©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Effects of Conserved Residues on the Regulation of Rabbit Muscle Pyruvate Kinase (*)

(Received for publication, August 18, 1995; and in revised form, December 8, 1995)

Xiaodong Cheng (§) Robert H. E. Friesen (§) J. Ching Lee (¶)

From the Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1055

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A cDNA encoding the complete rabbit muscle pyruvate kinase isozyme (RMPK) was cloned using the method of rapid amplification of cDNA ends. The sequence encodes a polypeptide chain of 530 amino acids which differs in three amino acid residues from a sequence reported by Larsen et al. (Larsen, T. M., Laughlin, T., Holden, H. M., Rayment, I., and Reed, G. H.(1994) Biochemistry 33, 6301-6309). Glu-Gln and Ala were identified instead of Asp-Glu and Ser, respectively. The recombinant RMPK was overexpressed in the Escherichia coli JM 105 cells. Purified recombinant pyruvate kinase displayed identical physical and enzymatic properties as the authentic enzyme.

Three point mutants of RMPK were constructed using site-directed mutagenesis. Like the wild type RMPK, sedimentation, and CD spectroscopic studies show that purified R119C and T340M are tetrameric proteins with similar secondary and tertiary structures. Mutant R119C enzyme exhibits 0.6% of the value of k and an order of magnitude decrease in the apparent affinity for ADP as compared to the wild type PK. The overall response to inhibitor and activator, Phe and FBP, respectively, were not affected by the R119C mutation. The T340M mutant enzyme is only half as active as the wild type PK. T340M is more susceptible to inhibition by Phe but apparently is not responsive to the activator FBP. The kinetic behavior of the Q377K mutant enzyme is in between that of the R119C and T340M mutants exhibiting 5% of the wild type enzymatic activity and an enhanced sensitivity to the inhibitor, Phe, while maintaining the same responsiveness to FBP and apparent affinities for substrates. The significant decrease in activity in all three mutants mimics the exact consequences of the same mutations in human erythrocyte PK from hemolytic anemia patients. Thus, this study demonstrates not only the effects of these conserved residues in the regulatory properties of mammalian PK but also that the observed effects are most likely applicable to all isozymic forms of PK.


INTRODUCTION

Mammalian pyruvate kinase (ATP:pyruvate-2-O-phosphotransferase, EC 2.7.1.40) (PK) (^1)which catalyzes the transphosphorylation from PEP to ADP is a key regulatory enzyme and is the subject of investigation in this laboratory because the regulatory properties of a metabolic pathway are highly responsive to the variations in metabolic requirements demanded by the different tissues in which the pathway functions. In many cases, the key regulatory enzymes of the metabolic pathway are the principal factors responsible for the differences. Hence, studying the same regulatory enzyme from different tissues may provide information for relating a change in regulatory pattern to a change in protein structure that confers the special regulatory features in the enzyme. Indeed, the clinical condition of hereditary nonspherocytic hemolytic anemia has its origin in aberrations of the control of cellular functions in erythrocyte PK (1, 2, 3) . In response to the specific demands of metabolic flow in specific tissues, PK is expressed in four isozymic forms, namely, muscle (M(1)), kidney (M(2)), erythrocyte (R), and liver (L). In the absence of inhibitor, M(1)-PK exhibits hyperbolic steady-state kinetic behavior whereas the rest of the other isozymes show sigmoidal kinetic behavior and are regulated by a host of allosteric inhibitors and activators (4, 5) although the amino acid sequences of these isozymes are known to be at least 65% identical (5) .

The mechanism of regulation of rabbit muscle PK (RMPK) isozyme, has been extensively studied(6, 7, 8, 9) . The results of these studies are consistent with the model of Monod-Wyman-Changeux(10) . The model indicates that PK has two conformational states (R and T) which are in equilibrium, and that each state exhibits differential affinity for any ligand. Our study of the structure of PK indicates that there are no significant changes in the secondary and tertiary structures of RMPK when it is converted from the inactive T-state to the active R-state, although the T R reaction is highly concerted. (^2)Hence, the transmission of information must be through intersubunit interfaces in this tetrameric enzyme. But, what are the structural elements that are involved and which one is the switch? Results from studying PK isozymes provide the most valuable information on this issue(8) . One set of intersubunit contacts that is functionally important is localized in a helical region in the C-domain, one of the three domains identified in each PK subunit. A change in the sequence in this region leads to a conversion of the RMPK with hyperbolic kinetics to the kidney PK with sigmoidal kinetics(8) . Besides this region, the rest of the amino acid sequences between M(1) and M(2) are identical(5) . The functional significance of these and other important residues involved in regulating PK is yet to be defined but can be addressed by studying mutants generated by site-directed mutagenesis. A prerequisite to generation of site-directed mutants is the availability of a recombinant mammalian PK in a bacterial expression system. At present, mammalian PK has not been cloned.

In the present paper, the cloning and expression of RMPK in E. coli is reported. Three important residues of PK, identified by genetic studies in hereditary nonspherocytic hemolytic anemia patients, were analyzed by site-directed mutagenesis. The structural and functional properties of these mutants and wild type recombinant RMPK were investigated further to reveal the modes of inactivation of RMPK by these mutations. Such biochemical and biophysical studies are necessary for understanding the mechanisms that lead to the disease of PK deficiency as well as the allosteric properties of this important enzyme in general.


EXPERIMENTAL PROCEDURES

Materials

Pyruvate kinase from rabbit muscle, lactate dehydrogenase, disodium salt of ADP, phosphoenolpyruvate, Tris base, and Tris-HCl were purchased from Boehringer Mannheim. Trypsin, reduced nicotinamide adenine dinucleotide (NADH), L-phenylalanine (Phe), potassium chloride, sodium chloride, and phenylmethylsulfonyl fluoride were all obtained from Sigma. Mono- and dibasic potassium phosphate were purchased from Fisher. [S]dATP was purchased from Amersham Life Science. Oligonucleotides were purchased from Genosys Biotechnologies, Inc.

RNA Extraction and cDNA Synthesis

After homogenization using a Tekmar Tissuemizer, the rabbit muscle tissue was extracted by the combined disruptive and protective properties of guanidine thiocyanate and beta-mercaptoethanol(11) . Extraction of mRNA was performed using the PolyATtract mRNA isolation system from Promega. cDNA was synthesized using random hexamer primers and avian myeloblastosis virus reverse transcriptase from the 5`-AmpliFinder RACE kit (Clontech).

Rabbit Muscle Pyruvate Kinase Cloning

After mRNA isolation and cDNA synthesis, the RMPK gene was amplified by PCR. To design primers for the PCR amplification, it is necessary to know the coding sequences at the 5` and 3` ends. To obtain the 5` and the 3` end sequences of the RMPK cDNA, 5`-AmpliFinder Rapid Amplification of cDNA Ends kit (Clontech) and 3` RACE kit (Life Technologies, Inc.) were used according to the manufacturer's instructions. The procedure of the 5` and 3` end RACE methods (12) are schematized in the first three steps of Fig. 1.


Figure 1: Strategy for cloning RMPK. The 5` and 3` RACE methods are schematized in steps 1 to 3 (see ``Experimental Procedures''). The 5` and 3` RACE methods are two distinct sets of reactions, schematized in A and B, respectively. The primers used for the PCR amplifications are represented by arrows, indicating their orientation and relative position within the RMPK gene. In C, step 4 represents the PCR amplification of the RMPK coding sequence. The open box represents the translated region of the RMPK gene and is shown in the 5` 3` direction. Direction and extension of the sequencing reactions are depicted by the arrows. The relative location of the nucleotide sequence different from that reported by Larsen et al.(22) are indicated by asterisks. Noncorresponding nucleotide sequences which result in a change in amino acid sequence are indicated above the asterisks with their three-letter code.



In the first step of the 5` RACE method, cDNA was synthesized with the antisense gene-specific primer P1 (5`-CACAGGATGTTCTCGTC-3`). In the second step, a single-stranded anchor oligonucleotide provided by Clontech was ligated to the 3` end of the cDNA. The extension introduced an upstream binding site for the PCR amplification in step 3. The PCR amplification in step 3 employed an anchor primer (AP), which is complementary to the anchor, and a nested gene-specific primer P2 (5`-GCAAAGCTTTCCGTGGCTGTGCGCAC-3`) to increase the specificity. The double-stranded DNA resulting from step 3 contained part of the coding and the noncoding sequence of 5` end of the RMPK gene.

PCR amplifications were performed according to the manufacturer's instructions, using a DNA Thermal Cycler (Perkin-Elmer Cetus), except that 2.5 units of Pfu DNA Polymerase (Stratagene) was used. The sample was subjected to a ``hot start'' PCR step at 94 °C for 3 min, followed by a temperature-step cycle of 94 °C (1 min), 70 °C (30 s), and 78 °C (105 s) for a total of 30 cycles, followed by a 7-min extension at 72 °C after the final cycle.

The first step of the 3` RACE (Fig. 1) involved the first strand cDNA synthesis employing an oligo(dT)-containing adapter primer (OAP) (5`-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3`). The OAP contains a sequence, complementary to the universal adapter primer of step 2 (dashed lines in Fig. 1), both provided by the manufacturer. The resulting cDNA-contained sequence extended to the 5` end. The RNA template was then removed by RNase H digestion, resulting in full-length single-stranded cDNA sequences. In step 2, double-stranded cDNA was synthesized by PCR amplification, employing the universal adapter primer, UAP (Life Technologies, Inc.), and the gene-specific primer P3 (5`-GTCCAGGAGGCCTGGGCT). The third step involved another PCR amplification using the UAP, except that a gene-specific primer P4 (5`-GGCAAGGCCCGAGGCTTCTTC-3`) was used to increase the specificity. The temperature step cycles for the two PCR amplifications were the same as for the 5` RACE, except the annealing step was at 55 °C for 1 min. The PCR fragment after the third step contained part of the coding and the noncoding sequence of 3` end of RMPK. The amplified 3` and 5` ends from the RACE method were sequenced using primers P2 and P4, respectively.

Subcloning and DNA Sequencing

Based on the 5` and 3` end sequences obtained with the RACE methods, a set of primers (P5, 5`-CTAAGAATTCATGTCGAAGTCCCACAGTGA-3`; P6, 5`-CTAAGAATTCATCGGTGGCACACTACAGC-3`) was generated bearing both the N- and C-terminal ends of the RMPK coding sequence flanked by EcoRI sites. These primers were used to generate a 1.6-kilobase PCR fragment containing the entire open reading frame, which was then subcloned into the multiple cloning site of pKK223-3 (Pharmacia Biotech Inc.). Recombinant plasmids (pRMPK) were introduced into E. coli JM105 by electroporation (Gene Pulser; Bio-Rad), in a 0.1-cm cuvette at 1.8 kV.

DNA sequencing of double-stranded DNA was performed by the dideoxynucleotide chain termination method (13) using Sequenase Version 2.0 (United States Biochemical Corp.). Oligonucleotides were synthesized to fit the strategy for sequencing (Fig. 1).

Site-directed Mutagenesis

The mutagenic oligonucleotides used for constructing the specific point mutants are as follows:

The underlined sequences are the mutagenic nucleotides with the corresponding wild type sequences labeled below.

Mutagenesis of the PK gene was performed directly with the double-stranded plasmid pRMPK. Two oligonucleotide primers, a mutagenic primer and a selection primer, were simultaneously annealed to one strand of the denatured pRMPK. The mutagenic primer containing one of the sequences shown above introduced the desired site-specific mutation into the PK gene, while the selection primer with the nucleotide sequence of 5`-TTTCA CACCG CAGCT GGTGC ACTCT C-3` mutated a unique NdeI restriction site in the pRMPK construct. The complementary strand, with both primers to be incorporated, was then synthesized by T4-DNA polymerase and circularized by T4-DNA ligase. Having been propagated in a repair-deficient E. coli strain, BMH 71-18 mutS, plasmids were isolated and digested with NdeI. Wild type plasmids, containing an intact NdeI site, were linearized while mutant plasmids, lacking the NdeI site, were resistant to the digestion. The reaction mixture was transformed into the JM 109 cells. Plasmids were isolated from culture of individual transformants and screened by NdeI digestion. More than 50% of the plasmids lacking the NdeI site also contained the desired point mutation as confirmed by DNA sequencing of these plasmids.

Overexpression of the Wild Type and Mutant RMPK

E. coli JM105 cells containing the plasmid pRMPK were grown overnight in M9 minimal medium containing 100 µg/ml Ampicillin and were inoculated 1/100 (v/v) into LB-medium containing 100 µg/ml Ampicillin. Culture was grown at 37 °C until the optical density at 600 nm reached 0.7 to 1.0, at which point expression was induced by the addition of IPTG to a final concentration of 60 µg/ml. After 5 h of induction, the cells were harvested by centrifugation at 6000 rpm for 20 min. in the GS-3 rotor of a Sorvall RC-5C centrifuge. Improvement on the expression of the PK mutants can be accomplished by using NZCYM broth as growth medium.

Protein Purification and Preparation

All purification steps for recombinant wild type and mutant PK were performed at 4 °C except the SP-Sepharose chromatography which was performed at room temperature. The cells were resuspended in 10 volumes of buffer A (20 mM Tris base, 100 mM KCl, 15% (v/v) glycerol, 6 mM beta-mercaptoethanol, 0.1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride, pH 7.9) and disrupted with a Piranha Press (Tesla Inc.) set at 9000 p.s.i. An equal volume of buffer A was added to the cell suspensions and centrifuged for 1 h at 10,000 rpm in the GSA rotor of a Sorvall RC-5C. A 10% (v/v) polyethyleneimine (pH 7.9) solution was added to the supernatant, with stirring, to a final concentration of 0.3%. After stirring for 5 min, the mixture was centrifuged for 15 min at 6000 rpm in a GSA rotor. Solid ammonium sulfate was added to the supernatant in small portions with stirring to 45% saturation. Stirring was continued for 30 min, and the precipitated proteins were removed by centrifugation at 8000 rpm in a GSA rotor for 45 min. The supernatant was brought up to 70% ammonium sulfate saturation with stirring and spun as above. The two chromatographic steps were performed on a FPLC system (Pharmacia Biotech Inc.) employing columns made for this system. The pellet was dissolved in up to 5 ml of buffer B (5 mM KP(i), 1 mM EDTA, 2 mM beta-mercaptoethanol, 5 mM MgSO(4), 100 mM KCl, pH 7.5) and applied to a HiLoad 16/60 (1.6 times 60 cm) Superdex 200 column. Fractions, typically containing more than 95% of the total activity, were pooled and dialyzed against buffer C (same as buffer B with no KCl and at pH 6.0). The dialyzed sample was applied to a 1-ml HiTrap SP-Sepharose (0.4 times 2.5 cm) column, and the nonadsorbed proteins were removed by washing the column with 25 column volumes of buffer C. The RMPK was eluted with a 30-ml linear salt gradient from 0 M NaCl to 1 M NaCl. The eluted RMPK was collected, precipitated with 70% ammonium sulfate, and stored at 4 °C.

RMPK was desalted before all experiments. The ammonium sulfate precipitate was concentrated by centrifugation at 14,000 rpm in an Eppendorf tabletop centrifuge for 5 min, and the pellet was resuspended in the appropriate buffer. The resuspended pellet was dialyzed extensively against the appropriate buffer. The protein concentration was determined by absorbance at 280 nm, using an absorptivity of 0.54 ml/mgbulletcm(14) .

SDS-Gel Electrophoresis

SDS-10% polyacrylamide gel electrophoresis was performed according to the method of Laemmli(15) , followed by staining with Coomassie Blue. The markers used for molecular weight determination were: phosphorylase b, 97,400; bovine serum albumin, 66,200; ovalbumin, 45,000 carbonic anhydrase, 31,000.

Amino Acid Compositional Analysis

Samples were hydrolyzed with 5.7 N HCl in vacuo at 107 °C for 24, 48, and 98 h. After drying in vacuo, amino acid analysis was performed on a Beckman 121 MB analyzer(16) . Tryptophan was determined after hydrolysis with mercaptoethanesulfonic acid(17) . Oxidation of half-cystinyl residues to cysteic acid was achieved with dimethyl sulfoxide(18) .

Automated Peptide Sequence Analysis

Primary structural analysis was performed using an Applied Biosystems 475A protein/peptide microsequencer with on-line Model 120A microbore phenylthiohydantion amino acid analyzer and a Model 900A data processor as described previously(19) . The software version employed was 1.41.

Circular Dichroism

CD spectra of the wild type and mutant RMPK were measured with an Aviv 62 DS Circular Dichroism Spectrometer. To acquire a full range of near- and far-UV CD spectrum, fused quartz cuvettes with pathlengths of 0.01 (220-190 nm), 0.1 (270-200 nm), and 1 cm (360-240 nm) and protein solutions with concentration around 1 mg/ml were used. Each spectrum was recorded with a 0.5 nm increment and 1-s interval. For each sample, five repetitive scans were obtained and averaged.

Sedimentation Velocity

Experiments were performed in a Beckman Model E analytical ultracentrifuge equipped with an ultraviolet scanner, electronic speed control, and RTIC temperature control. Data were acquired electronically by direct interfacing between the scanner and an IBM-PC computer. Observed sedimentation coefficients were determined from the centroids of the sedimentation boundaries analogous to the method of Arisaka and Van Holde(20) . All experiments were performed in TKM buffer (50 mM Tris, 72 mM KCl, 7.2 mM MgS0(4), pH 7.5) at 20 °C unless otherwise indicated.

Sedimentation Equilibrium

The quaternary structure of PK was monitored by sedimentation equilibrium in a Beckman-Spinco Model E analytical ultracentrifuge. The high-speed, meniscus-depletion procedure was employed(21) . The loading PK concentrations were between 0.5 and 0.8 mg/ml. Sedimentation data were acquired (10 scans) and then averaged after reaching equilibrium. Density of the solution was determined with a Mettler-Paar Precision DMA-02D density meter. Values of the partial specific volume of the wild type and mutant RMPKs were calculated based on the amino acid composition of PK using the procedure of Cohn and Edsall(22) .

Enzyme Kinetics

The enzymatic activity of wild type and mutant RMPK was determined by the lactate dehydrogenase-coupled enzyme assay(23) . The reaction occurs in a solution consisting of 50 mM Tris base, 72 mM KCl, 7.2 mM MgSO(4), 0.3 mM NADPH, 10 µg/ml lactate dehydrogenase, and varying amounts of Phe and FBP as indicated. The final concentration of ADP or PEP in the assay mixture was fixed at 4 or 2 mM, respectively, while varying the concentration of the other substrate. After adjusting the pH of the assay mixture to 7.5 at 23 °C, lactate dehydrogenase that was equilibrated with TKM buffer was added, and the assay mixture was finally brought to the desired volume. The reaction was started by the addition of 2-4 µl of PK to a 1-ml assay solution that had been equilibrated at 23 °C. The decrease in absorption at 340 nm was followed as a function of time with a Hitachi U-2000 spectrophotometer equipped with a six cell positioner. The data were collected and analyzed with the Hitachi Enzyme Kinetics Data System software program to obtain v, the observed steady-state kinetic velocity. All data sets were fitted to the modified version of the Hill equation as shown in (24)

where V(max) is the maximal velocity of each data set, [S] is the concentration of the variable substrate, n is the Hill coefficient, and K is a complex steady-state kinetic equilibrium constant that is equivalent to the K(m) in the Michaelis-Menten equation where n = 1.


RESULTS

Cloning and Nucleotide Sequence of Rabbit Muscle PK

Since the cloned RMPK gene is a result of a PCR amplification, alterations in the sequence might have been introduced. If indeed this is the case, variation in DNA sequences from different clones can be expected. However, identical nucleotide sequence was observed from two clones and is summarized in Fig. 2. Hence, it may be concluded that in this case PCR amplification did not result in alteration in the sequence. The open reading frame of 1593 base pairs runs from the ATG start codon to a TGA stop codon, with a deduced protein sequence of 530 amino acids. The nucleotide sequence also revealed that the EcoRI restriction site flanking the RMPK gene was cloned into the EcoRI site of pKK223-3 next to the putative Shine-Delgarno sequence with the correct orientation. The relative position, orientation, and extension of the oligonucleotides used for sequencing are depicted in Fig. 1. Sixteen differences were found between the published nucleotide sequence and these clones (Fig. 2), twelve of which do not result in a change in the amino acid sequences, and four differences in the nucleotide sequence led to three differences in the amino acid sequence (Fig. 2). The amino acid sequence differences are: Asp Glu; Glu Gln; Ser Ala.


Figure 2: Nucleotide and deduced amino acid sequence of RMPK. The predicted amino acid sequence is shown by the single-letter code under the nucleotide sequence, and numbers are indicated on the right for the amino acids and on the left for the nucleotides. Base pairs and amino acids diverging from the sequence reported by Larsen et al.(32) are doubly underlined. Amino acids verified with the automated sequencer are underlined.



Overexpression and Purification of Wild Type RMPK

The direction of ligation, after subcloning into pKK223-3, was determined by an asymmetric cut with HindIII. Only the E. coli which contained pRMPK with the correct direction of ligation showed an IPTG-inducible protein as indicated by a band observed by SDS-PAGE. This IPTG-inducible band comigrates with the authentic RMPK isolated directly from rabbit muscle. The PK activity detected in the homogenate of the overproducing E. coli was 1 unit/ml cell culture, a 50-fold higher activity than that of the control bacteria which do not contain the plasmid pRM-PK. The PK activity did not increase in the presence of 3 mM FBP, the activator of some PK enzymes. Furthermore, 76% of its activity was retained after heating at 60 °C for 10 min. The lack of activation by FBP and stability at high temperature are characteristics of RMPK(25, 26) . Thus, results from these activity measurements suggest that the PK activity measured in the overexpressing E. coli is predominantly the result of RMPK and not of the two PK isozymes of E. coli since the two endogenous PK isozymes of E. coli are either heat-stable and activated by FBP or heat-inactivated and insensitive to activation by FBP(27) .

Purification of recombinant RMPK from E. coli JM105 was performed by polyethyleneimine precipitation, ammonium sulfate fractionation, and gel filtration followed by ion-exchange chromatography. Under low ionic strength, polyethyleneimine precipitates a subset of negatively charged cellular components such as proteins, nucleic acids, and, presumably, phospholipids(28) . The polyethyleneimine precipitation step removes most of the nucleic acids and results in a 4-fold increase of the specific activity. Most importantly, polyethyleneimine precipitation selectively separated the RMPK from E. coli isozymes. A precipitation in the presence of 0.3% polyethyleneimine removed 89% of the E. coli PK activity. The remaining 11% of measured E. coli PK activity was separated from RMPK by the SP-Sepharose column because of their different isoelectric points(29, 30) .

After washing the nonabsorbed proteins from the SP-Sepharose column, a single peak was eluted between 130 and 185 mM NaCl, with a 280:260 ratio of 1.82 and a specific activity of 300 units/mg. The purity of the recombinant RMPK was 97% according to a densitometric analysis of SDS-PAGE gel. The results of a typical purification process using 1L of culture are summarized in Table 1.



Physical and Kinetic Characterization of Recombinant RMPK

The primary structure of the first 27 amino acids of the purified cloned RMPK was determined. The obtained primary structure was identical with the amino acid sequence predicted from the nucleotide sequence (Fig. 2). The start codons of prokaryotic expression systems encode for a formylmethionine; however, no methionine was detected at the first position.

The composition analysis of the purified RMPK is consistent with the deduced amino acid composition established by cDNA nucleotide sequence analysis. The distribution of aromatic amino acids in the recombinant protein is the same as that of the authentic enzyme since the amount of protein determined by the composition analysis is 97% of that determined by absorbance at 280 nm using the absorptivity established for the authentic RMPK.

The secondary structure of the recombinant and authentic RMPK were determined by CD. The maximum difference in molar ellipticity is 2.5%, which is insignificant under the experimental conditions (see Table 3in (31) ). The identity of the CD spectra for these two proteins indicates that the recombinant PK is folded into the same motif as the authentic enzyme as determined by this spectroscopic technique.



Limited trypsin digestion is a sensitive method to monitor the overall conformation of pyruvate kinase(9) . Recombinant and authentic RMPK were digested with trypsin under the same conditions(9) . The cleavage patterns for recombinant and authentic RMPK paralleled each other with respect to time dependence and sizes of cleavage products.

To compare the quaternary structure of the recombinant with the authentic RMPK, sedimentation velocity experiments were employed. A 2% difference in the sedimentation coefficient was observed, but such a change is not significant under the conditions of this experiment and, therefore, it can be concluded that the quaternary structure of the recombinant and authentic RMPK tetramers are indistinguishable.

Steady-state kinetics of recombinant RMPK were compared to authentic RMPK as a function of PEP concentration, in the absence or presence of 12 mML-phenylalanine, and the results are shown in Fig. 3A. In the absence of Phe, a plot of the velocity versus substrate concentration was hyperbolic, whereas in the presence of 12 mM Phe the plot was sigmoidal. Results are summarized in Table 2. There is no difference in k and K between the recombinant and authentic RMPK. As a result of the presence of 12 mM Phe, the value for K increased about 5-fold, and the kinetic behavior becomes cooperative as indicated by the Hill coefficient (n). The inhibitory effect of 12 mM Phe under nonsaturating substrate conditions (0.08 mM PEP) could be reversed by the addition of 3 mM FBP for both the recombinant and the authentic RMPK (data not shown). The quantitative analysis of all these kinetic data are similar to published values(7, 8) .


Figure 3: Effects of allosteric effectors on the kinetic properties of RMPK. A, recombinant and authentic; B, R119C; C, T340M; and D, Q377K mutants. The symbols and experimental conditions are as follows. A, recombinant RMPK with () and without (bullet) 12 mM Phe. circle and up triangle, authentic RMPK under corresponding conditions. B, C, and D, no effectors (circle), 10 mM FBP (bullet), 12 mM Phe (down triangle), 12 mM Phe and 10 mM FBP (). PEP was the variable substrate, and the ADP concentration was fixed at 4 mM. The lines represent the best fits to as described under ``Experimental Procedures.''





Construction, Expression, and Purification of RMPK Mutants

Point mutants of RMPK were constructed using site-directed mutagenesis that was performed directly with the pRMPK plasmid. Mutations were selected by a simultaneous elimination of a unique NdeI restriction site in the pRMPK construct. Mutagenic efficiency of greater than 50% was observed.

Mutant proteins were expressed and purified from E. coli using the same protocol for wild type RMPK except one modification: NZCYM broth instead of LB medium was used for the growth of E. coli cells harboring mutant PK plasmid. Mutant R119C and T340M were purified to greater than 97% homogeneity with yields of 6.3 and 8.7 mg/liter of culture, respectively. Mutant Q377K was expressed consistently at a much lower level, estimated to be at least 20-fold less than that of the R119C and T340M mutants. This observation indicates that Q377K is unstable when expressed in E. coli. After the SP-Sepharose column, the last step of purification, less than 0.5 mg of Q377K with a purity of about 60% was obtained from 1 liter of culture.

Physical Characterization of RMPK Mutants

Proper interpretation of mutant protein data relies on structural information; hence, purified R119C and T340M mutants were subjected to further biochemical and biophysical characterization. The secondary and tertiary structures of these proteins were monitored by CD. Near- and far-UV CD spectra of these mutants are identical with that of the wild type MPK, as shown in Fig. 4. These results indicate that no major secondary and tertiary structural changes have been introduced in PK by these mutations.


Figure 4: CD spectra in the far- and near-UV (inset) region for the wild type (bullet, solid line), R119C (circle, long-dashed line), and T340M (, short-dashed line) RMPK in TKM buffer at 23 °C.



Sedimentation velocity experiments were performed to probe the quaternary structure of the R119C and T340M mutants. To exclude the possibility of minor deviations between individual runs, protein solutions of identical concentrations of 0.75 mg/ml were loaded onto one ANF rotor and subjected to a centrifugal force of 30,000 rpm at 20 °C. The measured sedimentation coefficients, s(w), for the wild type RMPK, R119C, and T340M mutants are 10.66, 10.53, and 10.54 S, respectively. These values correspond well to a globular protein with a mass of 232,000 Da. Differences of less than 2% in s(w) are insignificant, and, therefore, these results suggest that the R119C and T340M mutant proteins exist in a tetrameric state, similar to wild type RMPK. Sedimentation equilibrium measurements were conducted to further confirm the quaternary structure of the R119C and T340M mutants. Equilibrium data were collected at 16,000 and 10,000 rpm at 20 °C. Using the same loading concentrations, the equilibrium sedimentation profiles of the wild type and mutant proteins are essentially superimposable, as shown in Fig. 5. Upon further analysis, the equilibrium data fit to a model of a single homogeneous sedimenting species, with an apparent molecular weight of approximately 230,000. Combining the results of sedimentation velocity and equilibrium studies, it can be concluded that, as in the case of the wild type, both mutant PK are tetrameric in TKM buffer.


Figure 5: Sedimentation equilibrium profiles of the wild type RMPK (bullet), R119C (), and T340M (circle) mutants at 10,000 rpm, 20 °C. The solid lines represent the best fits of the data to a scheme of single homogeneous species. The calculated apparent molecular weights are 230,000 (208,000, 253,000), 229,000 (218,000, 240,000), and 239,000 (227,000, 252,000) for the wild type PK, R119C, and T340M mutants, respectively. The values in parentheses represent the limits of 95% confidence intervals.



Kinetic Behaviors of Mutant R119C, T340M, and Q377K

All three mutants have low activity compared to the wild type RMPK. The values of k for R119C, T340M, and Q377K in the presence of saturating ADP and PEP are 1.79, 131, and 14.6 s, respectively. (^3)The most active mutant, T340M, is half as active as the wild type protein which has a k of about 288. Thus, these muscle PK mutants exhibit the same basic characteristics of decreased activity that are observed in human erythrocyte PK proteins that are mutagenized in these specific residues. A detailed kinetic study was employed to further dissect the enzymatic properties of the mutants. The effect of varying ADP concentration while keeping the concentration of PEP fixed at 2 mM was tested. Mutant R119C showed a normal dependence of v on ADP concentration, as shown in Fig. 6. Nevertheless, the curve is significantly shifted to higher concentrations of ADP yielding a high value of 6.0 mM for K, as indicated in Table 3. Mutants T340M and Q377K, however, exhibit v versus ADP concentration curves similar to that of the wild type PK, as shown in Fig. 6. Consequently, the values for K are also very similar, as summarized in Table 3. These observations indicate that mutation of residue Arg to Cys affects significantly the binding of ADP and also the efficiency of the enzyme for chemical catalysis while mutations at Thr and Gln do not.


Figure 6: Kinetic properties of the wild type (circle), R119C (bullet), T340M (down triangle), and Q377K () RMPK. ADP was the variable substrate in the presence of fixed 2 mM PEP in TKM buffer at 23 °C. The lines represent the best fits to as described under ``Experimental Procedures.''



Additional kinetic studies were conducted with PEP as the variable substrate. Furthermore, the responses of mutant PK to allosteric effectors were also tested. Four different sets of measurements were made for each mutant, namely, the relationship between activity and PEP concentration, the ability of 10 mM activator FBP to enhance the enzyme activity, the ability of 12 mM inhibitor Phe to decrease the enzyme activity, and the ability of FBP to reverse the inhibitory effect of Phe.

The dependence of the enzyme activity of the R119C mutant on PEP concentration is shown in Fig. 3B. The curve is characterized by values of 116 µM and 1.36 for K and Hill coefficient, respectively. The presence of 10 mM activator FBP has only a modest effect in reducing K to 103 µM, the Hill coefficient to 1.15 and no effect on k. The presence of 12 mM Phe shifts the curve of v versus PEP concentration to the right, as shown in Fig. 3B, and an increase in the Hill coefficient to 2.4 with a decrease in k. The inhibitory effect of Phe can be reversed by the presence of 10 mM FBP. Table 2summarizes the kinetic parameters of the R119C mutant under these various experimental conditions.

Mutant T340M showed a significantly different set of kinetic properties. In the absence of effectors, the curves for v versus PEP concentration is shifted to higher PEP concentration, as shown in Fig. 3C. Values of 0.47 mM and 2.0 for K and the Hill coefficient, respectively, were observed. These are both higher than that of the wild type PK. The presence of Phe significantly changed the value for K to 8.4 mM and raised the Hill coefficient further to 2.63. The presence of FBP only marginally reversed the effect of Phe by reducing K to 8.0 mM and the Hill coefficient to 1.96, as shown in Fig. 3C and summarized in Table 2. Thus, mutant T340M apparently is rendered more sensitive to Phe but significantly less responsive to FBP.

In the absence of effectors the kinetic properties of mutant Q377K is similar to that of the R119C, i.e. low turnover number, K and Hill coefficient assuming values similar to that of the wild type. The effect of FBP was minimal, as shown in Fig. 3D and Table 2. 12 mM Phe induced a shift of the relationship between enzyme activity and PEP concentration, leading to values of 2.2 mM and 1.68 for K and Hill coefficient, respectively. This inhibitory effect can be reversed significantly by 10 mM FBP, yielding values of 0.97 mM and 1.60 for K and Hill coefficient, respectively.


DISCUSSION

The deduced amino acid sequence of the RMPK gene is different from the sequence reported by Larsen et al.(32) in three residues. Instead of the dipeptide Asp-Glu, Glu-Gln were determined, and, instead of Ser, Ala was identified. Larsen et al.(32) obtained the RMPK clone by screening the cDNA library, whereas in this study the RMPK cDNA was obtained by PCR amplification employing specific oligonucleotides. Knowing that PCR amplification can lead to sequence alterations, Pfu DNA polymerase was used in the PCR reactions to minimize sequence errors in the RMPK clone. Furthermore, two different clones were sequenced to ascertain the accuracy of the cloned sequence. An identical sequence was obtained. A comparison was made with 16 known sequences of other PK isozymes (5) and amino acid composition of RMPK(34) . The Glu-Gln dipeptide reported in this study is identical with the sequence in all other known muscle PK isozymes, whereas the Asp-Glu reported by Larsen et al.(32) is not found in any PK isozyme at that position. The Ala determined in this study is identical with the rat muscle PK, human and rat liver PK, and rat red blood cell PK isozymes, whereas the Ser found by Larsen et al.(32) has not been reported in any PK isozyme at that position. It is interesting to note that the N-terminal methionine residue incorporated during the translation step by E. coli is excised. This observation is consistent with the report by Hirel et al.(33) that terminal methionine is excised efficiently.

The recombinant RMPK behaves like the tissue-derived RMPK with regard to the steady-state kinetic properties and its responses to effectors. The primary, secondary, and quaternary solution structures monitored by different methods have substantiated that the recombinant PK is indeed RMPK. This accomplishment enables the further study of the regulatory mechanisms of RMPK at the molecular level by perturbation analysis using site-directed mutagenesis.

A prerequisite for site-directed mutagenesis is the selection of site and the identity of the replacement amino acid residue. The adopted strategy in this study is to refer to natural mutants, in which cases the identities of sites and replacement amino acid residues are specified. In this study, the natural mutants are the erythrocyte PK mutants PK Linz, PK Tokyo/Nagasaki/Beirut, and PK Fukushima/Maebashi, which were identified by genetic studies of erythrocyte PK in hereditary nonspherocytic hemolytic anemia patients(1, 2, 3, 35) . The most outstanding characteristic of these mutants is the deficiency of PK activity. The sites and nature of mutations are R163C in PK Linz, T384M in PK Tokyo/Nagasaki/Beirut, and Q421K in PK Fukushima/Maebashi. The residue number refers to that of the erythrocyte PK sequence, and these correspond to Arg, Thr, and Gln of the RMPK sequence. Sequence homology analysis reveals that Arg and Thr are conserved in all PK isozymes while Gln is highly conserved. Thus, the issues that need to be resolved are as follows. 1) Would mutations of these sites in the RMPK isozyme yield the same phenotypic observations of low enzyme activity? 2) Do these sites target one specific step in the regulatory mechanism of PK or do they manifest their effects by different pathways? 3) Would a given mutation exert such an effect on the enzyme that the basic characteristics of this particular isozyme, such as binding constants of all ligands, are altered?

All three of the mutants presented in this study exhibit significantly reduced enzyme activity. The turnover number ranges from only 50 to 0.6% of the wild type value; thus, mutations of these specific sites in RMPK do generate mutant enzymes that mimic the basic defective characteristics observed in a different isozymic form of PK, namely, erythrocyte PK. These results attest to the importance of these highly conserved residues which most likely play the same role within the context of different forms of PK. The identities of these roles are currently being investigated.

The observed deficiency in enzyme activity for any of these mutant enzymes could have been caused by alterations in the structures of the enzyme. The CD and sedimentation data indicate that the R119C and T340M mutations do not induce any observable changes in the secondary, tertiary, and quaternary structures. The expression level and the ability to purify the RMPK indicates that these mutant PKs are as stable as the wild type enzyme. However, this is not the case with the Q377K mutant. Consistently, the expression level of the Q377K mutant is about 5 to 10% of the other mutants, and multiple bands that were not present in preparation of other mutants were observed in SDS-PAGE even after the last column of the purification procedure.

The Arg Cys mutation at residue 119, the location of which is shown in Fig. 7, significantly incapacitates the catalytic efficiency of the enzyme with a concomitant reduction in the apparent binding affinity for ADP. This is accomplished without much perturbation of the enzyme's ability to respond to effectors. Residue 119 is positioned near the cleft between the B and A domains. The active site is located in this cleft. Furthermore, according to the x-ray crystallographic data(32) , pyruvate, a product, is located in the near vicinity of residue 119. It is, therefore, reasonable to speculate that Arg plays an important role in the catalytic mechanism and binding of ADP.


Figure 7: RMPK atoms within a radius of 13.5 Å of pyruvate. A ball and stick model of a pair of spheres residing within subunit A (red) and subunit C (blue). Residues Arg (purple), Thr (white), Gln (yellow), and pyruvate (violet) are presented in a space-filling model. For clearer demonstration of the interdigitating nature of residue 340, atoms from residues 338-349 that are within a radius of 16 Å of pyruvate were included.



The Thr Met mutation at residue 340 leads to yet another set of effects. There is a modest 50% decrease in catalytic efficiency without significant effects on the apparent binding affinity for ADP. The observed change in K for PEP is most likely a consequence of an increase in the cooperativity as reflected by the Hill coefficient of 2.0 rather than an actual change in binding affinity for PEP. There is a significant increase in the Hill coefficient from 0.84 of the wild type to 2.0 in the T340M mutant in the absence of effectors. This observation implies that the distribution of this mutant between the active R-state and inactive T-state is in favor of the T-state, whereas the wild type RMPK has been shown to be essentially all in the R-state under these experimental conditions(8, 36) . Another significant consequence of this mutation is the apparent inability to respond to the presence of activator FBP. Combining the observations of an increase in the Hill coefficient and lack of response to FBP, one may speculate that residue 340 plays an important role in defining the equilibrium governing the R T transition and binding of the activator FBP. The locations of the sites of mutations are summarized in Fig. 7.

The roles of these residues in the vast number of linked equilibria which govern the allosteric regulatory behavior of PK have yet to be defined. Nevertheless, a few interesting structural correlations can be established. First, all three of these residues are within a 13.5-Å radius of the product, pyruvate. Second, while residues 119 and 377 are within the 13.5-Å sphere of pyruvate for a given subunit, residue 340 within each sphere is derived from a neighboring subunit.

In spite of the apparently different effects on RMPK elicited by these mutations, the response of these mutants to effectors, when they do, is characteristic of the muscle isozymic form, i.e. mM concentrations of effectors are required. Other isozymic forms of PK are more responsive since much lower concentrations of these effectors are needed to elicit a significant effect. Apparently, the specific sequence of RMPK imparts the quantitative characteristics of the isozymic form of PK, although these three specific residues may be conserved in other isozymic forms for well defined functions.

In summary, this study has shown that residues 119, 340, and 377 in RMPK are important for the normal functioning of PK. Equivalent mutations at these sites in human erythrocyte PK lead to the same net result, namely, significant deficiency in PK activity. Nevertheless, the specific mechanism that leads to the observed inactivation is dependent on the location of these residues. These mutants provide an excellent opportunity to dissect the complex mechanism of allosteric regulation of this key regulatory enzyme.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants GM-45579 and DK-21489 and Robert A. Welch Foundation Grants H-0013 and H-1238. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U44751[GenBank].

§
These two authors contributed equally to the work and both are considered as first authors.

To whom correspondence should be addressed. Tel.: 409-772-2281; Fax: 409-772-4298.

(^1)
The abbreviations used are: PK, pyruvate kinase; RMPK, rabbit muscle pyruvate kinase; M(1)-PK, muscle PK; M(2)-PK, kidney PK; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; IPTG, isopropyl-1-thio-beta-D-galactopyranoside; PEP, phosphoenolpyruvate; FBP, fructose 1,6-bisphosphate; PAGE, polyacrylamide gel electrophoresis.

(^2)
J. C. Lee et al., unpublished data.

(^3)
The value of k for the Q377K mutant has taken into account the heterogeneity in the preparation by densitometric scan of an SDS-PAGE gel of the Q377K sample employed in the kinetic measurement.


ACKNOWLEDGEMENTS

Compositional and protein analysis were performed in the UTMB Cancer Center Protein Chemistry Core Facility. The initial cloning experiments were conducted by M. Sloboda. J. C. L. thanks Dr. Scott Fredricksen for Fig. 7and all the discussion during the writing of this manuscript and Drs. Scott Fredricksen, E. Czerwinski, and B. Luxon for the preparation of Fig. 7.


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