(Received for publication, August 18, 1995; and in revised form, December 8, 1995)
From the
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.
Mammalian pyruvate kinase
(ATP:pyruvate-2-O-phosphotransferase, EC 2.7.1.40) (PK) ()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
),
kidney (M
), erythrocyte (R), and liver (L). In the absence
of inhibitor, M
-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. (
)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
and M
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.
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.
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).
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.
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/mgcm(14) .
where V 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
in the Michaelis-Menten equation where n = 1.
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.
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.
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 (
) 12 mM Phe.
and
, authentic RMPK under corresponding
conditions. B, C, and D, no effectors
(
), 10 mM FBP (
), 12 mM Phe (
), 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.''
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.
Figure 4:
CD spectra in the far- and near-UV (inset) region for the wild type (, solid
line), R119C (
, 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, 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
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 (), R119C (
), and T340M (
) 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.
Figure 6:
Kinetic properties of the wild type
(), R119C (
), T340M (
), 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.
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U44751[GenBank].