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J. Biol. Chem., Vol. 281, Issue 41, 30717-30724, October 13, 2006

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Dichotomic Phylogenetic Tree of the Pyruvate Kinase Family

K⁺-DEPENDENT AND -INDEPENDENT ENZYMES^*

From the Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, 04510 México, D. F., México

Received for publication, June 2, 2006 , and in revised form, August 9, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
K⁺ dependence was assumed to be a feature of all pyruvate kinases until it was discovered that some enzymes express K⁺ -independent activity. Almost all the K⁺-independent pyruvate kinases have Lys at position 117, instead of the Glu present in the K⁺-dependent muscle enzyme. Mutagenesis studies show that the internal positive charge substitutes for the K⁺ requirement (Laughlin, L. T. & Reed, G. H. (1997) Arch. Biochem. Biophys. 348, 262–267). In this work a phylogenetic analysis of pyruvate kinase was performed to ascertain the abundance of K⁺ -independent activities and to explore whether the K⁺ activating effect is related to the evolutionary history of the enzyme. Of the 230 studied sequences, 46% have Lys at position 117, and the rest have Glu. Pyruvate kinases with Lys¹¹⁷ and Glu¹¹⁷ are separated in two clusters. All of the enzymes of the Glu¹¹⁷ cluster that have been characterized are K⁺-dependent, whereas those of the Lys¹¹⁷ cluster are K⁺-independent. Thus, there is a strict correlation between the dichotomy of the tree and the dependence of activity on K⁺. 77% of the pyruvate kinases that possess Lys¹¹⁷ have Lys¹¹³/Gln¹¹⁴; they also have Ile, Val, or Leu at position 120. These residues are replaced by Glu¹¹⁷ and Thr¹¹³/Lys¹¹⁴/Thr¹²⁰ in 80% of K⁺-dependent pyruvate kinases. Structural analysis indicates that these residues are in a hinge region involved in the acquisition of the catalytic conformation of the enzyme. The route of conversion from K⁺-independent to K⁺-dependent pyruvate kinases is described. A plausible explanation of how enzymes developed K⁺ dependence is put forth.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
The dependence of enzyme activity on monovalent cations is widespread. Indeed more than 100 enzymes exhibit structural and catalytic requirement for either K⁺ or Na⁺ (1–4). In some enzymes, the monovalent cations participate in the formation of a ternary complex of enzymes and substrates, whereas in other, cations act allosterically or stabilize the protein structure (3–7). Structural information on the molecular origin of these effects has begun to emerge; however, there are still many questions that remain to be solved. Pyruvate kinase, the first enzyme in which an absolute requirement for K⁺ has been documented (8), is an excellent model enzyme for studying the effect of monovalent cations particularly because in the rabbit muscle enzyme the activating effect of K⁺ is about 10,000-fold (9, 10), which, to our knowledge, is the highest reported thus far. In regard to the mechanism of action of K⁺, it is known that this cation changes the ultraviolet and fluorescence spectra of pyruvate kinase (11–13) as well as its immunoelectrophoretic pattern (14). K⁺ also changes the structure of the active site (15–18). In this respect, NMR studies of Tl⁺ bound to pyruvate kinase show that the alkali metal ion binds within 0.8 nm of the Mn²⁺-binding site (16) and that this distance shifts to 0.49 nm upon binding of the substrate phosphoenolpyruvate (19). In addition, it has been shown that K⁺ changes the kinetic mechanism of rabbit muscle pyruvate kinase and is involved in the acquisition of the active conformation of the enzyme (20). The K⁺-binding site is highly conserved in all crystallographic structures reported to date. K⁺ is coordinated by O-1 of Asn⁷⁴, O- of Ser⁷⁶, O-2 of Asp¹¹², the carbonyl oxygen of Thr¹¹³ (21), a water molecule, and a phosphate oxygen of phosphoenolpyruvate analogs (22), or an oxygen from the -phosphate of ATP (5).

For a long time, it was thought that the absolute dependence of K⁺ was a common feature to all pyruvate kinases (8, 23–38). However, as the number of characterized enzymes increased, it became apparent that the activity of several pyruvate kinases was K⁺-independent, for example those from Escherichia coli (Type II) (39), Phycomyces blakesleeanus (40), Corynebacterium glutamicum (41), Zymomonas mobilis (42), Thermoproteus tenax (43), Synechococcus pcc 6301 (44), Archaeoglobus fulgidus, Pyrobaculum aerophilum, Aeropyrum pernix, and Thermotoga maritima (45). To explore the molecular basis of this different behavior, Laughlin and Reed (46) compared the amino acid sequence of rabbit muscle pyruvate kinase with those of E. coli type II and of C. glutamicum and found that Glu¹¹⁷ of the rabbit enzyme, which is close to the K⁺-binding site, is a Lys in the bacterial enzymes. The authors constructed the E117K mutant of the rabbit enzyme and found that the mutant is >200-fold more active than the wild type in the absence of K⁺ and exhibits no stimulation by monovalent cations. They also constructed two other mutants, E117A and E117D; these enzymes were activated by K⁺. The data clearly indicate that the positive charge of Lys mimics the action of K⁺ (46). Mimicry of monovalent cation activation by a Lys has also been reported in chaperon HSC70 (47) and H⁺-translocating inorganic pyrophosphatase (48). In consonance with the data of Laughlin and Reed (46), the sequences of five of the characterized K⁺-independent enzymes also contain Lys in position 117 (39, 41, 42, 44, 45). However, pyruvate kinase from T. tenax (43) and P. aerophilum (44) exhibit K⁺-independent activity, albeit with a Ser at position 117. These observations thus indicate that the expression of K⁺-independent activity is not completely explained by the charge of residue 117 and that other residues in the vicinity of the active site may be involved in conversion of the K⁺-independent to K⁺-dependent pyruvate kinase.

In this work we performed an extensive phylogenetic study of the pyruvate kinase family. The purpose of the study was to ascertain the abundance of K⁺-independent activities and to explore whether the K⁺ activating effect is related to the evolutionary history of the enzyme. Phylogenetic analyses of pyruvate kinase family have been reported previously (43, 45, 49). Schramm et al. (43) and Johnsen et al. (45) found a dichotomic structure of the phylogenetic tree that does not coincide with the universal tree topology (Bacteria, Archaea, and Eukarya) (50, 51); this unusual topology may be the result of ancient gene duplication or lateral transfer events (43). The latter studies were an attempt to correlate the major subfamilies or clusters to the type of allosteric effector of the enzymes. According to Schramm et al. (43), cluster I mostly includes enzymes activated by sugar phosphates, whereas cluster II comprises enzymes regulated by nucleotides. Given that some enzymes of cluster I are regulated by effectors of cluster II and vice versa and some even lack heterotropic allosteric regulation, a classification based on allosteric effectors of the enzymes may be more complex.

Our phylogenetic data show that there are numerous enzymes that have Lys at position 117 (the numbering used is that of rabbit muscle pyruvate kinase). Because pyruvate kinases that contain Lys¹¹⁷ and the E117K mutant of the rabbit enzyme are K⁺-independent, it may be safely assumed that K⁺-independent pyruvate kinases are broadly present in nature (106 sequences from 230 have Lys). In accordance with Schramm et al. (43), we found a dichotomic topology of the pyruvate kinase phylogenetic tree. However, in our case, cluster I groups sequences that include Glu¹¹⁷ correspond to K⁺-dependent enzymes, whereas cluster II comprise sequences that have Lys¹¹⁷ and are therefore K⁺-independent enzymes. In addition, we found that changes in the residue at position 117 are consistently accompanied by conservative changes in residues 113, 114, and 120. In all likelihood these residues are mechanistically important, because they localize in a hinge bending region that participates in the acquisition of the active catalytic conformation of the enzyme.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
The search for pyruvate kinase sequences was performed at the World Wide Web site of the National Center for Biotechnology Information (52). The integrated data base retrieval system ENTREZ (52) was used to access the National Center for Biotechnology Information data base. The search for homologous sequences was performed with gapped BLASTP and PSI-BLAST using default gap penalties and BLOSUM62 substitution matrix (53). The sequence of rabbit muscle pyruvate kinase was the starting point in the search for homologous sequences. Translated nucleotide sequences were retrieved from the GenBank^TM of the National Center for Biotechnology Information.

Progressive multiple sequence alignment was calculated with the ClustalX package (54), using secondary structure-based penalties. The alignment was manually corrected according to the results of gapped BLASTP and three-dimensional alignments obtained from the three-dimensional structure data base of Entrez (55). Phylogenetic analyses were performed with MEGA 3.1 software (56) using maximum parsimony, minimum evolution, and neighbor-joining methods with the aid of the empirical Jones-Taylor-Thornton amino acid substitution model. Differences between amino acid sequences were corrected for multiple substitutions assuming gamma distribution for rate variations among sites. The gamma-shaped parameter (a = 1.0) was estimated with the Whelan-Goldman matrix of substitutions and the eight-category discrete-gamma model using TREE-PUZZLE 5.2 (57). Confidence limits of branch points were estimated by 500 bootstrap replications.

Structural analysis of domain movements and hinge bending regions of pyruvate kinase were performed with DynDom software. DynDom is a program that determines dynamic protein domains, hinge axes, and amino acid residues involved in hinge bending (58).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Phylogenetic Analysis—The search for sequences of pyruvate kinase comprised the data reported up to August 2004. A total of 230 sequences were retrieved: 69 eukarya (21 animals, 17 fungi, 16 plants and 15 protists), 143 bacteria, and 18 archaea (Fig. 1). Position 117 is occupied by Glu in 121 sequences and by Lys, Ser, and Arg in 106, 2, and 1 sequence, respectively. The requirement for K⁺ by 21 pyruvate kinases that have Glu at position 117 has been examined, and the activity of all of them is K⁺-dependent (8, 23–38). The activities of eight pyruvate kinases that do not have Glu at that position have been characterized; in all cases the activity is K⁺-independent. Therefore, there is an excellent correlation between the dependence of activity on K⁺ and the existence of Glu at position 117. About half of all the sequences analyzed (46%) have Lys¹¹⁷; thus, the K⁺-independent activity is as frequent as the K⁺-dependent activity.

Our phylogenetic analysis shows a dichotomic tree structure (Fig. 1) that, similar to a previous phylogenetic study (43), does not coincide with the universal tree topology (Bacteria, Archaea, and Eukarya) (50, 51). As discussed by Schramm et al. (43), this unusual topology may be caused by an early gene duplication event. However, a remarkable feature of our phylogenetic data is the high correlation between the dichotomic nature of the tree and the dependence on K⁺. As shown in Fig. 2, the red branch corresponds to the enzymes that contain Glu¹¹⁷, which include the 21 experimentally characterized K⁺-dependent enzymes. The only exception found in this branch is pyruvate kinase from the anaerobic bacteria Clostridium perfringens, (GenBank^TM accession number Q46289 [GenBank] ) that has Lys at position 117. To gain insight into the peculiar position of pyruvate kinase of C. perfringens, we examined the sequences that had been deposited (in GenBank^TM) in the last year. We found four new sequences belonging to the genus Clostridium. One of these sequences corresponds to a pyruvate kinase of C. perfringens that surprisingly has Glu in position 117. We also found that Clostridium acetobutylicum has K117 pyruvate kinase, in addition to the E117 enzyme included in the phylogenetic tree. This property is also shared by Clostridium beijerinckii where both Glu¹¹⁷ and Lys¹¹⁷ pyruvate kinases were found. The data on the Clostridium genus are highly suggestive that the two enzymes resulted from recent gene duplication. In fact, the identity between the Glu and Lys enzymes of C. perfringens is 52.6%, whereas identity between early duplicated pyruvate kinases of E. coli in cluster I and cluster II is 36.5%.

View larger version (83K): [in this window] [in a new window]   FIGURE 1. Phylogenetic analysis of pyruvate kinase. Available protein sequences belonging to the pyruvate kinase family were used to construct the phylogenetic topology with the minimum evolution method. Similar topologies were obtained using the neighbor-joining and maximum parsimony methods. Trees were calculated using MEGA 3.1 (26). Dots indicate nodes supported in 70% (open), 80% (gray), or 90% (closed) of 500 random bootstrap replicates of all neighbor-joining, minimum evolution, and maximum parsimony trees. Taxonomic groups are indicated by the following colors: animalin red, fungi in brown, kinetoplastida in orange, apicomplexa in olive green, plants in green, -proteobacteria in blue, firmicutes in pink, chlamidiae in gray, euryarchaeota in navy blue, crenarchaeota in purple, diplomonanida in dark green, alfa-proteobacteria in magenta, actinobacteria in dark yellow, cyanobacteria in wine red, -proteobacteria in cyan, and single represented bacterial taxonomic groups in black. Sequence names are indicated according to a Swiss-Prot-like identifier (accession number + organism). Characterized K+-dependent and -independent enzymes are indicated by red and blue asterisks. A complete list of the names of the organisms included in the tree is provided in supplemental Appendix 1.

View larger version (100K): [in this window] [in a new window]   FIGURE 2. Correlation between the K+ dependence of pyruvate kinase and the dichotomic topology of the phylogenetic tree. The sequences between residues 107 and 127 are shown. Residues in positions 113, 114, 117, and 120 are highlighted in the following colors: Glu in red, Lys in blue, Thr in green, Gln in purple, Leu/Val/Ile in pink, Pro in brown, Ser in dark yellow, Asn in orange, Cys in yellow, Met in wine red, Arg in navy blue, and Ala in gray. The branches of the sequences with Glu, Lys, and Ser or Arg in position 117 are illustrated in red, blue, and black, respectively. Characterized K+-dependent (Glu117) and K+-independent enzymes (Lys, Ser, or Arg at position 117) are indicated by red and blue asterisks, respectively. As shown, residues 113, 114, and 120 vary depending on residue 117. The numbering used is according to the sequence of rabbit muscle pyruvate kinase. The phylogenetic topology and node dots are described in the legend for Fig. 1.

Co-evolving Amino Acids with Residue 117—The phylogenetic analysis also shows that there is a correlation between the residue that exists at position 117 and some of its neighboring residues (Table 1). When Glu is in position 117, in 99% of the cases the residue in position 113 is Thr. Likewise, when Lys is in position 117, position 113 is occupied by Leu in 98% of the enzymes. A similar, but slightly less strict pattern was observed between residue 117 and those at positions 114 and 120. The data show that: 1) pyruvate kinases that have Glu¹¹⁷ have Lys¹¹⁴ in 94% of the cases; 2) when Lys is in position 117, position 114 is occupied by Gln in 83% of the enzymes; 3) enzymes with Glu in position 117 have Thr in position 120 in 83% of the cases; 4) enzymes with Lys¹¹⁷ have a hydrophobic residue in position 120 (Leu, Val, or Ile) in 93% of the cases.

Concerning residue 114, Laughlin and Reed (46) found that in the rabbit muscle E117K mutant, the presence of Lys (the most abundant residue found in position 114 in K⁺-dependent enzymes) or Gln (found mostly in K⁺-independent enzymes) did not affect the kinetics of the enzyme. Therefore, it is possible that only the pair Leu¹¹³/Gln¹¹⁴ participates in K⁺-independent activity. In fact, there is not a single pyruvate kinase that has the pair Thr¹¹³/Gln¹¹⁴.

No functional role has been assigned to residue 120; however the co-evolution between this amino acid and the residue 117 (83% in cluster I and 93% in cluster II) makes it an attractive target to study. Finally, it is noted that in the transition between clusters I and II, there are about 30 sequences that do not show the conserved pattern in residues 114 and/or 120. This suggests that these positions are not as critical as positions 113 and 117 for monovalent cation dependence.

The overall analysis of K⁺-dependent pyruvate kinases (cluster I) shows that the sequence Thr¹¹³/Lys¹¹⁴/Glu¹¹⁷/Thr¹²⁰ is conserved in 80% of the cases, whereas in the K⁺-independent enzymes (cluster II) the sequence Leu¹¹³/Gln¹¹⁴/Lys¹¹⁷/(Leu-Val-Ile)¹²⁰ is conserved in 77% of the enzymes. The co-evolution of residues 113, 114, and 120 with that in position 117 suggest that these residues are involved in the activating effect of K⁺ in the K⁺-dependent enzymes or in the expression of the K⁺-independent activity. In this regard, it is relevant that the data presented of Laughlin and Reed (46) show that in the rabbit muscle enzyme, Lys¹¹⁷ may replace K⁺; however the activity of the mutant is about 10% of the activity of the wild type with K⁺. Therefore, further substitutions are needed to attain full enzymatic activity.

Transition from K⁺ Independence to K⁺ Dependence—The broad phyletic distribution of K⁺-independent pyruvate kinases (cluster II) in the three domains of life (Archaea, Bacteria, and Eukarya) in comparison with K⁺-dependent pyruvate kinases (cluster I, found in Bacteria and Eukarya only) suggest that K⁺-independent activity preceded the appearance of the K⁺-sensitive activity. To explain the possible route through which K⁺-independent are converted to K⁺-dependent pyruvate kinases, we analyzed the amino acid codons for positions 113, 114, 117, and 120. As shown in Fig. 3, all of the changes in positions 113, 114, 117, and 120 took place by single nucleotide substitutions, except in the Antarctic hair grass, Deschampsia antarctica (GenBank^TM accession number AAM22747 [GenBank] ), and the sulfate-reducing proteobacteria, Desulfovibrio desulfuricans (GenBank^TM accession number ZP_00130421), where more than one substitution was needed. Because the frequency of mutations of amino acid codons occurs in the order of third > second > first nucleotide, it is possible to trace the probable series of events that led to the changes of the residues (Fig. 3). For example, in position 117 (Fig. 3A), the mutation in the third base would account for the conversion of Ser to Arg; likewise the mutation in the second nucleotide leads to the transformation of Arg to Lys and in the first nucleotide to the transformation of Lys to Glu. By the same rationale, Fig. 3, B and C, shows the routes of conversion of residues 113 and 120. In regard to residue 114 of group I, it is possible that variations of this residue (Arg, Ser, and Asn) stemmed from single nucleotide substitutions of the Lys codon, whereas the changes in group II (Pro, Ala) derived from single substitutions of the Gln codon (Fig. 3D).

View larger version (30K): [in this window] [in a new window]   FIGURE 3. Analysis of the use of amino acid codons in positions 113 (B), 114 (D), 117 (A), and 120 (C) of pyruvate kinase. The codons used for each amino acid are listed. The probability that a change takes place is indicated by arrows of different width. The widest arrow indicates the most probable event. The number of sequences that possess an amino acid in position 113, 114, 117, and 120 are indicated in parentheses.

View larger version (82K): [in this window] [in a new window]   FIGURE 4. DynDom analysis of pyruvate kinase. Ribbon representation of an overview of the rabbit muscle pyruvate kinase monomer (inset) and an amplification of the boundary region between domains A and B (main figure). The program assigned a fixed domain shown in blue, a moving domain in red, and two hinge regions in green and yellow. The fixed domain assigned by DynDom is formed by the N-terminal, A, and C domains; the B domain was assigned as the moving domain. Residues 114–118 form a short hinge (green), and residues 209–224 form a long hinge (yellow). Residues 113 and 120 are, respectively, at the beginning and end of the short hinge, whereas residue 114 and 117 are part of it. Thr113, Lys114, Glu117, and Thr120 are illustrated as sticks, and K+ is shown in yellow. The sequence from residues 109–123 is shown at the bottom of the figure with the same color code (fixed in blue, moving in red, and short hinge in green). The sequence from residues 209–224 is not shown. The positions of residues 113, 114, 117, and 120 with respect to the short hinge are illustrated.

The enzyme is formed by four structural domains: the N-terminal and the A, B, and C domains. The active site is in a cleft formed by A and B domains, which are linked by two loops. Rotation of the B domain over the A domain closes the active site cleft. DynDom assigns domain B as the mobile domain, and the N-terminal and A and C domains as the fixed domain. Two hinge bending regions were assigned: a short hinge formed by the sequence 114–118 (green in Fig. 4) and a long hinge formed by residues 209–224 (yellow in Fig. 4). Therefore residues 113 and 120 are, respectively, at the beginning and end of the short hinge, whereas residue 114 and 117 are part of the hinge (Fig. 4).

Crystallographic data have clearly demonstrated that K⁺ provides a positive charge to coordinate the phosphate group of phosphoenolpyruvate (22) or ATP (5). In addition, NMR studies (15–19) have shown that monovalent cations induce structural rearrangements in the active site of rabbit muscle pyruvate kinase. Similar findings have also been documented in yeast pyruvate kinase where the interaction of the monovalent cation with the wild type enzyme (59, 60) and Thr²⁹⁸ mutants (60) was investigated by ²⁰⁵Tl⁺ NMR. The main results indicate that monovalent cation induces conformational alterations at the active site of yeast pyruvate kinase (59, 60). Moreover, kinetic and intrinsic fluorescence anisotropy measurements suggest that K⁺ induces the closure of the active site and the acquisition of the active conformation of rabbit muscle pyruvate kinase (20). In K⁺-dependent enzymes, the high conservation of Glu¹¹⁷ and its flanking residues Thr¹¹³/Lys¹¹⁴/Thr¹²⁰, which are in a strategic structural location, indicates that these residues may be involved in the transduction of K⁺ binding to the movement of domain B. In the K⁺-independent enzymes, Lys¹¹⁷, along with Leu¹¹³/Gln¹¹⁴/(Val-Leu-Ile)¹²⁰ may induce similar conformational changes but in the absence of K⁺.

Why Do Enzymes Use Monovalent Cations?—There are a large number of enzymes for which activity is dependent on either Na⁺ or K⁺. A salient feature of these enzymes is that extracellular enzymes are Na⁺-dependent, whereas K⁺-dependent enzymes are intracellular. Thus it may be asked, what is the evolutionary advantage of having Na⁺ or K⁺ as essential activators of these enzymes? Di Cera (4) has put forth that Na⁺ and K⁺ cannot function as regulators of enzyme activity because of their tightly controlled concentrations. Instead, he considers that monovalent cations provide a driving force for substrate binding and catalysis by lowering energy barriers (4). Thus monovalent cation-activated enzymes evolved to optimize their catalytic function by taking advantage of the large concentrations of Na⁺ and K⁺ in their surrounding media.

An alternative or additional explanation for the existence of the numerous proteins that require monovalent cations could involve the chemical rescue of an inactive enzyme. That is, we hypothesize that the biological activity of a mutated protein can be restored by the introduction of a chemical group that compensates for or overcomes the detrimental effect of a mutation. Indeed, it has been reported that imidazole compensates the loss of histidines that are essential for catalysis (61) and that guanidinium ions restore the substitution of arginine residues (62). Accordingly, it is possible that the binding of monovalent cations compensate for the loss of essential positive charged amino acids followed by additional mutations as the result of functional adaptations to the presence of the new ligand. In pyruvate kinase, the substitution of Lys for Glu¹¹⁷ is compensated by the binding of K⁺. Mutations in residues 113, 114, and 120 could be the evolutionary adaptations required to convert the enzyme into an efficient K⁺-dependent catalyst. Concerning cation selectivity, cation-dependent pyruvate kinases emerged to select K⁺ over Na⁺ because of evolutionary pressure that led to the optimization of the interactions of the protein with the K⁺ that abounds in the intracellular milieu.

In sum, the phylogenetic analysis of pyruvate kinase showed that the K⁺-independent activity is broadly present in nature; about half of the reported sequences possess Lys in position 117, whereas the rest have Glu in that position and are K⁺-dependent. The K⁺ dependence feature clearly explains the dichotomic topology of the phylogenetic tree of pyruvate kinase family; the K⁺-dependent and -independent enzymes belong to each of the branches of the tree. Because organisms that evolved at a later time have K⁺-dependent pyruvate kinases, it may be suggested that they emerged from K⁺-independent enzymes through single nucleotide substitutions. The high association found between the amino acid in position 117 and those in positions 113, 114, and 120, along with their central location in the structure of the enzyme, indicates that these residues might be involved in the activating effect of K⁺ or in the expression of the K⁺-independent activity. Therefore, the primary structure of pyruvate kinases, related to the K⁺ requirement, provides a precise criterion for their classification.

	FOOTNOTES

 
^* This work was supported by Grant IN202906 (to L. R.-S.) from the Dirección General de Asuntos del Personal Académico de la Universidad Nacional Autónoma de México (DGAPA-UNAM), México. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Appendix 1.

¹ To whom correspondence should be addressed: Departamento de Bioquímica, Facultad de Medicina, Apartado Postal 70-159, Universidad Nacional Autónoma de México, 04510, D. F., México. Tel.: 525-6232510; Fax: 525-6162419; E-mail: lramirez{at}bq.unam.mx.

	ACKNOWLEDGMENTS

 
We thank Dr. Marietta Tuena de Gómez-Puyou and Dr. Armando Gómez-Puyou for valuable discussion and careful revision of the manuscript.

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