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Metabolic regulation: could Mn be involved? Vern L. Schramm It is possible that Mn ~" acts as a regulatory signal in metabolism. Phosphoenolpyruvate carboxykinase, a regulated enzyme o f hepatic gluconeogenesis, is considered as a prototypic enzyme for which Mn "2~may act as a regulatory ion. The manganous ion serves an essential role in the metabolism of mammalian liver as a cofactor which binds tightly to enzymes such as pyruvate carboxylase, superoxide dismutase and arginase. Several other enzymes are reported to be activated by Mn ~ or to give greater catalytic rates in the presence of Mn ~ than with Mg ~ . These effects have frequently been invoked as having the potential for metabolic regulation ~. However, regulation by Mn ~" ions has remained speculative owing to the lack of experimental data for intracellular concentrations of free and bound Mn ~" and the lack of kinetic constants for the effects of Mn z' on enzymes. Recently, we have developed magnetic resonance techniques which can distinguish free Mn ~" from bound Mn ~ in intact cells and have determined free and bound Mn ~" in rat hepatocytes z. With the availability of this information, it is opportune to summarize the factors which are required for Mn ~" effects in metabolic regulation. The properties of P-enolpyruvate carboxykinase are examined as a model system for considering enzymic regulation by intracellular concentrations of Mn ~ .
Necessary conditions for enzymic regulation by M n 2' Direct regulation of an enzyme by Mn ~ requires Mn z to bind to the target enzyme followed by a metabolically significant response in catalytic activity. This simple tenet, and the knowledge of Mn ~ concentrations in the cell allows us to predict the properties an enzyme must exhibit to be regulated by Mn ~ in vivo. To permit interaction between Mn ~" and a target enzyme; (1) 7he kinetic constant(s) for the interaction o f Mn ~ must approximate the intracellular free Mn ~ concentration at physiological substrate concentrations. The kinetic constants for Mn ~ dissociation can be evaluated by steady-state kinetic methods. Special precautions are required Vern I.. t;chramm is at the Department of Biochemistry, lemple University School o f Medicine, Philadelphia, PA 19140, U.S.A.
to regulate the concentration of free Mn 2 since substrates, especially those which contain pyrophosphates, are avid chelators of divalent cations. The kinetic constants for Mn 2" must be determined over a range of substrate concentrations to evaluate the effect of substrates on the interaction of Mn 2 . For example, the kinetic constant for Mn 2~ activation of galactosyltransferase changes from greater than 1 mM to less than 1 /~M as a function of substrate and Ca 2~ concentrations ~. The kinetic constants for Mn 2~ should be extrapolated to zero and saturating concentrations of substrate or other effectors in order to estimate the free Mn 2~ concentration required to alter the enzyme at given metabolite concentrations. Kinetic constants for Mn 2 obtained in this manner should be below 5 /xM since free Mn ~ is in the range 0.2-1 /.tM in isolated rat hepatocytesL (2) The binding site(s) for Mn 2~ must discriminate strongly against Mg 2~. Enzymes which are regulated by Mn z" must discriminate between Mg 2~ and Mn 2 to a degree which approximates or exceeds the intracellular ratio of free Mg 2' to free Mn 2~ . The similarity of the chelate structures of these ions accounts for the almost universal ability to substitute Mg 2' and Mn ~ , even though the affinities for the two ions can differ by orders of magnitude in proteins or in model chelating compounds. For example at pH 8, the stability constant for Mn 2 . E D T A is 10'~ times greater than that for Mg 2'.EDTA. With intracellular free Mg 2 of approximately 1 mM '~ and free Mn ~ of 0.2-1 /zM in hepatocytes, a binding site specific for Mn z" must bind Mn 2" at least I(P times tighter than it binds Mg ~ . Steady-state kinetic measurements to determine constants for Mn 2~ must therefore be performed in the presence of Mg 2 . When it is possible to overcome the effects of Mn ~ by excess Mg ~ , direct inhibition experiments can establish the KM~,/KM~. This ratio must equal or exceed 1000 for Mn z specific effects to occur in the presence of physiological concentrations of Mg 2 . (3) 1"he amount o f intracellular, exchangeable Mn 2 must approximate or exceed the molarity o f target
enzymes required for observed metabolic flux. Enzymes or other molecules which contain tightly bound Mn 2~ represent a nonexchangeable pool of Mn 2~ which cannot participate in rapidly reversible binding to target enzymes. The quantity of enzyme protein which could be affected by the reversible binding of Mn 2~ is therefore limited by the amount of exchangeable Mn 2". Estimates of exchangeable ions in the extramitochondrial compartment can be made by the addition ofdigitonin to isolated hepatocytes. Low concentrations of digitonin make the plasma membrane permeable, allowing the release of ions which are freely exchangeable in the cytosolic compartmenP. Estimation of exchangeable Mn 2~ by this technique gives approximately 13 #M Mn 2~ (13 nmol Mn 2" per ml of cell H20)". If the average cytosolic protein is assumed to be of mol. wt 50,000, 13/~M Mn 2~ could saturate 650/xg/ml protein, or 10 such proteins which are present at 65 /xg/ml of cell 1-120. (4) 7he intracellular free Mn 2~ must change in response to altered physiological or hormonal states. Regulation of enzymes by Mn 2~ requires changes in the intracellular concentration of the ion. Such changes could result from the active transport of Mn 2 into the cell, or from redistribution of Mn 2~ between subcellular compartments in response to a metabolic change and/or a homonal signal. Changes in the concentration of free Mn 2" could also occur in response to the induction of proteins which bind Mn 2~ or in response to factors which increase the affinity of target proteins for Mn ~ . The observed change in Mn 2 concentration must be accompanied by a change in the catalytic activity of the target enzymes. These four conditions are necessary to permit Mn2~-mediated regulation of an enzyme. The remainder of this article will consider a specific enzyme, P-enolpyruvate carboxykinase from rat liver cytosol. Each of the four conditions will be evaluated to the extent possible with the experimental evidence currently available.
Could Mn 2~ regulate P-enolpyruvate carboxykinase? The activation of P-enolpyruvate carboxykinase by Mn 2~ was one of the earliest observations of the kinetic properties of the enzyme 7. The physiological role of Mn 2~ activation was later discounted because of the relatively low concentration of total cytosolic Mn ~ in fractionated rat liverL Recent studies of Mn 2 interaction with both mitochondrial and cytosolic P-enolpyruvate carboxykinases~' " and the demonstration that Mn 2 can be rapidly
JIBS - October 1982
370 accumulated by mitochondria ~ suggested that Mn ~' requires re-investigation as a potential effector of P-enolpyruvate carboxykinase. Activation constant for Mn 2 The kinetic constant for activation of P-enolpyruvate carboxykinase by Mn 2~ is 1.7/~M, using the physiological nucleotide (MgGTP 2 ) and maintaining free Mg z~ in excess. P-enolpyruvate carboxykinase can be activated at least 10-fold by Mn 2" , and this effect is predominantly a V ..... effect 9.~°''~. Changes in MgGTW and oxaloacetate over the concentration ranges thought to exist in v i v o have no significant effect on the activation constant for Mn ~ (Ref. 9). For Mn 2' to affect P-enolpyruvate carboxykinase in v i v o , the free Mn 2~ must approximate the activation constant. The free Mn 2 of hepatocytes is estimated directly by placing freshly prepared cells in an electron paramagnetic resonance (EPR) instrument. This is a non-destructive technique and the cells remain viable throughoutL The Mn 2~ EPR spectra from such an experiment are shown in Fig. 1. The experiments indicate that free Mn 2~ is approximately 1.0 p.M and 0.2 p.M in hepatocytes from fed and fasted rats, respectively2.''. These values are sufficiently near the activation constant of 1.7 /xM for Mn 2~ to permit substantial interaction of Mn z~ and P-enolpyruvate carboxykinase at i n - v i v o concentrations of Mn 2~ and the enzyme. Mn ~ / M g 2~ discrimination factor Mg 2' is a competitive inhibitor of the Mn 2~ activation of P-enolpymvate carboxykinase. This relationship allows a direct comparison of the dissociation constants for Mg 2" and Mn 2" at the site which leads to activation. The inhibition constant of 5 mM for Mg ~' gives a Mn2~/Mg 2 discrimination of approximately 3000, sufficient to allow Mn 2" interaction with the enzyme in the presence of free Mg 2 in the millimolar rangeL
E x c h a n g e a b l e M n 2, The total Mn 2~ of rat liver has been reported by a number of investigators to be approximately 35 nmol/ml cell H202. Treatment of cells with digitonin allows release of 13 nmol Mn ~' per ml of cell t-120 (Fig. 1 )% These findings indicate that about one-third of the total Mn 2" is extramitochondrial and is not tightly bound to protein. The cellular concentrations of pyruvate carboxylase, arginase and superoxide dismutase can be calculated to be approximately 20/.~M, thereby accounting for most of the remaining, non-
1 IJM Mn 2+ STANDARD
HEPATOCYTES- FED
TOTAL Mn2+, HEPATOCYTES
TOTAL Mn2+,DIGITONIN TREATED HEPATOCYTES
k~g. l. EPR spectra of Mn ~ in hepatocTtes and hepato¢ yte extracts. Instrument gain in the upper three spectra is approximately 10 times greater than far the lower two spectra. Spectra 2 and 3 are the free Mn ~ in hepatoo'tes. Spectra 4 and 5 are perchloric acid extracts oJ hepato¢yws before and after treatment with digitonin (see Ref. 2 for the methodsof these determinations).
exchangeable Mn ~ . Since the concentration of P-enolpyruvate carboxykinase varies from approximately 3/zM in de-induced liver to 10 /J.M in induced liver '4, a stoichiometeric quantity of exchangeable Mn ~ is available. The catalytic capacity of P-enolpyruvate carboxykinase in induced liver is approximately 10 times greater than the maximum rates of gluconeogenesis. Thus, no more than 10% of the total P-enolpyruvate carboxykinase need be active to account for the observed rates of gluconeogenesis. Approximately 1 p.M Mn 2 would be required to bind to the enzyme for the observed rates of gluconeogenesis. These considerations are consistent with the postulate that adequate extramitochondrial Mn 2 is present to account for Mn 2 activation of P-enolpyruvate carboxykinase as well as other enzymes in rat hepatocytes.
Mn ~ in these cells shows no significant change under the same conditions. The free Mn ~ of rat liver is therefore in dynamic equilibrium with cellular proteins and exhibits sufficient variation to influence target proteins. In the case of P-enolpyruvate carboxykinase, the observed decrease of free Mn 2 in hepatocytes from fasted rats could represent an increased affinity of the enzyme for Mn z thus decreasing the free Mn ~ of the cells. The mechanism for the observed change in free Mn ~ in response to dietary state is unknown, but could be caused by Mn 2 uptake by mitochondria or by induction of molecules which bind Mn 2 . Although no direct experimental evidence is yet available to address these possibilities, there is good evidence that P-enolpymvate carboxykinase can exist in forms with differing responses to Mn 2~ activation'" ':~.
Mn 2" in metabolic states The concentration of flee Mn 2 varies three-fold in hepatocytes depending on whether the rats are fed or fasted 2. Total
Conclusions Four conditions are necessary for the regulation of enzyme steps by Mn z .
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I l B S - October 1982 Determination of Mn 2 activation constants for P-enolpyruvate carboxykinase and the concentration of free and bound Mn 2 in rat hepatocytes has demonstrated that the necessary conditions have been met for an enzyme-Mn~ interaction. Even though the necessary conditions for Mn 2 activation of P-enolpyruvate carboxykinase have been met, additional information is required to determine whether these factors are sufficient for regulation. The relatively large amount of exchangeable Mn ~ , compared to that required for activation of P-enolpyruvate carboxykinase, suggests that more than one protein is regulated by Mn 2 . Another candidate for such regulation is arginase, the cytosolic enzyme responsible for urea formation. Arginase also appears to contain binding sites for
Mn z which may influence the catalytic activity 1~. Acknowledgement This work was supported by research grant AM 25551 from the National Institutes of Health. References 1 Williams, R. J. P. (1982) Flz BS Lett. 140, 3-10 2 Ash, D. E. and Schramm, V. L J. Biol. (hem. (in press) 3 Morrison, J. F. and Ebner, K. E. (1971)J. Biol. (hem. 246. 3977-3984 4 Veloso, D., Guynn, R. W , Oskarsson, M. and Veech, R. L. (19731 .L Biol. (hem. 248, 4811~-819 5 Zuurendonk, P. F. and Tager, J. M 11974) Biochem, Biophys. Acta 333, 39_3-399
Conversion of pyruvate to acetyl CoA under anaerobic conditions For strict and facultative anaerobes, two ahernatives exist to prevent excessive reduction of the NAD-POol. One possibility is to avoid pyruvate oxidation and to convert this substrate to acetyl CoA and formiate by the action of pyruvate formiate lyase. When, on the other hand, pyruvate is oxidized by anaerobes, electrons are transferred to acceptors with a more negative redox potential than NAD, in most cases to ferredoxin, in methanogens to the deazaflavin derivative F42oa. In some organisms flavodoxin may be used alternatively4. Thus the high reducing power of pyruvate can be used for reactions requiring stronger
reductants than NADH; for example, nitrogen fixation, synthesis of other 2-oxoacids, reduction of carbon dioxide to formiate, or the 'excretion' of redox equivalents by evolution of hydrogen. A great advantage for photosynthetic and chemolithotrophic organisms is the possibility to drive 2-oxacid oxidation (which is irreversible with NAD as oxidant) in the opposite direction, provided the reducing power of the electron carrier is negative enough. This reaction represents a net reduction of carbon dioxide and is the basis of carbon dioxide fixation via the reductive carboxylic acid cycles of green photosynthetic bacteria ~ and of not yet fully understood assimilatory pathways of methanogens% 2-Oxoacid : ferredoxin oxidoreductases were therefore often referred to as 2-oxoacid synthases. The example of Escherichia coli shows that facultative anaerobes have retained maximal flexibility by conserving all types of enzymes mentioned above. During aerobic growth they use the pyruvate dehydrogenase complex to channel electrons towards oxidative phosphorylation2. Under anaerobic conditions pyruvate formiate lyase is activated to avoid production of redox equivalents. Although anaerobic pyruvate catabolism is mainly performed by this enzyme, reducing power can be generated, as required, by pyruvate oxidation using flavodoxin or ferredoxin as electron acceptoP. For pyruvate : ferrodoxin oxidoreductases of different organisms mol. wts of 200-300 000 have been determined. Accordingly they are much smaller than dehydrogenase multienzyme complexes. Pyruvate : ferredoxin oxidoreductases have been purified from Clostridium acidi-urici and from Halobacterium halobium and proved to be thiamin diphosphate contain-
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ElsevierBiomedicalPress 1982 0376 5067!82/t'fd~K) ~hii$(ll O0
Pyruvate : ferredoxin o x i d o r e d u c t a s e - new findings on an ancient enzyme Lorenz Kerscher and Dieter Oesterhelt A simple globular enzyme present in arehaebacteria and anaerobic eubacteria served to oxidize pyruvate long before the evolution o f pyruvate dehydrogenase multienzyme complexes of respiratory eubacteria and mitochondria. Formation and breakdown of a stable free radical intermediate are the clues to understanding its unique catalytic mechanism. Oxidative decarboxylation of 2-oxoacids, such as pyruvate and 2-oxoglutarate, is a key reaction of intermediary metabolism. Most organisms exploit the high reducing power of these substratesI for the reduction of a low potential electron carrier and concomitant formation of an energy-rich thioester linkage between coenzyme A and the resulting carboxylic acid. In mitochondria and many respiratory eubacteria this sequence of reactions is catalysed by a highly developed enzyme system, the well-studied 2-oxoacid dehydrogenase multienzyme complexes which use NAD as the electron acceptor. They are large aggregates (mol. wts 3-7 million) of three different enzymes which catalyse the three partial reactions2. The 2-oxoacid dehydrogenase complexes are present in aerobic organisms and fulfil exactly the requirements of respiratory metabolism, i.e. unidirectional transfer of electrons to NAD, the ultimate electron donor of oxidative phosphorylaLorenz Kerseher and Dieter Oesterhelt are at the Max-Planck-lnstitut fiir Biochemie, Am KIopferspitz 18 a, D-8033 Martinsried. F.R.G.
6 Schramm, V. L. and Ash. D. E. (1982) k~d. Proc. 41, 5060 7 Utter. M F. and Kolenbrander, H. M. (19721 Enzymes 3rd edn. 6 pp, 136-154 8 Foster, D. O., Lardy, H. A.. Ray, P. D. and Johnston. J. B. 119671 Biochemi.~trv 6. 21211-2128 9 Schramm. V. L.. Fullin. F. A. and Zimmerman, M. D. 1198113. Biol. (_hem. 256, 10803-10808 I0 Colombo. G. and Lardy, H. A. ,119811 Biochemistry 20, 2758-2767 11 Lee, M. H., Hebda, C. A and Nowak. T. ( 1981 ) J. Biol. (_hem. 256, 12793-12801 12 Lehninger, A. L.. Carafoli. E. and Rossi, C S. (19671 Adv. Enzymol. 29,259-320 13 Brinkworth, R. I., Hanson, R. W., Fullin. F. A, and Schramm, V. L. (1981)J. Biol. (hem. 256, 11/795-10802 14 Ballard, F. J. and Hanson. R. W. (19691J. Bh)I. (hem. 244. 5625-5630 15 Hirsch-Kolb. H., Kolb. H. J. and Greenberg, D, M (1971)J. Biol. (hem. 246,395-401
tion. Under anaerobic conditions, NAD would tend to act as an electron trap with limited capacity and a redox potential unsuitable for easy removal of redox equivalents by the hydrogenase reaction. Other enzymes then have the important task of converting the pyruvate produced by carbohydrate metabolism into the acetyl CoA required for many important biosyntheses.