (v/v) glycerol. The synthase Was (Miziorko et al., 1976); and finally hydrolysis (step 5) slightly retarded by the column, and emerged after of this to liberate 3-hydroxy-3-methylglutaryl-CoA most of the haemoglobin. The active'fractions were and enzyme. The kinetics of the synthase are of applied to a column of cellulose phosphate equili- interest in view of the extremely low acetoacetyl-CoA brated as above, and a linear gradient of phosphate concentrations that may exist in vivo as a consequence (10-150mM) was applied. The synthase was normally of the very unfavourable equilibrium of the aceto- eluted at about 70mM-phosphate, but with some acetyl-CoA thiolase reaction (Wakil, 1963). It has batches of cellulose phosphate a higher concentration been suggested (Greville & Tubbs, 1968) that a was required. thiolase-synthase binary complex, with direct 'chan- The fractions containing HMG-CoA synthase nelling' ofacetoacetyl-CoA between the two enzymes, were pooled and concentrated at 4 °C by ultrafiltration may exist. We report here some kinetic properties of through an Amicon Diaflo PM 10 membrane the HMG-CoA synthase of ox liver mitochondria; (Amicon, Lexington, MA 02173, U.S.A.). Further these are in agreement with the reaction pathway in purification was achieved by passage through two Scheme 1 and confirm a very high affinity for columns (see below) of Sephadex G-150 equilibrated acetoacetyl-CoA. with 50mM-phosphate containing O.5mM-dithio- Abbreviation used: HMG-CoA synthase, 3-hydroxy- threitol and 15% glycerol. The enzyme was stored 3-methylglutaryl-CoA synthase (EC 4.1.3.5). at -20'C after addition of glycerol to 30 % (v/v). Vol. 173 926 M. A. PAGE AND P. K. TUBBS

AcCoA CoA AcAcCoA E AcCoA-E E-Ac AcAcCoA E-Ac 2

H20 {4 E+ HMGCoA ) E-HMGCoA 5 Scheme 1. Formation of3-hydroxy-3-methylglutaryl-CoA by a stepwise mechanism Abbreviations: E, enzyme; AcCoA, acetyl-CoA; AcAcCoA, acetoacetyl-CoA; HMGCoA, hydroxymethylglutaryl-CoA.

This procedure i's quite similar to that for the puri- Results and Discussion fication ofthe chicken liver mitochondrial HMG-CoA Fig. l(a) shows, in double-reciprocal form, the synthase (Reed et al., 1975). The main consideration effects of varying the substrate concentrations on the was the separation of the synthase from the several reaction rate at pH 7.8. The results show that the types of 3-oxoacyl-CoA thiolase (Middleton, 1973). Km values for acetoacetyl-CoA are exceedingly low The cytoplasmic and mitochondrial thiolases specific (below 1,pM), and that at high concentrations (over for acetoacetyl-CoA were removed by the DEAE- 10pM) this substrate is inhibitory; similar behaviour cellulose and cellulose phosphate columns respec- is found with the chicken liver enzymes (Clinkenbeard tively. The mitochondrial general-specificity thiolase et al., 1975b; Reed et al., 1975). The lines representing exists in at least two forms (Middleton, 1974), of different acetyl-CoA concentrations are apparently which one is like the synthase in its elution behaviour parallel, indicative of Ping Pong (Cleland, 1963) from cellulose phosphate but emerges rather earlier behaviour; this suggests that the first product (pre- from Sephadex G-150. The apparent molecular sumably CoA) leaves the enzyme before the second weights of this thiolase and of the synthase, deduced substrate (acetoacetyl-CoA) binds. The data in Fig. from their gel-filtration behaviour, were about l(a) also show that the Km value for acetyl-CoA is 125000 and 88000 respectively, and synthase free of very high (over 100UM), even when the concentration thiolase activity was obtained by passing 1.5ml of of acetoacetyl-CoA is low; again the chicken enzymes enzyme concentrated as above through two columns are similar in behaviour (Clinkenbeard et al., 1975b; of Sephadex G-150, each lm long and 2.5cm in Reed et al., 1975). diameter, mounted in series. The determination of the Km for acetoacetyl-CoA at pH 7.8 is difficult, since it is very small compared HMG-CoA synthase assay with experimentally convenient concentrations. The slopes in Fig. l(a) (essentially zero) are equal to the The assay system contained l00mm-Tris, 10mM- ratio K.etoacetYlCOA/Vmax.; similarly, if the v- MgC12 and 50mM-KCI; the pH was adjusted with values are plotted against [acetyl-CoA]- the result- HCI to the desired values at 30°C. Acetoacetyl-CoA ing slope is KmctYlcoA/Vmax.. Comparison of these and enzyme were added, the blank rate was observed, ratios showed that at pH7.8 K cetoacetyl coA is less and the reaction started with acetyl-CoA. Dis- than one five-hundredth of Kae'ty-coA. appearance of acetoacetyl-CoA was followed at At pH8.7 the Km values for acetoacetyl-CoA are 303nm. The specific activity of the purified enzyme considerably higher than at pH 7.8, so that the with 1001uM-acetyl-CoA and 5pM-acetoacetyl-CoA observed rates are not independent of this substrate's at pH7.8 was about 3,umol of 3-hydroxy-3-methyl- concentration (Fig. lb); at this pH the Km for aceto- glutaryl-CoA synthesized/min per mg of protein acetyl-CoA is only about one-hundredth that for (Fig. la). acetyl-CoA. The fact that the lines in Fig. l(b) appear parallel again suggests Ping Pong kinetic behaviour. Substrates As discussed by Middleton (1974) and Tsopanakis & Herries (1975), the use of mixtures in each of which Acetyl-CoA and acetoacetyl-CoA were prepared the concentration of the second substrate is in a by treating CoA with acetic anhydride or diketen; fixed ratio to that of the first can reveal the presence acetoacetyl-CoA was purified by chromatography on of a modified enzyme intermediate (in the present DEAE-cellulose at pH2.7 (Middleton, 1972). The case probably acetyl-enzyme). If such an intermediate absorption coefficients at 303 nm for acetoacetyl- occurs the double-reciprocal plots will be linear, CoA utilization are 11x 103 and 21.6x 103 litre' whereas they will be parabolic if the intermediate is molh cm- at pH7.8 and 8.7 respectively, under the kinetically insignificant; this method avoids the conditions used for the kinetic experiments. difficulty of deciding whether plots such as those in 1978 OX LIVER HYDROXYMETHYLGLUTARYL-CoA SYNTHASE 927

(a) (b) [Acetyl-CoA] (pM) 20 -, 16.3 E [Acetyl-CoA] (PM)

10.1 2 24.5 )E~0 . 15 0 0 U 0O2 in.= 0_ 40.8 0 0. 10 0. - * . 20.2 0 81.5 30.3 163 50.5 5 A *o*101 II 0 0.2 0.4 0.6 0.8 0 0.5 1.0 1.5 lAcetoacetyl-CoAl-' (#M-8) Fig. 1. Effect of different substrate concentrations on 3-hydroxy-3-methylglutaryl-CoA synthase activity: (a) at pH7.8, (b) at pH8.7 The assay method is described in the text.

0 0.4 0.8 1.2 0 0.5 1.0 1.5 [Acetoacetyl-CoA]-' (PM 1) Fig. 2. 3-Hydroxy-3-methylglutaryl-CoA synthase activity as afunction ofthe acetoacetyl-CoA in substrate mixtures in which the molar ratios (n) ofacetyl-CoA/acetoacetyl-CoA were as shown: (a) at pH7.7, (b) at pH8.7

Figs. 1(a) and l(b) are truly parallel or gradually condensation) in Scheme 1. The results in Fig. 2(a) converging. The results of such mixture experiments correspond to Case III of Middleton (1974), in which at pH7.8 and 8.7 are shown in Figs. 2(a) and 2(b). only one substrate is inhibitory, and the intercept It seems that at pH7.8 the proposed acetyl-enzyme shows that the inhibition by acetoacetyl-CoA has a does exist to a significant extent, but the curves in Ki value of about 12AM. Fig. 2(b) show that it is very transient at pH 8.7. The results suggest that the mechanism of the Presumably this difference is due to an effect of pH HMG-CoA synthase from ox liver mitochondria is on the relative rates of steps 2 and 4 (acetylation and similar to that of the yeast (Middleton, 1972; Vol. 173 928 M. A. PAGE AND P. K. TUBBS

Middleton & Tubbs, 1972) and chicken liver enzymes remains to be studied by using the enzyme uncon- (Clinkenbeard et al., 1975a,b; Reed et al,, 1975), and taminated by thiolase. may be represented by Scheme 1. We are grateful to the Science Research Council for As with those other enzymes, the ox liver synthase financial support. can be separated from the 3-oxoacyl-CoA thiolases. A thiolase-synthase complex may exist, which could overcome the difficulty ofachieving rapid ketogenesis References in spite of the presumed extremely low acetoacetyl- Bremer, J. & Aas, M. (1969) in Mitochondria: Structure CoA concentration in vivo, but if so it is readily and Function (Ernster, L. & Drahota, Z., eds.), pp. 127-135, Academic Press, New York and London disrupted. The very low Km for acetoacetyl-CoA (less Cleland, W. W. (1963) Biochim. Biophys. Acta 67, 104-137 than 1juM at pH7.8, and probably still lower at Clinkenbeard, K. D., Reed, W. D., Mooney, R. A. & neutral pH) shown by the synthase and the lack of Lane, M. D. (1975a)J. Biol. Chem. 250, 3108-3116 inhibition by acetyl-CoA are in accord with the Clinkenbeard, K. D., Sugiyama, T., Reed, W. D. & Lane, physiological situation, and may render a binary M. D. (1975b) J. Biol. Chem. 250, 3124-3135 enzyme complex unnecessary. The properties of the Greville, G. D. & Tubbs, P. K. (1968) Essays Biochem. synthase and of the thiolase reaction (Middleton, 4, 155-212 1974; Huth et al., 1975) are such that the rate of Huth, W., Jonas, R., Wunderlich, I. & Seubert, W. (1975) and Eur. J. Biochem. 59, 475-489 formation of 3-hydroxy-3-methylglutaryl-CoA, Middleton, B. (1972) Biochem. J. 126, 35-47 hence of acetoacetate, should vary as a sensitive Middleton, B. (1973) Biochem. J. 132, 717-730 function of the acetylation state of the mitochondrial Middleton, B. (1974) Biochem. J. 139, 109-121 CoA; this is indeed observed (Greville & Tubbs, Middleton, B. & Tubbs, P. K. (1972) Biochem. J. 126, 1968; Bremer & Aas, 1969). Free CoA strongly 27-34 inhibits the yeast HMG-CoA synthase (Middleton, Middleton, B. & Tubbs, P. K. (1974) Biochem. J. 137, 1972), and the amount offree CoA in liver is decreased 15-23 by starvation, diabetes or a fatty diet, conditions Miziorko, H. M., Clinkenbeard, K. D., Reed, W. D. & which are accompanied by rapid acetoacetate syn- Lane, M. D. (1975) J. Biol. Chem. 250, 5768-5773 not observe inhibition Miziorko, H. M., Shortle, D. & Lane, M. D. (1976) thesis. Reed et al. (1975) did Biochem. Biophys. Res. Commun. 69, 92-98 of the chicken liver mitochondrial synthase by CoA, Reed, W. D., Clinkenbeard, K. D. & Lane, M. D. (1975) but were using a high acetoacetyl-CoA concentration J. Biol. Chem. 250, 3117-3123 (50M). Inhibition of HMG-CoA synthase (and also Tsopanakis, A. D. & Herries, D. G. (1975) Eur. J. of thiolase; Huth et al., 1975) by free CoA may be Biochem. 53, 193-196 metabolically important in mammalian liver, but Wakil, S. J. (1963) Enzymes 2nd Ed. 7, 97-103

1978