Catalytic and Regulatory Properties of Pyruvate Kinases in Tissues of a Marine Bivalve*

Abstract Pyruvate kinase (EC 2.7.1.40) from oyster tissues occurs in at least two electrophoretically and kinetically distinguishable forms in mantle tissue and in adductor muscle. In mantle, pyruvate kinase activity appears as a single band having a pI value of 6.35 while adductor pyruvate kinase appears as a "doublet" having pI values of 5.6 and 6.5. Both forms of the enzyme have an essential requirement for Mg++ and a monovalent cation (K+ or NH4+). The activation of oyster enzymes was more efficient with K+. Neither of the enzymes is inhibited by Cu++. In the absence of fructose 1,6-diphosphate, the Km(adp) for adductor muscle pyruvate kinase is several-fold higher than the Km(adp) for the mantle enzyme. In the presence of fructose-1,6-P2, the Km(adp) for both enzymes are identical. The Km of the substrate phosphoenolpyruvate differs for the two enzymes, Km(pep) being several-fold lower for the adductor enzyme. In the absence of fructose-1,6-P2, the Km(pep) for both enzymes decreases markedly with increasing pH; the Km(pep) is strikingly decreased by FDP and becomes insensitive to pH in presence of fructose-1,6-P2. For oyster pyruvate kinases fructose-1,6-P2 stimulation is maximal at acidic pH. Both forms of the enzymes are subject to feedback inhibition by ATP, l-alanine, and phenylalanine, and these inhibitory effects are reversed by fructose-1,6-P2. Ki values of these metabolites for adductor pyruvate kinase are lower than the Ki values of mantle enzyme. ATP inhibition differs for the two enzymes, being noncompetitive for the mantle form and competitive for the adductor enzyme. l-Alanine inhibition of both enzymes is of the mixed competitive type. Phenylalanine inhibition is competitive with respect to P-enolpyruvate for the mantle enzyme, but not for the adductor pyruvate kinase. The data in this study suggest that, as in the mammalian case, the enzyme pyruvate kinase in the oyster occurs in tissue specific multimolecular forms and that the kinetic properties of each isozyme seem to gear in well with the over-all metabolism of the tissue.


SUMMARY
Pyruvate kinase (EC 2.7.1.40) from oyster tissues occurs in at least two electrophoretically and kinetically distinguishable forms in mantle tissue and in adductor muscle. In mantle, pyruvate kinase activity appears as a single band having a p1 value of 6.35 while adductor pyruvate kinase appears as a "doublet" having p1 values of 5.6 and 6.5. Both forms of the enzyme have an essential requirement for Mg++ and a monovalent cation (K+ or NH,+). The activation of oyster enzymes was more efficient with K+.
Neither of the enzymes is inhibited by Cu++. In the absence of fructose 1,6-diphosphate, the K,cADP) for adductor muscle pyruvate kinase is several-fold higher than the K,cADP) for the mantle enzyme.
In the presence of fructose-l, 6-P2, the K, cADP) for both enzymes are identical.
The K, of the substrate phosphoenolpyruvate differs for the two enzymes, KmcPEP) being several-fold lower for the adductor enzyme.
In the absence of fructose-1,6-P*, the Km (PEP) for both enzymes decreases markedly with increasing pH; the KmcPEF') is strikingly decreased by FDP and becomes insensitive to pH in presence of fructose-l ,6-PZ. For oyster pyruvate kinases fructose-l, 6-PZ stimulation is maximal at acidic PH.
Both forms of the enzymes are subject to feedback inhibition by ATP, L-alanine, and phenylalanine, and these inhibitory effects are reversed by fructose-1,6-Pz. Ki values of these metabolites for adductor pyruvate kinase are lower than the K; values of mantle enzyme. ATP inhibition differs for the two enzymes, being noncompetitive for the mantle form and competitive for the adductor enzyme. L-Alanine inhibition of both enzymes is of the mixed competitive type. Phenylalanine inhibition is competitive with respect to P-enolpyruvate for the mantle enzyme, but not for the adductor pyruvate kinase.
The data in this study suggest that, as in the mammalian case, the enzyme pyruvate kinase in the oyster occurs in tissue specific multimolecular forms and that the kinetic properties of each isozyme seem to gear in well with the over-all metabolism of the tissue.
* This work was supported by the National mesearch Council of Canada.
1 Recipient of a Canadian Commonwealth Scholarship of the Canadian International Development Agency.
In theory, pyruvate occupies a central crossroads position in energy metabolism since it may be metabolized by a number of different pathways.
At least six such pathways are present in oyster tissues. However, not all are equally functional in all tissues (1). In oyster adductor muscle, the major source of pyruvate is storage glycogen.
The pyruvate, which is produced in high quantities during periods of anaerobiosis, can have several metabolic fates, the most important one being conversion to malic acid. The latter is readily metabolized to succinate, which is a major end product of anaerobic metabolism in oysters. Lactate, in contrast, does not accumulate (1). Under aerobic conditions pyruvate can be oxidized by the usual Krebs cycle reactions. In mantle, on the other hand, the pyruvate branching point is more complex and the degree to which these pathways opera,te differs from that in t'he adductor muscle. During anaerobiosis glucose-6-l% is a better source of succinate than pyruvate@%, suggesting that the pathway to succinate branches off before pyruvate production.
Simpson and Awapara (2) suggest that carboxylation of P-enolpyruvate by P-cnolpyruvate carboxykinase is the major mechanism leading to succinate accumulation. Also, in contrast to adductor muscle, mantle is a major gluconeogenic tissue. Under gluconeogenic conditions, pyruvate and Penolpyruvate may be produced from noncarbohydrate sources as well as from Krebs cycle intermediates.
The pyruvate kinase step, which in most organisms is physiologically irreversible (3), may be bypassed durin g glucose (or glycogen) synthesis by the P-enolpyruvate carboxykinase which generates P-enolpyruvate for further metabolism to glucose. In mantle of most molluscs examined, however, pyruvate kinase activity exceeds P-enolpyruvate carbosykinase by a factor of about 10 (I). Hence if the data are correct, there are special requirements for holding pyruvate kinase in a "shut off" conformation in this tissue.
From the above consideration it is evident that the requirements for the regulation of the pyruvate kinase reaction in adductor and mantle tissues are quite distinct from each other. One obvious way of meeting t,hese different requirements is the elaboration of tissue-specific variants of the enzyme. 1Ve therefore initiated this study by examining mantle and adductor tissues for different electrophoretic forms of pyruvate kinase. In adductor muscle homogenates, two forms of pyruvate kinase can be resolved by electrophoresis on cellulose acetate or by isoelectrofocusing; in mantle tissue, a single form of pyruvate kinase is present.
The pyruvate kinase activities from these factors. Animals were brought to the laboratory in an icebox and opened quickly to excise the tissues. All tissues were washed thoroughly with cold homogenizing medium to remove exogenous algae and other microorganisms.
Mantle and adductor tissues were homogenized in a Sorvall Omnimixer for 1 to 2 min with 3 to 4 volumes of ice-cold 0.01 M Tris-HCl buffer, pH 7.5, containing 2 mM EDTA.
The homogenate was stirred for 1 hour at 4" and then centrifuged at 12,000 x g for 15 min and the pellet was discarded.
The supernatant was filtered through glass wool and then brought to 40% saturation with solid ammonium sulfate and stirred for 1 hour at 4". The suspension was then centrifuged as above, the pellet was discarded, and the supernatant was brought to 75% saturation with solid ammonium sulfate. After 1 hour with stirring, the solution was centrifuged at 37,000 X g for 20 min. The pellet was dissolved in a minimal volume of 0.01 M Tris-HCI, buffer, pH 7.5. The dissolved pellet was further centrifuged at 84,000 x g for 90 min in refrigerated Beckman model L preparative ultracentrifuge to remove glycogen and the high speed supernatant was used as the source of pyruvate kinase. Portions of enzymes were dialyzed before use against 0.05 M Tris-HCI buffer, pH 7.5. The enzyme was stable at O-4" for a few days and if frozen was stable for several weeks without causing any change in the K, of the substrate, P-enolpyruvate. Mantle enzyme was somewhat unstable to dialysis, showing a loss of activity of approximately 10% within 2 hours of dialyzing.
Enzyme was assayed by the methods of Bucher and Pfleiderer (5). Pyruvate formation was coupled to lactate dehydrogenase and the rate of pyruvate kinase activity was measured as the decrease in Es40 due to NADH.
Tris-HCl buffers were used in all assay reactions.
All reactions were started by the addition of pyruvate kinase preparation.
AI1 experiments were performed at 20" since KrncpEp)l was found temperature-independent over a temperature range of 5-30". 2 Mantle, gill, and adductor muscle pyruvate kinases were prepared and studied electrophoretically to determine whether tissue-specific forms are present in the oyster. These experi- ments were performed in collaboration with Dr. Walter Susor by the procedure given by Susor and Rutter (6).
The technique of electrofocusing was used to elaborate further the results of electrophoresis and to determine whether isoenzymes having different p1 values were present.
Electrofocusing experiments were performed according to the method of Haglund (7). Both mantle and adductor enzymes were run at pH 5 to 8 (LKB-8133) at 900 volts for 53 hours. The temperature of the apparatus was maintained at 3" + 0.05. For enzyme activity all fractions were assayed in presence of 2.5 X 10d6 M fructose-l ,6-Pz at pH 8.5.

RESULTS
Electrophoresis and Electrojocusing-.Electrophoretic resolution of pyruvate kinase activity in three different tissues is shown in Fig. 1. Mantle pyruvate kinase activity appears as a single band, moving toward the cathode; pyruvate kinase in gill tissue displays a similar electrophoretic mobility. Adductor pyruvate kinase, showing a different pattern, moves as a "doublet" toward the cathode.
The electrophoretic differences were confirmed by electrofocusing experiments (Fig. 2) Effect of Fructose-l, 6-Pz on pH Optima-As shown in Fig. 3 in the absence of fructose-l ,6-P2, both forms show pH optima at pH 8.5 but the shape of the activity curves differs for both, with adductor pyruvate kinase being more sensitive to pH changes.
In the presence of fructose-l ,6-P2, the activity of the mantle enzyme is approximately constant between pH 7.5 and 9 while the pH optimum for the adductor enzyme is displaced toward pH 7. EJect of Fructose-l ,6-P:! 0'1% K, of AUP-The effect of increas-the reaction velocity against ADP in the presence and absence of ing ADP concentration at a fixed P-enolpyruvate concentration fructose-l, 6-PZ have the Michaelis-Menten form. For mantle on the activity of the two enzymes in the absence and presence pyruvate kinase the KmcADP) in the absence of fructose-1,6-Pt of 2.5 x 10V6 nr fructose-l ,6-Pz at pI-I 8.5 are given in Fig. 4. is 6.1 X 10d4 M while fructose-1,6-Ps increases the KrncADP) at Both enzymes require 2 X IOU4 M ADP for maximal activity at least by 6-fold.
In contrast to this, fructose-l, 6-PZ has no effect 1 x lo-* M P-enolpyruvate.
For both enzymes the curves for on the KmcADP) of adductor pyruvate kinase. Effect of pH and Fructose-I ,6-Pz on K, of P-enolpyruvate-Double reciprocal plots for the initial velocities of P-enolpyruvate reaction rates at different pH values in the presence and absence of fructose-l, 6-Pz are shown in Fig. 5 (right panel).
For mantle enzyme (Fig.   5, left panel), the K, in the absence of fructose-1,6-Pz and at pII 8.5 is about 2 X lo+ M; the K, increases markedly below pH 8.5 to a value of about 6 X 10m4 M at pH 6.5.
In the presence of fructose-l, 6-PZ the K, for the mantle enzyme is markedly lowered to about 7 X low5 M and remains constant over the pH range of 6.5 to 8.5. Fig. 6  to fructose-l, 6-1'2 activation correspondingly decreases. In the case of oyster pyruvate kinases both enzymes are much more susceptible to fructose-1, 6-PZ activation at acidic pH than at alkaline PH.
ATP inhibition-Like pyruvate kinases from other sources (9, 10) ATP rilso inhibits both forms of the oyster enzymes. While ATP inhibits enzymes at both pH values examined, several pH differences can be noted. With 1 mM P-enolpyruvate at pH 8.5 both enzymes are less inhibited than at pH 7.5. The Ki values for the mantle enzymes are 4 mM and 2.65 mM at pH 8.5 and 7.5, respectively.
The adductor enxymc shows a similar behavior, although the Ki values for the adductor pyruvate kinase are lower, about 2.8 mM and 1.9 InM at $1 8.5 and 7.5, respectively.
The nature of inhibition is also different. Double reciprocal plots of the velocity of the pyruvate kinase reaction at different ATP concentrations (Fig. 7) indicate that the ATP inhibition of the mantle enzyme is noncompetitive with respect to P-enolpyruvate.
Thus for mantle enzyme ATP decreases the calculated V,,,, but does not affect the K, cpEp). This noncompetitive 4 inhibition of mantle pyruvate kinase is relatively unique, having been reported only once previously for the mouse brain enzyme by Lowry and Passonneau (11). Since P-enolpyruvate in mantle is not likely to reach saturating concentrations, these results suggest that BTP is not an important modulator of mantle enzyme.
In contrast, the adductor enzyme shows competitive inhibition (Fig. 8) and is inhibited by lower concentrations of ATP.
Thus, 2 rnnl ATP increases the KmcPEP) at least IO-fold with little or no effect on the V,,,.
Further, it was noted that the nature of inhibition of either enzyme could not be altered by increasing or decreasing the Mg* concentration of the assay medium, although the degree of inhibition clear1.y depends upon the amount of the Mg+f present.
However, Mg* even at very high concentrations does not completely abolish ATP inhibit.ion. Interacting Eflects of A TP and Fructose-l ,6-P-Rozengurt et al. (9) and Tanaka, Sue, and Morimura (10) have reported that ATP inhibition of liver pyruvate kinase is reversed by fructose-1,6-Pz.
Studies of this kinetic property with the oyster mantle enzyme at two different pH values at varying concentrations of P-enolpyruvate indicate a behavior similar to that of mouse liver pyruvate kinase. Figs. 9 and 10 show double reciprocal plots of P-enolpyruvate reaction rat,es of the mantle enzyme in the presence of fructose-l, B-P2 and ATP at two pH values. At pH 8.5 (Fig. 9) 10-G M fructose-l, 6-PZ releases the ATP inhibition (caused by 3 m&f ATP) by decreasing the Km to two-thirds of control and increasing the Tr,,,,, by 2-fold; thus, fructose- ATP and fructose-l ,6-P2 the behavior of adductor pyruvate kinase closely parallels that of the mantle enzyme. Search for Other Modulators-Since pyruvate occupies a central crossroads in oyster tissue metabolism, we felt it necessary to study the effects of other metabolites on pyruvate kinase activities.
Of the various compounds tested, 5'-AMP, acetyl-CoA, citrate, succinate, malate, and oxaloacetate have neither stimulatory nor inhibitory effects on the oyster enzymes. Only L-alanine and phenylalanine were found to affect the enzyme in an inhibitory manner. Kith 5 X low4 M P-enolpyruvate, both oyster enzymes were found more susceptible to alanine inhibition at, pH 7.5 than at 8.5 (Fig.  11). For mantle enzyme, the Ki values for alanine inhibition are 7.6 mM and 2.7 mM at pH 8.5 and 7.5, respectively.
The adductor enzyme (Fig. 1lA) is inhibited at lower concentrations of alanine (Xi values 3.6 and 0.6 mM, respectively). Fig. 12 shows Ki determination of phenylalanine for mantle and adductor pyruvate kinase activities.
Phenylalanine inhibition of mantle pyruvate kinase is pH-independent, having a Kc value around 6 mu at both pH values examined; again the Ki values for the adductor enzyme are somewhat lower.
Nature of Alanine and Phenylalakne Inhibition-L-Alanine is known to inhibit pyruvate kinase (12)(13)(14) in a manner competitive with respect to P-enolpyruvate.
In contrast, in the case of oyster pyruvate kinases, L-alanine inhibition of both enzymes involves changes in the apparent KmcPEP) and the V,,, at both pH values examined.
As in the case of ATP inhibition, fructose-1 , B-P2 reverses alanine inhibition.
The nature of phenylalanine inhibition differs for the two enzymes, being competitive for the mantle enzyme (Fig. 14, upper panel) and mixed competitive (involving changes in apparent K,cwp, and V,,,) for the adductor enzyme (Fig. 14, lower panel).
For the mantle enzyme, 6 to 10 InM phenglalanine doubles the K,QE~) while V,,, remains Vol. 246, So. 10 almost unaffected. Again fructose-l, 6-P* protects the mantle enzyme against phenylalanine inhibition, 2.5 x 10-e M fructose-1, 6-PZ reversing the inhibition caused by 6 mM phenylalanine. DISCUSSION The data in this study suggest that, as in the mammalian case, the enzyme pyruvate kinase in the oyster occurs in tissuespecific multimolecular forms and that the kinetic properties of each isozyme seem to gear in well with the over-all metabolism of the tissue in which it occurs. Thus, the K, values of P-enolpyruvate and ADP for mantle pyruvate kinase are 3 and 6 times higher than the corresponding values for adductor pyruvate kinase. Under conditions of gluconeogenesis, when P-enolpyruvate is being produced from pyruvate and C-4 acids of the Krebs cycle, any significant simultaneous pyruvate kinase activity would serve merely to recycle carbon at the expense of ATP (15). In mantle, this kind of recycling would not be favored at low P-enolpyruvate concentrations because of the high Michaelis constant for the mantle pyruvate kinase. Also, the mantle enzyme is strongly activated by fructose-l, 6-PZ (causing a large decrease in the Km(PEp)). This may reflect a physiological mechanism whereby pyruvate kinase activity can be increased during glycolysis and markedly decreased during gluconeogenesis, when fructose-l, 6-PZ .concentration may be reduced.
An entirely analogous situation occurs in mammalian tissues. Thus in liver, a major gluconeogenic tissue, the K, cpEp) for pyruvate kinase is an order of magnitude higher than in the highly glycolytic muscle (15,16) and indeed is comparable to the G(PEP) for the mantle enzyme. In this circumstance, too, P-enolpyruvate conversion to pyruvate would not be favored in the glucogenic tissue when P-enolpyruvate concentrations are low. Also, in this case, fructose-l, 6-P2 may act as a specific "on-off" switch on the liver enzyme, but it does not affect the mammalian muscle enzyme. Thus both liver and mantle enzymes seem well adapted for function in a metabolism that involves glycolytic and gluconeogenic function within a single tissue.
The Michaelis constants for P-enolpyruvate and BDP for pyruvate kinases of adductor muscle, fish muscle, and mammalian muscle are rather similar to each other and, as pointed out, are distinctly lower than for the mantle and liver enzymes. Thus, these enzymes would compete favorably for quite low concentrations of P-enolpyruvate for conversion to pyruvate. The adductor muscle pyruvate kinase differs from the mammalian muscle pyruvate kinases, however, in being strongly feed forward-activated by fructose-l, 6-Pz. This has been observed for fish muscle as well, and may be a general characteristic of muscle pyruvate kinase in poikilothermic organisms (17).
The role of fructose-1,6-Pz protection of both mantle and adductor pyruvate kinases is of interest.
In all cases thus far examined, fructose-1,6-Pt is able to reverse ATP inhibition of pyruvate kinase. In addition, in the oyster, fructose-l ,6-P* protects both enzyme forms against alanine and phenylalanine inhibition.
Thus far there is no adequate explanation available for these effects and this is clearly an important area for further research.
In mammalian systems, fructose-l, 6-PZ activation is greatest at alkaline pH values (9). An opposite pH dependence of the fructose-l ,6-PZ activation of the oyster pyruvate kinases is observed. For both enzymes, fructose-l, 6-Pz lowers the K, cpEp) and this effect is particularly striking at lower pH values (at pII 6.5 the K, is reduced from 5.8 X 10u4 M to 8 X 10-j M; at pH 8.5 the K, is reduced from 1.9 X lo+ M to 6.6 X lo-" M).
In consequence, in the presence of fructose-l ,6-PZ the K,cpEp) is essentially pie-illdependellt.
Since the oyster is a facultative anaerobe, the physiological function of fructose-1,6-Pz activation may be to allow I'-enolpyruvate conversion to pyruvate during extended periods of anaerobiosis, when intracellular pH might be reduced.
The adductor enzyme :~ppears to be under tight ATP regulation. Thus, 2 InM ATI', :L value probably within the physiological range (la), causes about a lo-fold increase in the K,(PEP).
Under conditions of low P-enolpyruvate concentrations, it is evident that adductor pyruvatc kinase would be unusually sensitive to ATP.
In this characteristic, the adductor enzyme resembles mammalian muscle pyruvate kinase (16) and adipose pyruvate kinase (19), all of which have similar KicATEa) values, but it differs from msrnlrlaliall brain pyruvate kinase (11) and the mantle enzyme.
In both of the latter, ATP inhibition is noncompetitive.
Because of the high Ki(ATP) for the mantle enzyme, and because ,4TI' does not alter the apparent, k', cPEP), ATP would not be an efficient inhibitor of this enzyme.
In this connection, it is interesting that both mantle enzyme and mammalian brain pyruvate kinase (20, 21) are competitively inhibited by phenylalanine and the Ki values are again similar for the enzymes from the two tissue types. In the mantle, pyruvate kinase activity is fairly sensitive to phenylalanine control since phenylalaninc (at K; concentrations) produces quite large increases in the K,cPEP).
Since phenylalanine concentrations are known to be unusually high in mollusc tissues (22), this amino acid may be an important physiological feedback inhibitor of pyruvate kinase activity in this tissue as it is in mammalian brain.
From the data presented it is speculated that coordinated changes in intracellular corlcerltr:ttiolls of Hf, P-enolpyruvate, fructose-l, 6-PZ, ATP, alanine, and phenylalanine control pyruvate kinase activity in viva.