Brief Critical Reviews
September 1992: 263-274
The Pyrroloquinoline Quinone (PQQ) Coenzymes: A Case of Mistaken ldent ity Recent evidence has failed to support the claim for a pyrroloquinoline quinone (PQQ) cofactor in mammalian enzymes previously reported to have PQQ. The validity of the original analysis now has been questioned, and a second cofactor, topa quinone, has been identified in at least one enzyme.
Enzymes that exchange electrons between substrates (collectively known as oxidoreductases) must have a specific organic cofactor or a metal ion to prevent electrons in transit from modifying the protein structure. Nutrition plays a role in this process in that two of the better known “redox” cofactor pairs, NAD(P)+/NAD(P)H and FAD/ FADH,, are derived from vitamins niacin and riboflavin, respectively. With the discovery of pyrroloquinoline quinone (PQQ), Salisbury et al.’ and Duine and Frank2 introduced a third redox cofactor to the family, along with the term “quinoprotein,” to specify enzymes bearing the novel PQQ cofactor.3 It seemed only a matter of time before PQQ would also be discovered in mammalian enzymesan event that came nearly two years later when two groups working independently reported a PQQ-like compound in mammalian enzymes.435Before long, PQQ had captured the attention of a world audience. Chemists rushed to confirm its structure, and nutritionists, eager to learn if PQQ qualified as a new vitamin,6 conducted whole-animal studies to determine whether PQQ deprivation under controlled conditions altered the growth and development of laboratory animals. Some of the excitement surrounding PQQ, however, was dampened at a recent worldwide conference in Italy. At least three laboratories were unable to confirm PQQ as a cofactor in three mammalian enzymes previously shown to contain From this meeting also came the announcement of another cofactor that could have been mistaken for PQQ. This compound, a derivative of tyrosine, has been identified as peptide-bound trihydroxyphenylalanine (6-hydroxydopa, or topa This review was prepared by Edward D. Harris, Ph.D., at the Department of Biochemistry and Biophysics, Texas A & M University, College Station, TX 77843-2128. Nutrition Reviews, Vol. 50, No. 9
quinone; Figure 1). Unlike PQQ, topa quinone is very much within the biosynthetic capabilities of animals. In this update we will examine the evidence for and against a role for PQQ in biologic systems and will, perhaps, learn why careful confirmatory research should never be compromised. Historical Perspective PQQ (Figure 2) was first observed’ in bacteria as a cofactor for an alcohol dehydrogenase, an enzyme that permitted Pseudomonas TPI to survive on methane, methanol, and other one-carbon sources. PQQ was bound firmly to the enzyme and positioned to receive electrons directly from the substrate in analogous fashion to flavin (FAD) and nicotinamide (NAD+) cofactors. A structural analysis showed that PQQ had a molecular weight of 424 Da and an empirical formula of C,,H,,N?O, 2H20. There was also evidence for an orthoquinone group in the molecule. The cofactor was given the trivial name “methoxatin” and, through more testing, was found to have a redox potential of +0.090 volts at pH 7.0, which made it very suitable to participate in the oxidation of methanol.’ Comparing fluorescence spectra from bacterial and mammalian enzymes, Duine and colleagues4 and Ameyama et al.’ independently concluded that beef plasma and hog kidney amine oxidase enzymes also could be classified as quin~proteins.~.’ Supporting evidence was obtained when compounds believed to be the dinitrophenyl (DNP) and other hydrazones of PQQ were isolated from the enzyme after reacting with phenylhydrazines. These experiments were followed by a broader examination of all amine oxidases, with the hope and perhaps expectation that PQQ would be the unifying cofactor giving each its catalytic property. Duine et al., had established the methodology and defined the experimental parameters to characterize PQQ cofactors
OH
Figure 1. 6-hydroxydopa (topa) quinone. 263
L .
I
\\
0 Figure 2. Pyrroloquinoline quinone (PQQ or methoxatin).
in enzymes. In due time, lysyl oxidase," dopamine P-monooxygenase," galactose oxidase,l* and choline dehydr~genase'~ were reported to have a PQQ cofactor. DNP derivation became a standard for analyzing PQQ in enzymes. PQQ Enzymes: The Doubt Is Raised
At a meeting in Manziana, Italy, in 1990, a session was set aside to discuss PQQ cofactors in copper enzymes. The session provided a forum for scientists to present evidence for and against PQQ. Experimentally, the focus was on the origin and structure of the phenylhydrazine derivative isolated from copper enzymes. Because of its link with amine groups, pyridoxal phosphate (PLP) was considered a candidate for the cofactor. One amine oxidase in particular, lysyl oxidase, had shown a PLP-like substance at its active site.I4 However, when a powerful new analytical tool, 3 1 ~ - n u c ~ emagnetic ar resonance (NMR), was applied to amine oxidases, no evidence for a phosphate group was discovered, thus raising doubts as to PLP's presence. Moreover, an analytical technique referred to as "reductive trapping" failed to detect a single carbonyl group in another amine oxidase." PLP was therefore removed from the list of cofactor candidates and attention was turned to PQQ. Three key mammalian enzymes-galactose oxidase, dopamine P-monooxygenase, and bovine serum amine oxidase-previously had been added to the list of PQQ-containing proteins. As the new evidence for each of the enzymes was discussed, it became clear that a cofactor role for PQQ was more likely to be disputed than confirmed. For example, a thorough chemical analysis of the functional galactose oxidase enzyme strongly supported the existence of a rare, stable tyrosyl radical in the structure, but no quinone-like structure.'6 Similarly, although dopamine P-monooxygenase reacted positively with phenylhydrazine, newer findings showed a reaction with the histidyl residues in the enzyme. l 7 There was no evidence for phenylhydrazine reacting with PQQ. These results cast strong doubts on the claims for PQQ and made suspect the evidence favoring PQQ in other enzymes. The case for PQQ was presented by Duine and colleague^,^ who reported on the interaction of the 264
bovine enzyme with hydrazines and other carbonyl reagents and the subsequent spectrographic analysis supporting PQQ as the hydrazone adduct. Phenylhydrazine and p-nitrophenylhydrazine adducts had been shown (before PQQ was known) to be powerful agents for detecting carbonyl groups in proteins. In the analyses of Duine and colleague^,^ the complex isolated from the bovine enzyme had spectroscopic and chromatographic properties identical to the PQQ-phenylhydrazone obtained from methylotropic bacteria (Figure 3). In both cases, the adduct was produced by treating the enzyme with moderately high amounts of phenylhydrazine in the presence of oxygen, followed by a rather harsh destruction of the protein with pronase. The organic factor released by the digestion and subsequently extracted by hexanol was identified by high-performance liquid chromatography (HPLC) and 'H-NMR. These analyses all supported the identity of the unknown as a PQQ adduct of phenylhydrazine. Despite a rather convincing display of analytical evidence, a lingering doubt still pervaded the research. The questions asked did not challenge the spectrographic evidence, but, rather, the origin of the factor that gave the spectra. Was this indeed PQQ, or did some unexpected chemical change in the protein lead to the formation of a compound that resembled PQQ, mimicking its absorption data? Could this unknown in fact arise as a result of the rather unfavorable conditions imposed? These questions formed the bases for legitimate concerns and led to adoption of a different protocol for determining PQQ's presence. Is the Cofactor Topa Quinone? Disputing the claim for one cofactor is only a partial solution to the PQQ problem. If the cofactor was not PQQ, what was it? Applying less rigorous conditions to the analysis, Klinman's group'* used [U-'4C]phenylhydrazine (to avoid excess phenylH,
H&$>
/ \
HOOC
N
COOH
N-
II
/GO
N
I NH I
NO,
Figure 3. 2,4-dinitrophenylhydrazoneof PQQ. Nutrition Reviews, Vol. 50, No. 9
hydrazine exposure) and thermolysin, a low-specificity protease, to prepare smaller I4C-labeled peptide fragments from bovine serum amino oxidase. The labeled peptides, produced with a 40% yield, gave rise to a series of absorption peaks dominated by a single peak that absorbed light at the same wavelength as a phenylhydrazone adduct. Using HPLC, Klinman’s group” succeeded in isolating a single peptide whose structure was shown by sequence and mass spectra analysis to be LEU-ASNX-ASP-TYR. The peptide’s unknown X residue became the object of intensive analysis. Was it an amino acid attached to PQQ? By applying an analytical technique called “liquid secondary ion mass spectrophotometry” (LSIMS), an ”N-labeled fragment of the peptide (referred to as a molecular ion or MH+) was shown to have a molecular mass of 808.3. Only two carboxyl groups were found-one associated with aspartic acid, while the other could only be the C-terminal of tyrosine. Thus, PQQ with three carboxyl groups in an underivatized state (Figure 2), could not possibly be part of residue X. What, then, was X? By subtracting the atomic weights of Leu, Asn, Asp, and Tyr, the molecular mass of X was deduced to be 283.0967. Since the molecular mass of PQQ is 470, there was further reason to eliminate PQQ from all consideration. Thus, X was a compound that had not been characterized previously. Based on elemental composition, it was determined that X must have the empirical formula C,SH,,N,O,. Considering the possibilities, only two structures ratio-
nally fit the formula for X. One was a serine residue linked to the phenylhydrazone through an uncommon ether linkage, and the second structure, and perhaps the more probable, was a derivative of tyrosine in which two oxygen groups had been added to the ring (Figure 1). Based on mass spectrometry and other evidence, this trihydroxyphenylalanine derivative, or 6-hydroxydopa (topa) quinone, was the leading candidate. To be certain, however, it was necessary to synthesize topa and compare its properties with the phenylhydrazone derived from the enzyme. These results showed that the spectra for the synthetic topa closely matched the enzymederived compound. For example, X in the pentapeptide showed an absorption maximum at 457 nm, and nitrophenylhydrazone of topa hydantoin absorbed maximally at 456 nm-almost exactly the same wavelength. An absorption at 456 nm was also close to the value of 468 nm reported for phenylhydrazones of PQQ. In both instances, the presence of peptide side chains may have caused a slight shift in the maximal wavelengths. A telling blow came when PQQ hydrazone and topa hydantoin (the respective adducts) were analyzed by Raman resonance spectrometry and the resulting vibrational spectra were compared with the bovine enzyme cofactor.’’ The results (Figure 4) clearly showed that the multiple peaks generated by the active site cofactor (panel B) aligned more closely with topa hydantoin than with PQQ or PLP. The pattern for PQQ (panel C) was distinct in giving a strong absorption at wavenumber 1159 and three additional moderately strong peaks between wave
I\
A
D
-cI
500
1000
Ramen S h i f t ( w a v e n u m b e r )
Nutrition Reviews, Vol. 50, No. 9
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Figure 4. Resonance Raman spectra for p-nitrophenylhydrazones of (A) topa-hydantoin; (B) activesite peptide of bovine plasma amine oxidase; (C) PQQ; (D) pyridoxal 5 ’ phosphate. Reprinted with permission of Brown et a1.19
265
numbers 1425 and 1335. These features were absent from topa hydantoin (panel A) and the enzyme. On the other hand, only topa quinone and the enzyme had components greater than 1600. The spectral evidence challenged the presence of PQQ (or pyridoxal S'phosphate) in the bovine enzyme. What Went Wrong?
The issue now turned to how PQQ could have been mistaken for topa in copper enzymes. One objection raised was that a prolonged treatment of the enzyme with phenylhydrazine in the presence of oxygen, followed by protein digestion, could create derivatives from amino acids in the protein, rather than from PQQ. Due to the reactive nature of the chemicals, such reactions could occur at any stage of the isolation process. Wanton oxygenation was considered an important variable to be controlled in all future experiments. As one of the more revealing experiments had shown earlier, a tyrosine ethyl ester was converted to a PQQ-like product by a simple oxidative reaction.20 Faced with mounting evidence against PQQ, Duine's group' retracted some of their original conclusions for amine oxidase and offered a conciliatory statement indicating that strong evidence supported the view that amine oxidases contain a covalently bound cofactor with a quinone character and that the hydrazine method would not discriminate between PQQ and amino acids in the protein that react with hydrazine. They noted, moreover, that reactions occurring after the hydrazine had reacted would form the final products and that these might not be the compounds originally bound. The authors concluded that 6-hydroxydopaquinone, identified in the bovine serum enzyme, was a good candidate for the cofactor with quinone-like properties, although the absorption spectra and chromatographic behavior of topa dinitrophenylhydrazone and PQQ phenylhydrazone would need to be significantly different to permit distinguishing one from the other. Neither PQQ or TOPA Quinone Is Confirmed
Unfortunately, the PQQ cofactor debate has not been completely settled. A recent paper describes PQQ in the reproductive organs of higher plants and as part of the composition of pollen grains.21These findings suggest that PQQ's presence is not limited strictly to microorganisms. Indeed, while the presence of PQQ in bovine serum amine oxidase has been unequivocally disproved, the cofactor in at least two copper oxidases-lysyl oxidase and benzylamine oxidase, both suspected of containing bound PQQ-has not yet been identified. An early report that PQQ was present in lysyl oxidase had 266
been based on the phenylhydrazine analysis" and a negative finding for pyridoxal phosphate.22 In a rather startling nutritional discovery, Rucker and coworkers23 have shown that rats and mice fed PQQ-deficient diets develop classical symptoms of lathyrism, a defect based on lysyl oxidase impairment. Moreover, the levels of lysyl oxidase in the tissues are significantly lower in animals fed the PQQ-deficient diets. Thus, the PQQ vitamin hypothesis is still alive today, although barely. As a consequence of the discovery of topa quinone in the structure of proteins, our understanding of how amino acids already existing within a protein can be derivatized to a functional cofactor has been enhanced, as has our knowledge of enzyme cofactors in general. As for PQQ, anyone wishing to further the understanding of this compound can draw on the considerable experience resulting from this intriguing controversy. 1. Salisbury SA, Forrest HS, Cruse WBT, Kennard 0. A novel coenzyme from bacterial primary alcohol dehydrogenases. Nature 1979;280:843-4 2. Duine JA, Frank J. The prosthetic group of methanol dehydrogenase: purification and some of its properties. Biochem J 1980;187:221-6 3. Duine JA, Frank J. Quinoproteins: a novel class of dehydrogenases. Trends Biochem Sci 1981 ;6: 278-80 4. Lobenstein-Verbeek CL, Jonjegan JA, Frank J, Duine JA. Bovine serum amine oxidase: a mammalian enzyme having covalently bound PQQ as a prosthetic group. FEBS Lett 1984;170:305-9 5. Ameyama M, Hayashi M, Matshshita K, Matsushita K, Shinagawa E, and Adachi 0. Microbial production of pyrroloquinoline quinone. Agric Biol Chem 1984 ;481561-5 6. Is pyrroloquinoline quinone a cofactor derived from an undiscovered vitamin? Nutr Rev 1988;46: 139-42 7. Beinert H. Copper in biological systems. A report from the 6th Manziana Conference, September 23-27, 1990. J lnorgan Biochem 1991;44:173-218 8. Klinman JP, Dooley DM, Duine JA, Knowles PF, Mondovi B, Villafranca JJ. Status of the cofactor identity in copper oxidative enzymes. FEBS Lett 1991;282:1-4 9. Duine JA, Frank J, Jongejan JA. Detection and determination of pyrroloquinoline quinone, the coenzyme of quinoproteins. Anal Biochem 19833 33: 239-43 10. Williamson PR, Moog RS, Dooley DM, Kagan HM. Evidence for pyrroloquinoline quinone as the carbony1 cofactor in lysyl oxidase by absorption and resonance Raman spectroscopy. J Biol Chern 1986;261:16302-5 11. van der Meer RA, Jongejan JA, Duine JA. Dopamine @-hydroxylasefrom bovine adrenal medula contains covalently bound PQQ. FEBS Lett 1988; 231 :303-7 Nutrition Reviews, Vol. 50, No. 9
12. van der Meer RA, Jongejan JA, Duine JA. Pyrroloquinoline quinone as cofactor in galactose oxidase (EC 1.1.3.9). J Biol Chem 1989;264:7792-4 13. Ameyama M, Shinagawa E, Matsushita K, Takimot0 K, Nakashima K, Adachi 0. Mammalian choline dehydrogenase is a quinoprotein. Agric Biol Chem 1985;49:3623-6 14. Levene CI, O’Shea MP, Carrington MJ. Protein lysine 6-oxidase (lysyl oxidase) cofactor: methoxatin (PQQ) or pyridoxal? Int J Biochem 1988;20: 1451-6 15. Harmann C, Klinman JP. Reductive trapping of substrate to bovine plasma amine oxidase. J Biol Chem 1987;262:962-5 16. Whittaker MM, Whittaker JW. The active site of galactose oxidase. J Biol Chem 1988;263:6074-80 17. Robertson JG, Kuman A, Mancewicz JA, Villafranca JJ. Spectral studies of bovine dopamine P-hydroxylase. Absence of covalently bound pyrroloquinoline quinone. J Biol Chem 1989;264: 19916-21 18. Janes SM, Mu D, Wemmer D, et al. A new redox cofactor in eukaryotic enzymes: 6-hydroxydopa at
19.
20.
21.
22.
23.
the active site of bovine serum amine oxidase. Science 1990;248:981-7 Brown DE, McGuir MA, Dooley DM, Janes SM, Mu D, Klinman JP. The organic functional group in copper-containing amine oxidases. Resonance Raman spectra are consistent with the presence of TOPA quinone (6-hydroxy quinone) in the active site. J Biol Chem 1991;266:4049-51 Buchi J, Botkin JH, Lee GCM, Yakushijin K. A synthesis of methoxatin. J Am Chem SOC1985;107: 5555-6 Xiong LB, Sekiya J, Shimose N. Occurrence of pyrroloquinoline quinone (PQQ) in pistils and pollen grains of higher plants. Agric Biol Chem 1990; 54:249-50 Williamson PR, Kittler JM, Thanassi JW, Kagan HM. Reactivity of a functional carbon moiety in bovine aortic lysyl oxidase. Evidence against pyridoxal 5’-phosphate. Biochem J 1986;235:597605 Kilgore J, Smidt C, Duich L, et al. Nutritional importance of pyrroloquinoline quinone. Science 1989;245:850-2
Body Weight, Fat Storage, and Alcohol Metabolism Ethanol account for a significant fraction of the energy intake of persons consuming even moderate amounts of alcohol. A recent study has shown that although alcohol does not reveal itself as a layer floating at the top of a drink, metabolically it behaves more like oil than sugar.
Alcohol accounts for slightly more than 5% of the nutrient energy consumed in the United States,’ and for individuals regularly consuming alcoholic beverages, it frequently provides more than 10% of their energy needs. The acute dose of alcohol required to raise blood alcohol levels to the legal limit of 0.1% is roughly equivalent to 20% of a person’s daily resting energy expenditure. Clearly, consumption of alcoholic beverages can provide a significant influx of energy, which can promote accumulation of body fat. Conventional wisdom has indeed led to recognition of the cause of the “beer belly syndrome.” But emaciation is also common among heavily addicted alcoholics, who may consume alcohol in doses equivalent to half of their daily enThis review was prepared by J.P. Flatt, Ph.D., at the Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655. Nutrition Reviews, Vol. 50, No. 9
ergy needs. How then should one rate the impact of alcohol consumption on body weight? Research on the regulation of body weight has begun to focus on the regulation of nutrient balances, instead of merely considering the overall energy balance.233It has, of course, been long established from nitrogen balance studies that amino acid oxidation adjusts itself effectively to protein intake. Glycogen reserves are not much greater than one day’s habitual carbohydrate intake. These reserves are spontaneously maintained at a level high enough to prevent hypoglycemia but below the point at which fat synthesis becomes an important process for carbohydrate d i ~ p o s a lThis . ~ shows that carbohydrate oxidation also adjusts to carbohydrate intake and that food intake is spontaneously inhibited well before glycogen stores are saturated. Fat balance, on the other hand, is much less well regulated, with deviations from energy balance primarily translated into gains or losses of body fat.5 Since fat oxidation is not influenced by the fat content of a meal, fatty foods are most likely to induce fat accumulation.6 The high-fat content of the “mixed diets” consumed in affluent societies is thus believed to be an important factor in promoting the incidence of obesity. In considering the impact of alcohol on bodyweight regulation, an important question is, therefore, whether alcohol should be counted as a car267
September 1992: 263-274
The Pyrroloquinoline Quinone (PQQ) Coenzymes: A Case of Mistaken ldent ity Recent evidence has failed to support the claim for a pyrroloquinoline quinone (PQQ) cofactor in mammalian enzymes previously reported to have PQQ. The validity of the original analysis now has been questioned, and a second cofactor, topa quinone, has been identified in at least one enzyme.
Enzymes that exchange electrons between substrates (collectively known as oxidoreductases) must have a specific organic cofactor or a metal ion to prevent electrons in transit from modifying the protein structure. Nutrition plays a role in this process in that two of the better known “redox” cofactor pairs, NAD(P)+/NAD(P)H and FAD/ FADH,, are derived from vitamins niacin and riboflavin, respectively. With the discovery of pyrroloquinoline quinone (PQQ), Salisbury et al.’ and Duine and Frank2 introduced a third redox cofactor to the family, along with the term “quinoprotein,” to specify enzymes bearing the novel PQQ cofactor.3 It seemed only a matter of time before PQQ would also be discovered in mammalian enzymesan event that came nearly two years later when two groups working independently reported a PQQ-like compound in mammalian enzymes.435Before long, PQQ had captured the attention of a world audience. Chemists rushed to confirm its structure, and nutritionists, eager to learn if PQQ qualified as a new vitamin,6 conducted whole-animal studies to determine whether PQQ deprivation under controlled conditions altered the growth and development of laboratory animals. Some of the excitement surrounding PQQ, however, was dampened at a recent worldwide conference in Italy. At least three laboratories were unable to confirm PQQ as a cofactor in three mammalian enzymes previously shown to contain From this meeting also came the announcement of another cofactor that could have been mistaken for PQQ. This compound, a derivative of tyrosine, has been identified as peptide-bound trihydroxyphenylalanine (6-hydroxydopa, or topa This review was prepared by Edward D. Harris, Ph.D., at the Department of Biochemistry and Biophysics, Texas A & M University, College Station, TX 77843-2128. Nutrition Reviews, Vol. 50, No. 9
quinone; Figure 1). Unlike PQQ, topa quinone is very much within the biosynthetic capabilities of animals. In this update we will examine the evidence for and against a role for PQQ in biologic systems and will, perhaps, learn why careful confirmatory research should never be compromised. Historical Perspective PQQ (Figure 2) was first observed’ in bacteria as a cofactor for an alcohol dehydrogenase, an enzyme that permitted Pseudomonas TPI to survive on methane, methanol, and other one-carbon sources. PQQ was bound firmly to the enzyme and positioned to receive electrons directly from the substrate in analogous fashion to flavin (FAD) and nicotinamide (NAD+) cofactors. A structural analysis showed that PQQ had a molecular weight of 424 Da and an empirical formula of C,,H,,N?O, 2H20. There was also evidence for an orthoquinone group in the molecule. The cofactor was given the trivial name “methoxatin” and, through more testing, was found to have a redox potential of +0.090 volts at pH 7.0, which made it very suitable to participate in the oxidation of methanol.’ Comparing fluorescence spectra from bacterial and mammalian enzymes, Duine and colleagues4 and Ameyama et al.’ independently concluded that beef plasma and hog kidney amine oxidase enzymes also could be classified as quin~proteins.~.’ Supporting evidence was obtained when compounds believed to be the dinitrophenyl (DNP) and other hydrazones of PQQ were isolated from the enzyme after reacting with phenylhydrazines. These experiments were followed by a broader examination of all amine oxidases, with the hope and perhaps expectation that PQQ would be the unifying cofactor giving each its catalytic property. Duine et al., had established the methodology and defined the experimental parameters to characterize PQQ cofactors
OH
Figure 1. 6-hydroxydopa (topa) quinone. 263
L .
I
\\
0 Figure 2. Pyrroloquinoline quinone (PQQ or methoxatin).
in enzymes. In due time, lysyl oxidase," dopamine P-monooxygenase," galactose oxidase,l* and choline dehydr~genase'~ were reported to have a PQQ cofactor. DNP derivation became a standard for analyzing PQQ in enzymes. PQQ Enzymes: The Doubt Is Raised
At a meeting in Manziana, Italy, in 1990, a session was set aside to discuss PQQ cofactors in copper enzymes. The session provided a forum for scientists to present evidence for and against PQQ. Experimentally, the focus was on the origin and structure of the phenylhydrazine derivative isolated from copper enzymes. Because of its link with amine groups, pyridoxal phosphate (PLP) was considered a candidate for the cofactor. One amine oxidase in particular, lysyl oxidase, had shown a PLP-like substance at its active site.I4 However, when a powerful new analytical tool, 3 1 ~ - n u c ~ emagnetic ar resonance (NMR), was applied to amine oxidases, no evidence for a phosphate group was discovered, thus raising doubts as to PLP's presence. Moreover, an analytical technique referred to as "reductive trapping" failed to detect a single carbonyl group in another amine oxidase." PLP was therefore removed from the list of cofactor candidates and attention was turned to PQQ. Three key mammalian enzymes-galactose oxidase, dopamine P-monooxygenase, and bovine serum amine oxidase-previously had been added to the list of PQQ-containing proteins. As the new evidence for each of the enzymes was discussed, it became clear that a cofactor role for PQQ was more likely to be disputed than confirmed. For example, a thorough chemical analysis of the functional galactose oxidase enzyme strongly supported the existence of a rare, stable tyrosyl radical in the structure, but no quinone-like structure.'6 Similarly, although dopamine P-monooxygenase reacted positively with phenylhydrazine, newer findings showed a reaction with the histidyl residues in the enzyme. l 7 There was no evidence for phenylhydrazine reacting with PQQ. These results cast strong doubts on the claims for PQQ and made suspect the evidence favoring PQQ in other enzymes. The case for PQQ was presented by Duine and colleague^,^ who reported on the interaction of the 264
bovine enzyme with hydrazines and other carbonyl reagents and the subsequent spectrographic analysis supporting PQQ as the hydrazone adduct. Phenylhydrazine and p-nitrophenylhydrazine adducts had been shown (before PQQ was known) to be powerful agents for detecting carbonyl groups in proteins. In the analyses of Duine and colleague^,^ the complex isolated from the bovine enzyme had spectroscopic and chromatographic properties identical to the PQQ-phenylhydrazone obtained from methylotropic bacteria (Figure 3). In both cases, the adduct was produced by treating the enzyme with moderately high amounts of phenylhydrazine in the presence of oxygen, followed by a rather harsh destruction of the protein with pronase. The organic factor released by the digestion and subsequently extracted by hexanol was identified by high-performance liquid chromatography (HPLC) and 'H-NMR. These analyses all supported the identity of the unknown as a PQQ adduct of phenylhydrazine. Despite a rather convincing display of analytical evidence, a lingering doubt still pervaded the research. The questions asked did not challenge the spectrographic evidence, but, rather, the origin of the factor that gave the spectra. Was this indeed PQQ, or did some unexpected chemical change in the protein lead to the formation of a compound that resembled PQQ, mimicking its absorption data? Could this unknown in fact arise as a result of the rather unfavorable conditions imposed? These questions formed the bases for legitimate concerns and led to adoption of a different protocol for determining PQQ's presence. Is the Cofactor Topa Quinone? Disputing the claim for one cofactor is only a partial solution to the PQQ problem. If the cofactor was not PQQ, what was it? Applying less rigorous conditions to the analysis, Klinman's group'* used [U-'4C]phenylhydrazine (to avoid excess phenylH,
H&$>
/ \
HOOC
N
COOH
N-
II
/GO
N
I NH I
NO,
Figure 3. 2,4-dinitrophenylhydrazoneof PQQ. Nutrition Reviews, Vol. 50, No. 9
hydrazine exposure) and thermolysin, a low-specificity protease, to prepare smaller I4C-labeled peptide fragments from bovine serum amino oxidase. The labeled peptides, produced with a 40% yield, gave rise to a series of absorption peaks dominated by a single peak that absorbed light at the same wavelength as a phenylhydrazone adduct. Using HPLC, Klinman’s group” succeeded in isolating a single peptide whose structure was shown by sequence and mass spectra analysis to be LEU-ASNX-ASP-TYR. The peptide’s unknown X residue became the object of intensive analysis. Was it an amino acid attached to PQQ? By applying an analytical technique called “liquid secondary ion mass spectrophotometry” (LSIMS), an ”N-labeled fragment of the peptide (referred to as a molecular ion or MH+) was shown to have a molecular mass of 808.3. Only two carboxyl groups were found-one associated with aspartic acid, while the other could only be the C-terminal of tyrosine. Thus, PQQ with three carboxyl groups in an underivatized state (Figure 2), could not possibly be part of residue X. What, then, was X? By subtracting the atomic weights of Leu, Asn, Asp, and Tyr, the molecular mass of X was deduced to be 283.0967. Since the molecular mass of PQQ is 470, there was further reason to eliminate PQQ from all consideration. Thus, X was a compound that had not been characterized previously. Based on elemental composition, it was determined that X must have the empirical formula C,SH,,N,O,. Considering the possibilities, only two structures ratio-
nally fit the formula for X. One was a serine residue linked to the phenylhydrazone through an uncommon ether linkage, and the second structure, and perhaps the more probable, was a derivative of tyrosine in which two oxygen groups had been added to the ring (Figure 1). Based on mass spectrometry and other evidence, this trihydroxyphenylalanine derivative, or 6-hydroxydopa (topa) quinone, was the leading candidate. To be certain, however, it was necessary to synthesize topa and compare its properties with the phenylhydrazone derived from the enzyme. These results showed that the spectra for the synthetic topa closely matched the enzymederived compound. For example, X in the pentapeptide showed an absorption maximum at 457 nm, and nitrophenylhydrazone of topa hydantoin absorbed maximally at 456 nm-almost exactly the same wavelength. An absorption at 456 nm was also close to the value of 468 nm reported for phenylhydrazones of PQQ. In both instances, the presence of peptide side chains may have caused a slight shift in the maximal wavelengths. A telling blow came when PQQ hydrazone and topa hydantoin (the respective adducts) were analyzed by Raman resonance spectrometry and the resulting vibrational spectra were compared with the bovine enzyme cofactor.’’ The results (Figure 4) clearly showed that the multiple peaks generated by the active site cofactor (panel B) aligned more closely with topa hydantoin than with PQQ or PLP. The pattern for PQQ (panel C) was distinct in giving a strong absorption at wavenumber 1159 and three additional moderately strong peaks between wave
I\
A
D
-cI
500
1000
Ramen S h i f t ( w a v e n u m b e r )
Nutrition Reviews, Vol. 50, No. 9
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Figure 4. Resonance Raman spectra for p-nitrophenylhydrazones of (A) topa-hydantoin; (B) activesite peptide of bovine plasma amine oxidase; (C) PQQ; (D) pyridoxal 5 ’ phosphate. Reprinted with permission of Brown et a1.19
265
numbers 1425 and 1335. These features were absent from topa hydantoin (panel A) and the enzyme. On the other hand, only topa quinone and the enzyme had components greater than 1600. The spectral evidence challenged the presence of PQQ (or pyridoxal S'phosphate) in the bovine enzyme. What Went Wrong?
The issue now turned to how PQQ could have been mistaken for topa in copper enzymes. One objection raised was that a prolonged treatment of the enzyme with phenylhydrazine in the presence of oxygen, followed by protein digestion, could create derivatives from amino acids in the protein, rather than from PQQ. Due to the reactive nature of the chemicals, such reactions could occur at any stage of the isolation process. Wanton oxygenation was considered an important variable to be controlled in all future experiments. As one of the more revealing experiments had shown earlier, a tyrosine ethyl ester was converted to a PQQ-like product by a simple oxidative reaction.20 Faced with mounting evidence against PQQ, Duine's group' retracted some of their original conclusions for amine oxidase and offered a conciliatory statement indicating that strong evidence supported the view that amine oxidases contain a covalently bound cofactor with a quinone character and that the hydrazine method would not discriminate between PQQ and amino acids in the protein that react with hydrazine. They noted, moreover, that reactions occurring after the hydrazine had reacted would form the final products and that these might not be the compounds originally bound. The authors concluded that 6-hydroxydopaquinone, identified in the bovine serum enzyme, was a good candidate for the cofactor with quinone-like properties, although the absorption spectra and chromatographic behavior of topa dinitrophenylhydrazone and PQQ phenylhydrazone would need to be significantly different to permit distinguishing one from the other. Neither PQQ or TOPA Quinone Is Confirmed
Unfortunately, the PQQ cofactor debate has not been completely settled. A recent paper describes PQQ in the reproductive organs of higher plants and as part of the composition of pollen grains.21These findings suggest that PQQ's presence is not limited strictly to microorganisms. Indeed, while the presence of PQQ in bovine serum amine oxidase has been unequivocally disproved, the cofactor in at least two copper oxidases-lysyl oxidase and benzylamine oxidase, both suspected of containing bound PQQ-has not yet been identified. An early report that PQQ was present in lysyl oxidase had 266
been based on the phenylhydrazine analysis" and a negative finding for pyridoxal phosphate.22 In a rather startling nutritional discovery, Rucker and coworkers23 have shown that rats and mice fed PQQ-deficient diets develop classical symptoms of lathyrism, a defect based on lysyl oxidase impairment. Moreover, the levels of lysyl oxidase in the tissues are significantly lower in animals fed the PQQ-deficient diets. Thus, the PQQ vitamin hypothesis is still alive today, although barely. As a consequence of the discovery of topa quinone in the structure of proteins, our understanding of how amino acids already existing within a protein can be derivatized to a functional cofactor has been enhanced, as has our knowledge of enzyme cofactors in general. As for PQQ, anyone wishing to further the understanding of this compound can draw on the considerable experience resulting from this intriguing controversy. 1. Salisbury SA, Forrest HS, Cruse WBT, Kennard 0. A novel coenzyme from bacterial primary alcohol dehydrogenases. Nature 1979;280:843-4 2. Duine JA, Frank J. The prosthetic group of methanol dehydrogenase: purification and some of its properties. Biochem J 1980;187:221-6 3. Duine JA, Frank J. Quinoproteins: a novel class of dehydrogenases. Trends Biochem Sci 1981 ;6: 278-80 4. Lobenstein-Verbeek CL, Jonjegan JA, Frank J, Duine JA. Bovine serum amine oxidase: a mammalian enzyme having covalently bound PQQ as a prosthetic group. FEBS Lett 1984;170:305-9 5. Ameyama M, Hayashi M, Matshshita K, Matsushita K, Shinagawa E, and Adachi 0. Microbial production of pyrroloquinoline quinone. Agric Biol Chem 1984 ;481561-5 6. Is pyrroloquinoline quinone a cofactor derived from an undiscovered vitamin? Nutr Rev 1988;46: 139-42 7. Beinert H. Copper in biological systems. A report from the 6th Manziana Conference, September 23-27, 1990. J lnorgan Biochem 1991;44:173-218 8. Klinman JP, Dooley DM, Duine JA, Knowles PF, Mondovi B, Villafranca JJ. Status of the cofactor identity in copper oxidative enzymes. FEBS Lett 1991;282:1-4 9. Duine JA, Frank J, Jongejan JA. Detection and determination of pyrroloquinoline quinone, the coenzyme of quinoproteins. Anal Biochem 19833 33: 239-43 10. Williamson PR, Moog RS, Dooley DM, Kagan HM. Evidence for pyrroloquinoline quinone as the carbony1 cofactor in lysyl oxidase by absorption and resonance Raman spectroscopy. J Biol Chern 1986;261:16302-5 11. van der Meer RA, Jongejan JA, Duine JA. Dopamine @-hydroxylasefrom bovine adrenal medula contains covalently bound PQQ. FEBS Lett 1988; 231 :303-7 Nutrition Reviews, Vol. 50, No. 9
12. van der Meer RA, Jongejan JA, Duine JA. Pyrroloquinoline quinone as cofactor in galactose oxidase (EC 1.1.3.9). J Biol Chem 1989;264:7792-4 13. Ameyama M, Shinagawa E, Matsushita K, Takimot0 K, Nakashima K, Adachi 0. Mammalian choline dehydrogenase is a quinoprotein. Agric Biol Chem 1985;49:3623-6 14. Levene CI, O’Shea MP, Carrington MJ. Protein lysine 6-oxidase (lysyl oxidase) cofactor: methoxatin (PQQ) or pyridoxal? Int J Biochem 1988;20: 1451-6 15. Harmann C, Klinman JP. Reductive trapping of substrate to bovine plasma amine oxidase. J Biol Chem 1987;262:962-5 16. Whittaker MM, Whittaker JW. The active site of galactose oxidase. J Biol Chem 1988;263:6074-80 17. Robertson JG, Kuman A, Mancewicz JA, Villafranca JJ. Spectral studies of bovine dopamine P-hydroxylase. Absence of covalently bound pyrroloquinoline quinone. J Biol Chem 1989;264: 19916-21 18. Janes SM, Mu D, Wemmer D, et al. A new redox cofactor in eukaryotic enzymes: 6-hydroxydopa at
19.
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the active site of bovine serum amine oxidase. Science 1990;248:981-7 Brown DE, McGuir MA, Dooley DM, Janes SM, Mu D, Klinman JP. The organic functional group in copper-containing amine oxidases. Resonance Raman spectra are consistent with the presence of TOPA quinone (6-hydroxy quinone) in the active site. J Biol Chem 1991;266:4049-51 Buchi J, Botkin JH, Lee GCM, Yakushijin K. A synthesis of methoxatin. J Am Chem SOC1985;107: 5555-6 Xiong LB, Sekiya J, Shimose N. Occurrence of pyrroloquinoline quinone (PQQ) in pistils and pollen grains of higher plants. Agric Biol Chem 1990; 54:249-50 Williamson PR, Kittler JM, Thanassi JW, Kagan HM. Reactivity of a functional carbon moiety in bovine aortic lysyl oxidase. Evidence against pyridoxal 5’-phosphate. Biochem J 1986;235:597605 Kilgore J, Smidt C, Duich L, et al. Nutritional importance of pyrroloquinoline quinone. Science 1989;245:850-2
Body Weight, Fat Storage, and Alcohol Metabolism Ethanol account for a significant fraction of the energy intake of persons consuming even moderate amounts of alcohol. A recent study has shown that although alcohol does not reveal itself as a layer floating at the top of a drink, metabolically it behaves more like oil than sugar.
Alcohol accounts for slightly more than 5% of the nutrient energy consumed in the United States,’ and for individuals regularly consuming alcoholic beverages, it frequently provides more than 10% of their energy needs. The acute dose of alcohol required to raise blood alcohol levels to the legal limit of 0.1% is roughly equivalent to 20% of a person’s daily resting energy expenditure. Clearly, consumption of alcoholic beverages can provide a significant influx of energy, which can promote accumulation of body fat. Conventional wisdom has indeed led to recognition of the cause of the “beer belly syndrome.” But emaciation is also common among heavily addicted alcoholics, who may consume alcohol in doses equivalent to half of their daily enThis review was prepared by J.P. Flatt, Ph.D., at the Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655. Nutrition Reviews, Vol. 50, No. 9
ergy needs. How then should one rate the impact of alcohol consumption on body weight? Research on the regulation of body weight has begun to focus on the regulation of nutrient balances, instead of merely considering the overall energy balance.233It has, of course, been long established from nitrogen balance studies that amino acid oxidation adjusts itself effectively to protein intake. Glycogen reserves are not much greater than one day’s habitual carbohydrate intake. These reserves are spontaneously maintained at a level high enough to prevent hypoglycemia but below the point at which fat synthesis becomes an important process for carbohydrate d i ~ p o s a lThis . ~ shows that carbohydrate oxidation also adjusts to carbohydrate intake and that food intake is spontaneously inhibited well before glycogen stores are saturated. Fat balance, on the other hand, is much less well regulated, with deviations from energy balance primarily translated into gains or losses of body fat.5 Since fat oxidation is not influenced by the fat content of a meal, fatty foods are most likely to induce fat accumulation.6 The high-fat content of the “mixed diets” consumed in affluent societies is thus believed to be an important factor in promoting the incidence of obesity. In considering the impact of alcohol on bodyweight regulation, an important question is, therefore, whether alcohol should be counted as a car267
