Research: Science and Education edited by
Concepts in Biochemistry
William M. Scovell Bowling Green State University Bowling Green, OH 43403
Lactate Dehydrogenase Catalysis: Roles of Keto, Hydrated, and Enol Pyruvate J. E. Meany Department of Chemistry, Seattle University, Seattle, WA 98122-4340; *
[email protected] Carbonyl compounds are substrates for many oxidoreductase enzymes. In their aqueous solutions in vivo, these compounds exist as equilibrated mixtures of keto, hydrated (gem-diol), and enol species:
CH2 C(OH)CO2− enol
−
CH3COCO2 keto
+H2O −H2O
−
CH3C(OH)2CO2 hydrate
Interconversions between these compounds are catalyzed by acids and bases (1–15), as well as by certain enzymes (16– 18). Some oxidoreductases utilize one form (keto, hydrate, enol) as the enzyme substrate but are inhibited by another form (19). Thus recognition of the roles of keto, hydrated, and enol forms in certain oxidoreductase reactions may lead to an understanding of an important mode of metabolic regulation, a topic of major importance in both introductory and advanced biochemistry courses. It is hoped that this article will encourage advanced biochemistry students to assess the physiological importance of this type of metabolic regulation by lactate dehydrogenase when asked to critically evaluate pertinent journal articles or biochemistry text passages. Numerous pyruvate-converting enzymes exist (17, 18– 24). Pyruvate is the product of aerobic glycolysis and forms a bridge between the metabolism of carbohydrates and several amino acids. Under anaerobic conditions, pyruvate is reduced to lactate by the coenzyme NADH, in a reaction catalyzed by lactate dehydrogenase (LDH:EC1.1.1.27). Under aerobic conditions, the same enzyme catalyzes the oxidation of lactate by NAD + . The catalytic efficiency of pyruvate-converting enzymes may depend on the distribution or rates of interconversion between keto, hydrated and enol pyruvate. Keto pyruvate has been shown to be the preferential substrate for LDH (21, 22). It is also known that LDH is sensitive towards “substrate” inhibition by some form of pyruvate and it has been reported that hydrated pyruvate (21, 24) or enol pyruvate (25, 26) inhibits the enzyme by forming an abortive ternary complex with the stoichiometry LDH–NAD+–“pyruvate”.
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The original research suggesting that enol pyruvate was involved in the formation of the abortive ternary complex was reported by Coulson and Rabin (25) and has been cited extensively and perpetuated in the literature. However, advanced biochemistry students may be invited to critically evaluate the data included in that article in light of the evidence presented in ref 24 in this article that supports the involvement of hydrated pyruvate in forming the abortive complex. Under physiological conditions, pyruvate in aqueous solution reversibly undergoes enolization and hydration (27), as shown at the left. In neutral or acidic aqueous solutions, the enol form accounts for only a minute fraction of the total concentration of the pyruvate system (Table 1). On the other hand, substantial quantities of the hydrated species exist in pyruvate solutions; their concentrations vary with temperature and pH (Table 1). It is known that the manifestation of “substrate” inhibition for LDH is considerably more pronounced for the isoenzymes in heart muscle than those in smooth or skeletal muscle (12, 19, 23, 26, 28). The physiological importance of substrate inhibition for the heart isoenzymes has been questioned (29, 30) and many biochemical texts have included discussions of its mechanism and physiological relevance. This article will address two important questions concerning the LDH-catalyzed reduction of pyruvate by NADH: (i) what roles do the keto, hydrated, and enol pyruvate species play in this enzymatic process and (ii) when, if ever, is “substrate” inhibition physiologically important in LDH catalysis? Literature Data The equilibria between the chemical species of the pyruvic acid–pyruvate system in aqueous solution and the LDHcatalyzed reduction of pyruvate are shown in Scheme I. Acidic solutions of pyruvic acid (pKa = 2.18) are extensively hydrated (e.g., hydrate = 86.3% at 5.0 ⬚C), whereas solutions of pyruvate anion are hydrated to a lesser extent (hydrate = 13.6% at 5.0 ⬚C) (Table 1) (27). The extent of the enolization of pyruvic acid (enol = 0.002% at 5.0 ⬚C and pyruvate anion (enol = 0.001% at 5.0 ⬚C) are both very small (Table 1) (24). Earlier experiments (21) utilized small quantities of pre-acidified solutions of pyruvic acid (pH < 2.18) to initiate the LDH-catalyzed reduction of pyruvate in buffered solutions (pH final = 6.8). Under these conditions, the initial pyruvic acid hydrate (86.3%) instantly was deprotonated to hydrated
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pyruvate anion, which began its first-order dehydration. The rate of the enzymatic reduction of pyruvate began slowly and increased as the dehydration of pyruvate commenced. Furthermore, it was observed that the increase in enzymatic reduction rate exactly paralleled the rate of dehydration of the highly hydrated pyruvic acid molecule to the less hydrated keto pyruvate anion. Thus it was demonstrated by this work that keto pyruvate (rather than hydrated pyruvate) serves as the substrate for LDH (21). Earlier work was also carried out to identify the “substrate inhibitor” of the heart isoenzymes of LDH, that is, keto, hydrated, or enol pyruvate (23). In that study, initial rates of NADH oxidation were determined at various percentages of keto, hydrated, and enol pyruvate, which were controlled by pH and temperature. The degree of “substrate inhibition” was determined as a function of each of these chemical species. A typical set of experiments utilized the addition of small quantities of four different pyruvate stock solutions as the last reaction component to phosphate buffer, pH 6.8 at 5.0 ⬚C: (i) prepared in pH 6.8 phosphate buffer and preincubated at 41 ⬚C (initial hydrated pyruvate = 2.7%), (ii) prepared in pH 6.8 phosphate buffer and preincubated at 5.0 ⬚C (initial hydrated pyruvate = 13.6%), (iii) prepared in HCl and preincubated at 41 ⬚C, resulting in an aqueous pyruvic acid solution, pH = 0.45 (initial hydrated pyruvic acid = 45.7%), and (iv) prepared in HCl and preincubated at 5.0 ⬚C, resulting in an aqueous pyruvic acid solution, pH = 4.5 (initial hydrated pyruvic acid = 86.3%).1 When solutions i–iv were added to the reaction cuvet, initial velocities were determined from kinetic runs carried out under identical conditions of pH = 6.8 and temperature of 5.0 ⬚C. Thus, these aqueous solutions of pyruvate anion initially were equilibrated systems containing the percentages of keto, hydrated, and enol forms indicated in Table 1. The results of such experiments are given in Table 1, which correlates the initial percentages of the keto, hydrated, and enol forms of pyruvate and the resultant per-
Table 1. Substrate Inhibition of LDH in the Presence of Var ying Percentages of Keto, Hydrated, and Enol Pyruvate
T/ºC
41
5.0
41
Keto (%)
13.7
54.3
86.4
97.3
Hydrate (%)
86.3
45.7
13.6
2.7
Enol (%)
0.002
0.004
0.001
0.003
Inhibition (%)
~100
80
30
15
NOTE: Values calculated from Michaelis–Menten plots of velocity vs pyruvate concentration in a typical set of experiments described in text (i–iv). Percentages of inhibition were determined at the points on the graphs where the pyruvate concentration was 5 x 10−4 M.
centages of “substrate inhibition”. The calculation of the percentages of inhibition is described in ref 23. The data in Table 1 show that LDH inhibition is increased by the same experimental conditions that increase the percent of hydration and that substrate inhibition is diminished when the equilibrium position favors keto pyruvate. It will be noted that these observations cannot be explained in terms of inhibition by enol pyruvate since the solutions containing lower enol but higher hydrate content cause greater percentages of inhibition (see, especially, the data in the first column of the table). Pyruvate inhibition of LDH, as observed from initial velocity studies similar to those illustrated by the data in Table 1, has been attributed to the formation of a relatively loosely bound (and rapidly formed) abortive ternary complex consisting of LDH, NAD+, and the inhibitory form of pyruvate (30). The data in Table 1 are consistent with the identification of the inhibitory form of pyruvate as hydrated pyruvate.
−
+H+
pyruvic acid hydrate −H2O
Pyruvate Anion As Last Component (pH = 6.8)
5.0
−H+
CH3C(OH)2CO2H
Pyruvic Acid Solution As Last Component (pH = 0.45)
Parameter
CH3C(OH)2CO2 pyruvate hydrate −H2O
+H2O −H
CH3COCO2H
+H2O
+
pyruvic acid
−H+
NADH NAD+
L-lactate
−
+H+
pyruvic acid enol
CH3CH(OH)CO2−
CH3COCO2 pyruvate
CH2 C(OH)CO2H
LDH
−
+H+
CH2 C(OH)CO2 pyruvate enol
Scheme I. Equilibria between the chemical species of the pyruvic acid–pyruvate system in aqueous solution and the LDH-catalyzed reduction of pyruvate.
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In other experiments, it was shown that the aforementioned substrate inhibition of heart LDH more than doubled when the buffered solutions of pyruvate were incubated for various times along with LDH and the oxidized form of the coenzyme, NAD+. The observed increase in the extent of LDH inhibition by these preincubated solutions was a firstorder process with a half-life of approximately 12 min (23). This observation is consistent with earlier work that postulated the existence of a second mode of LDH inhibition that results from the relatively slow reversible formation of a more stable inhibitory ternary complex, again, with the stoichiometry LDH–NAD+–“pyruvate”(30, 31). In a related reaction, LDH catalyzes the oxidation of glyoxylate hydrate to oxylate (32):
HC(OH)2CO2− + NAD+ HOCOCO2− + NADH It has been shown that this bi bi ordered reaction involves the formation of the ternary complex LDH–NAD + – HC(OH)2CO2−, which is directly analogous to the structure proposed in the present article for the abortive ternary complex LDH–NAD+–CH3C(OH)2CO2− for the enzymatic reduction of pyruvate. For the bi bi ordered oxidation of L-lactate, the complex LDH–NAD+–CH3C(OH)CO2− exists. In this regard, it is interesting to compare the structures of lactate and hydrated pyruvate. While it is noted that hydrated pyruvate has two hydroxyl groups at C2 and lactate has only one, the sp3 hybridized nature of C2 is common for both 2,2-dihydroxypropanoate (hydrated pyruvate) and 2-hydroxypropanoate (lactate). Given this structural similarity and the fact that the enzyme–NAD+ complex accommodates glyoxylate hydrate, it is easily conceivable that the enzyme– NAD+ complex would also accommodate hydrated pyruvate.
A number of researchers have considered the physiological significance of the phenomenon of “substrate inhibition” for the LDH-catalyzed reduction of pyruvate and several authors have declared that any such significance is negligible (29, 30). However, in light of the discussion in this article, instructors of biochemistry courses and their students may recognize that further consideration should be given to this question in view of the following: (a) The substrate inhibition involves a ternary abortive complex, the stoichiometric composition of which is LDH–NAD+–“pyruvate” (probably hydrate). (b) The slowly formed and more stable inhibitory ternary complex causes more potent inhibition of LDH. It is this inhibitory complex that would be operative under physiological conditions since LDH, both forms of the coenzyme (NADH and NAD+), and pyruvate coexist together and incubate in aqueous solution. (c) For cold-blooded animals, the phenomenon of “substrate inhibition” may indeed be physiologically more important at lower temperatures since the exothermic hydration of the pyruvate system greatly increases the percentage of inhibitory pyruvate hydrate under these conditions (Table 1). Acknowledgments The author wishes to thank Vicky Minderhout (Biochemistry program at Seattle University) for her valuable suggestions in the preparation of this manuscript. Special thanks also to P. J. Alaimo at this institution for proof reading the MS and for his helpful insights. Note 1. It should be noted that when these pyruvic acid solutions were added to the reaction mixture, appropriate quantities of sodium hydroxide were also added to neutralize the acid.
Discussion
Literature Cited
The dual interaction of the carbonyl and gem-diol forms of substrates with oxidoreductase enzymes is not without precedent. For example, in the oxidation of acetaldehyde catalyzed by xanthine oxidase, it was demonstrated that while acetaldehyde serves as preferential substrate, acetaldehyde hydrate inhibits the enzymatic reaction (33). Somero (34) has demonstrated that the pyruvate inhibition observed for muscle LDH isolated from the fish Gillichtgys mirabilis is more pronounced at colder temperatures than at warmer temperatures. He suggested that this phenomenon is a property of the enzyme itself and further that it is beneficial for such fish since oxygen tensions in water are higher at colder water temperatures. He reasoned that the energetically more efficient aerobic glycolytic pathway (allowing pyruvate to move directly towards the TCA cycle) is favored in colder waters while anaerobic glycolysis (involving the conversion of pyruvate into lactate) is slowed by the “substrate inhibition” of LDH. In another study, however, it was shown that for trout muscle LDH, the increased sensitivity of substrate inhibition with decreasing temperatures is not a property of the enzyme itself, but is actually due to the equilibrium shift that increases the fraction of hydrated pyruvate at lower temperatures (35).
1. Damitio, J.; Smith, G.; Meany, J. E.; Pocker, Y. J. Am. Chem. Soc. 1992, 114, 3081. 2. Craig, M. M.; Baff, M.; Gresser, M. J. J. Am. Chem. Soc. 1988, 110, 3302. 3. Rendina, A. R.; Hermes, J. D.; Cleland, W. W. Biochemistry 1984, 23, 5148. 4. Sorensen, P. E.; Jencks, W. P. J. Am. Chem. Soc. 1987, 109, 4675. 5. Gregory, D.; Goodman, P. A.; Meany, J. E. Biochemistry 1972, 11, 4472. 6. Miller, B. A.; Leussing, D. L. J. Am.. Chem. Soc. 1985, 107, 7146. Leussing, D. L. J. Org. Chem. 1990, 55, 666. Emly, M.; Leussing, D. L. J. Am. Chem. Soc. 1981, 103, 628. Leussing, D. L.; Emly, M. J. Am. Chem. Soc. 1984, 106, 443. 7. Pocker, Y.; Meany, J. E. J. Phys. Chem. 1967, 71, 3113. Pocker, Y.; Dickerson, D. G. J. Phys. Chem. 1969, 73, 4005. 8. Schellenberger, A.; Hubner, G. Chem. Ber. 1965, 98, 1938. 9. Meany, J. E. J. Phys. Chem. 1969, 73, 3421. 10. Hegazi, M.; Meany, J. E. J. Phys. Chem. 1972, 76, 3121. 11. Albery, W. J.; Bell, R. P. Trans. Faraday Soc. 1965, 61, 1194. 12. Burgner, J. W., II; Ray, W. J. Biochemistry 1974, 13, 4229. 13. Germay, T.; Meany, J. E.; Pocker, Y. 45th Northwest Regional Meeting, American Chemical Society, Salt Lake City, UT, 1990, Abstract No. 255.
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27. Pocker, Y.; Meany, J. E.; Nist, B. J.; Zadorojny, C. J. Phys. Chem. 1969, 73, 2879. Strehlow, H. Z. Elektrochem. 1962, 66, 392. Eigen, M.; Kustin, K.; Strehlow, H. Z. Phys. Chem. 1962, 31, 140. Gold, V.; Socrates, G.; Crampton, M. R. J. Chem. Soc. 1964, 5888. Griffiths, V. S.; Socrates, G. Trans. Faraday Soc. 1967, 673. Becker, M. Ber. Bunset-Ges. Phys. Chem. 1964, 68, 669. 28. Plagemann, P. G. W.; Gregory, K. F.; Wrobiewski, F. Biochem. Z. l961, 334, 37. Stambaugh, R.; Post, P. J. Biol. Chem. 1966, 441, 1462. Pesce, A.; Fondy, T. P.; Stolzenbach, D.; Castillo, D.; Kaplan, N. O. J. Biol. Chem. 1967, 241, 2151. Berg, Jeremy M.; Tymoczko, John L.; Stryer, L. Biochemistry, 5th ed.; Freeman and Company: New York, 2002; p 460. 29. Manchester, K. L. Biochem. Ed. 1994, 22, 91. 30. Burgner, J. W.; Ainslie, G. R.; Cleland, W. W.; Ray, W. I. Biochemistry 1978, 17, 1646. 31. Everse, J.; Barnett, R. E.; Thorne, C. J. R.; Kaplan, N. O. Arch. Biochem. Biophys. 1971, 143, 444. 32. Warren, W. A. J. Biol. Chem. 1970, 245, 1675. 33. Gregory, D.; Goodman, P. A.; Meany, J. E. Biochemistry 1972, 11, 4472. Goodman, P. A.; Meany, J. E. Biochemistry 1974, 13, 3254. 34. Somero, G. N. Comp. Biochem. Physiol. 1973, 44B, 205. 35. Robinson, D. H.; Ju, J.; Meany, J. E. 41st Northwest Regional Meeting, American Chemical Society, Portland, OR, 1986, Abstract No. 195.
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