(Received for publication, October 4, 1996, and in revised form, November 26, 1996)
From the Doris and Bertie Black Center for
Bioenergetics in Life Sciences, Ben Gurion University of the Negev,
Beer Sheva 84105, Israel, § Institut für Biochemie
der Pflanzen, Heinrich Heine Universität Düsseldorf,
Düsseldorf, Federal Republic of Germany, and
¶ Department of Plant Genetics, Weizmann Institute of Science,
Rehovot 76100, Israel
The participation of the amino acid
83 in determining the sensitivity of chloroplast ATP synthases to
tentoxin was reported previously. We have changed codon 83 of the
Chlamydomonas reinhardtii atpB gene by site-directed
mutagenesis to further examine the role of this amino acid in the
response of the ATP synthase to tentoxin and in the mechanism of ATP
synthesis and hydrolysis. Amino acid
83 was changed from Glu to Asp
(
E83D) and to Lys (
E83K), and the highly conserved tetrapeptide
T82-E83-G84-L85 (
TEGL) was deleted. Mutant strains were produced
by particle gun transformation of atpB deletion mutants
cw15
atpB and FUD50 with the mutated atpB genes. The
transformants containing the
E83D and
E83K mutant genes grew well
photoautotrophically. The
TEGL transformant did not grow
photoautotrophically, and no CF1 subunits were detected by
immunostaining of Western blots using CF1 specific
antibodies. The rates of ATP synthesis at clamped
pH with thylakoids
isolated from cw15 and the two mutants,
E83D and
E83K, were
similar. However, only the phosphorylation activity of the mutant
E83D was inhibited by tentoxin with 50% inhibition attained at 4 µM. These results confirm that amino acid
83 is critical in determining the response of ATP synthase to tentoxin. The
rates of the latent Mg-ATPase activity of the CF1s isolated from cw15,
E83D, and
E83K were similar and could be enhanced by
heat, alcohols, and octylglucoside. As in the case of the
membrane-bound enzyme, only CF1 from the
E83D mutant was
sensitive to tentoxin. A lower alcohol concentration was required for
optimal stimulation of the ATPase of the
E83K-CF1 than
that of CF1 from the other two strains. Moreover, the
optimal activity of the
E83K-CF1 was also lower. These
results suggest that introduction of an amino acid with a positively
charged side chain in position 83 in the "crown" domain affects the
active conformation of the CF1-ATPase.
The eukaryotic unicellular green algae Chlamydomonas reinhardtii constitutes a powerful experimental model system for the study of the photosynthetic machinery. It is accessible to genetic analysis and grows photoautotrophically on minimal medium or heterotrophically with acetate as the sole carbon source. These properties have been used to isolate numerous photosynthetic mutants which have helped to examine the function of the photosynthetic apparatus (1).
The chloroplast of C. reinhardtii contains approximately 80 copies of its 196-kb1 circular genome (2). Due to recent progress in the molecular genetics of C. reinhardtii, chloroplast proteins can be altered by site-directed mutagenesis of the corresponding genes followed by transformation into the chloroplast (3-7). Chloroplast transformation was first demonstrated in 1988 by Boynton and co-workers (3), by complementation of an atpB deletion mutant with the cloned wild type gene. The transforming DNA integrates into the recipient chloroplast DNA by homologous recombination. Goldschmidt-Clermont (5) has constructed a chimeric selectable marker using the chloroplast atpA promoter and the bacterial gene aminoglycoside adeninetransferase (aadA), which confers resistance to spectinomycin and streptomycin in Escherichia coli.
Tentoxin, a cyclic tetrapeptide
(cyclo-L-leucyl-N-methyl-(z)-dehydrophenylalanyl-glycyl-N-methyl-alanyl)
produced by the fungus Alternaria tenuis, is a potent
inhibitor of the chloroplast ATP synthase of certain sensitive plant
species (8, 9), which acts as an uncompetitive inhibitor of the
CF1-ATPase with respect to nucleotide substrates. It was
proposed that 1 mol of tentoxin/mol of CF1, bound at a site
near the interface between the N-terminal domains of subunits and
, interferes with the cooperative interaction between nucleotide
binding sites inhibiting the enzyme activity (10). Binding of tentoxin
to a second, low affinity site stimulates the ATPase activity of
CF1 (11-13). Avni et al. (7) have shown that
amino acid
83 is involved in conferring tentoxin sensitivity. Nicotiana species having a glutamic acid residue at position
83 are resistant, whereas species with an aspartic acid residue in this
position are sensitive to tentoxin. The peptide sequence around
position 83 in C. reinhardtii
subunit was replaced by the corresponding sequence from a tentoxin-sensitive tobacco
subunit. This exchange of five amino acids including Glu83
to Asp83 converted the normally tentoxin-resistant to a
tentoxin-sensitive C. reinhardtii ATP synthase.
We investigated in more detail the role of the acidic amino acid
residue at position 83 of the subunit of the ATP synthase by
site-directed mutagenesis. The atpB deletion mutant
cw15
atpB was prepared from the cell wall deficient strain cw15 by
replacing the atpB open reading frame by the aadA
cassette (14). The use of the cell wall deficient strain cw15 allowed
us to isolate well preserved thylakoids and to characterize electron
transport and photophosphorylation activities of these membranes (15,
16).
C. reinhardtii cw15 was cultivated in high salt
medium (1). The cell wall deficient atpB deletion mutant
cw15atpB (14) was cultivated in high salt medium containing 0.4%
acetate and 150 µg/ml spectinomycin (high salt medium containing
acetate/spectinomycin) at low light intensity (~50 lux). The
atpB deletion mutant FUD50 (17) was cultivated in
Sager-Granick medium (1) containing 0.2% acetate.
All cloning procedures were done using commercially available
restriction endonucleases and DNA-modifying enzymes according to the
suppliers instructions. DNA fragments were isolated from agarose gels
using the Glass Select DNA Isolation Kit (5 Prime 3 Prime, Boulder,
CO) or by freezing the sliced agarose fragments in the presence of 80%
phenol, followed by chloroform extraction, and ethanol precipitation of
the DNA from the supernatant. Transformation into E. coli
JM109 or DH5
was done according to the instructions supplied in the
pAlter site-directed mutagenesis kit (Promega Biotech). Plasmid DNA was
isolated by alkaline miniprep as described in the T7 DNA sequencing
protocol (Promega Biotech) or by the Qiagen plasmid kit (Qiagen GmbH,
Hilden, Germany). DNA sequencing was done using Sequenase version 2
(USB) and denatured plasmid DNA as template. Site-directed mutagenesis
was done using the pAlter site-directed mutagenesis kit (Promega
Biotech) according to the suppliers instructions, except that T7 DNA
polymerase was used for second strand synthesis instead of T4 DNA
polymerase. The mutagenesis procedure was performed using
single-stranded DNA from plasmid pAlter containing a 2.8-kb
HindIII-KpnI fragment with the complete
atpB gene (18) and the mutagenic primers shown in Fig. 1.
Introduction of the mutation into the atpB gene was confirmed by PCR, restriction analysis, and DNA sequencing using atpB-specific primers. The 0.9-kb
EcoRV-PstI fragments containing the mutant sites
were cloned back into suitable transformation vectors containing
extended 5
- and 3
-flanking regions and antibiotica resistance
cassettes as markers for successful transformation.
Chloroplast transformation was done by vortexing in the presence of glass beads (6) or with the PDS1000-He apparatus of Bio-Rad. Cells were spread in 0.2% soft agar onto high salt medium-agar plates and bombarded with DNA-coated M10 tungsten particles. After incubation overnight at low light, cells were transferred to full light intensity (500-1000 lux) in CO2-enriched atmosphere for selection of transformants. To select for spectinomycin or kanamycin resistance, cells were bombarded on acetate containing medium, incubated overnight at low light intensity, and then spread onto acetate containing plates containing 150 µg/ml spectinomycin or kanamycin. C. reinhardtii total DNA was isolated as described (19) except that cells were scraped from agar plates and directly resuspended in lysis buffer instead of grown in liquid medium. Proteins were separated by SDS-polyacrylamide gel electrophoresis (20) and stained with Coomassie R250 or transferred to nitrocellulose and immunostained using a CF1 specific rabbit antiserum and protein A alkaline phosphatase.
C. reinhardtii CF1 was prepared by chloroform extraction and anion exchange chromatography (21) with some modifications. Cells were harvested, washed, and resuspended in lysis buffer (50 mM Tricine/NaOH, pH 8.0, 10 mM NaCl, 10 mM sodium ascorbate, and 250 mM sucrose) to a concentration of 1 mg/ml chlorophyll. The cells were broken by passage through a Yeda press (2 times at 106 N/m2), and the membranes were precipitated and washed 4 times in 2 mM Tricine/NaOH, pH 8.0, 50 mM NaCl. Membranes were resuspended at room temperature to 3 mg/ml chlorophyll in 20 mM Tricine/NaOH, pH 8.0, 1 mM EDTA, 1 mM ATP, 2 mM dithiothreitol, and 10% glycerol. A 1/2 volume of chloroform was added, and the suspension was vigorously vortexed for 20 s. The suspension was centrifuged for 30 min at 12,000 × g at room temperature, and the upper phase was subjected to a second centrifugation under the same conditions. The chloroform extract was mixed with Fractogel TSK DEAE-650 (M) from Merck preequilibrated with loading buffer (20 mM Tricine/NaOH, pH 8.0, 1 mM NaEDTA, 1 mM ATP, 2 mM dithiothreitol containing 10 mM (NH4)2SO4) at a ratio of 1 ml of gel per 2 mg of chlorophyll extracted and incubated for 30 min at room temperature. This mixture was loaded onto a bed of 5 ml of Fractogel TSK DEAE-650 (M) in a chromatography column, rinsed with loading buffer, and eluted with a 10-300 mM (NH4)2SO4 gradient in loading buffer. The purest fractions, corresponding to a protein peak eluting at about 120 mM (NH4)2SO4, were collected, supplemented with 1 mM ATP, precipitated by addition of (NH4)2SO4 to 50% saturation, and stored at 4 °C. For enzymatic assays, the CF1 pellet was collected by 15 min centrifugation at 12,000 × g, dissolved in 20 mM Tricine/NaOH, pH 8.0, to a concentration of 0.5-1 mg/ml. Salts were removed by column centrifugation through Sephadex G-25 equilibrated with 20 mM Tricine/NaOH, pH 8.0.
Mg-ATPase activity of these preparations was measured by photometric determination of released inorganic phosphate (Pi) as described (22). 5 µg of enzyme were incubated for the indicated times in 20 mM Tricine/NaOH, pH 8.0, with 4 mM MgCl2 and 6 mM ATP present, and the additions indicated in the legends of figures and tables. Reaction mixes lacking either enzyme or ATP were preincubated at the indicated temperature, and the reactions were started by adding enzyme or substrate. The reaction was stopped by addition of 3% trichloroacetic acid, and inorganic phosphate released was determined photometrically.
Active thylakoid membranes were prepared as described (15, 16). Cells were grown at 20 °C in high salt medium containing 0.2% acetate at a light intensity of 900 lux in a 14-h light to 10-h dark cycle, in 400-ml cultures bubbled with sterile air. The cultures were grown to a cell density of 6 × 106 cells/ml and then harvested by centrifugation (10 min, 1000 × g), washed with medium A (0.3 mM sucrose, 10 mM Tricine, pH 8.0, 50 mM NaCl, and 5 mM MgCl2), and resuspended in the same medium at 4 °C. All further steps were carried out at this temperature. The cells were passed twice through a Yeda Press at 5 × 105 N/m2 and 5.5 × 105 N/m2, respectively, and harvested by a 1-min centrifugation step at 3000 × g. The resulting pellet was resuspended and washed twice in 2 mM Tricine, pH 8.0, 50 mM NaCl, 1 mM MgCl2 and resuspended in a small volume of solution A.
ATP formation was measured at clamped proton gradient by employing the
pH clamp device (23). The proton gradient was controlled by
monitoring the fluorescence quench of 9-aminoacridine and adjusting the
light intensity correspondingly. The reaction medium contained in a
final volume of 2.5 ml: 25 mM Tricine, pH 8.0, 50 mM KCl, 5 mM MgCl2, 50 µM phenazine methosulfate, 10 mM
dithiothreitol, 50 nM valinomycin, 10 mM
glucose, 12 units/ml hexokinase, 5 µM 9-aminoacridine,
and thylakoid vesicles to give a final concentration of 25 µg Chl/ml.
After preillumination at 20 °C for 2 min, the
pH was adjusted to
60% fluorescence quench and after 1 min 1 mM ADP and 5 mM [32P]Pi were added. Rates of
photophosphorylation were calculated from the organic
32P-labeled phosphate determined in the samples taken after
10, 20, 30, and 40 s according to the method of Sugino and Miyoshi (24).
The oligonucleotide primers presented in Fig. 1
were used to introduce the desired changes (bold letters)
into the atpB gene by site-directed mutagenesis as follows:
(a) change of codon 83 from glutamic acid to aspartic acid
(E83D), (b) change of codon 83 from glutamic acid to
lysine (
E83K), and (c) deletion of the tetrapeptide
Thr82-Glu83-Gly84-Leu85
(
TEGL). In the
E83D and
E83K oligonucleotides a second, silent point mutation, creating a new AseI restriction site
(underlined), was introduced to facilitate transformant
analysis. The mutant atpB genes were cloned into the
transformation vectors pT7atpB5
L3
LaadA/Kan (Fig.
2A) or pBSatpB
aadA/Kan (Fig. 2B).
The extended 5
- and 3
-untranslated regions of these plasmids
significantly increased transformation efficiency. The kanamycin or
aadA cassettes were added to select for successful
transformation with mutant atpB genes that do not restore
photoautotrophic growth. The constructs with the atpB genes
containing the
E83D,
E83K, and the
TEGL mutations shown in
Table I were used to transform the cell wall deficient
deletion mutant cw15
atpB (14) and the atpB deletion mutant FUD50 (17). Selection for successful transformation was done
under autotrophic growth conditions on high salt medium or Sager-Granick minimal medium plates or for spectinomycin or kanamycin resistance on acetate-containing media containing 150 µg/ml of the
corresponding antibiotica. The
E83D and
E83K mutant plasmids yielded photoautotrophic transformants, but the spectinomycin- or
kanamycin-resistant transformants obtained by transformation with the
TEGL deletion mutant plasmid did not grow photoautotrophically (Table I). The DNA fragments containing the mutant sites were isolated
from total DNA by PCR, and in the case of the
E83D and
E83K
transformants the mutations were confirmed by restriction analysis
(Fig. 3). Subsequently, in all three types of
transformants, the mutations were verified by sequence analysis of the
corresponding PCR products. The presence of the
CF0CF1 complex was examined by
SDS-polyacrylamide gel electrophoresis of the thylakoid proteins followed by immunostaining of Western blots with a
CF1-specific antiserum. The thylakoids from the
spectinomycin- and kanamycin-resistant transformants containing the
TEGL mutation were completely devoid of the ATP synthase complex
(not shown). Thus, this highly conserved peptide segment seems to be
important for the assembly or stability of the ATP synthase
complex.
|
Thylakoids prepared from cw15 and the transformants cw15D221 and
cw15K221 containing the mutations E83D and
E83K, respectively, were assayed for their photophosphorylation capacity. All three thylakoid preparations yielded similar rates at a clamped
pH (60%
fluorescence quench). Photophosphorylation activity of cw15 and
E83K
was not inhibited by tentoxin, whereas that of
E83D was sensitive to
tentoxin with 50% inhibition attained at 4 µM (Fig.
4A). The degree of inhibition was enhanced by
preincubation of thylakoids with tentoxin and maximal inhibition was
attained after 1 h with 5 µM tentoxin (Fig.
4C). Fig. 4B shows the response to tentoxin of
the ethanol-enhanced Mg-ATPase activity of CF1 isolated
from the wild type and the two transformants. The ATPase activity of
cw15 and
E83K CF1 was not inhibited by tentoxin, whereas
that of
E83D was inhibited with 50% inhibition attained at a
concentration of 0.5 µM tentoxin. Preincubation with
tentoxin was also required for maximal inhibition (Fig. 4
C). Hence, the isolated CF1-ATPase is about
10-fold more sensitive to tentoxin than photophosphorylation.
We examined whether the mutations in codon 83 had additional effects on
the catalytic properties of the mutant ATPases. The general
characteristics of all three CF1 preparations were similar to those reported before for C. reinhardtii (21) although
the rates with the latent enzyme were lower. As shown in Table
II, the low basal rates of the Mg-ATPase activities of
all three CF1 preparations were enhanced by elevating the
assay temperature, by octylglucoside, by ethanol, and by methanol. In
the presence of 20% ethanol the apparent
Km MgATP determined for all three
enzymes was 1 mM. Full stimulation of the Mg-ATPase
activity by octylglucoside was attained at a concentration of about 1% (Fig. 5A). While the basal, heat- and
detergent-stimulated ATPase activities were similar for the wild type
and mutant enzymes, the ATPase activity of the E83K CF1
elicited by alcohols was significantly lower (Fig. 5, B and
C). Maximal stimulation of the Mg-ATPase activities of the
cw15 and
E83D enzymes was obtained at an ethanol concentration of
23% (v/v) and a methanol concentration of 30% (v/v), whereas the
maximal ATPase activity of the
E83K enzyme was attained at 20%
ethanol and 25% methanol (Fig. 5, B and C). The
degree of stimulation of the
E83K enzyme by alcohols was less than
half of that with the cw15 and the
E83D enzymes (Fig. 5,
B and C).
|
The lower activity and the shift in the concentration of alcohols
needed for maximal stimulation of ATPase with the E83K enzyme could
be caused by rapid time-dependent inactivation of this
enzyme, in particular at higher organic solvent concentrations. We
tested this possibility by measuring the time course of the Mg-ATPase
activity of all the three preparations at 17 and 25% ethanol. The
specific activities of the
E83K enzyme at both ethanol concentrations were equal and about 30% lower than its maximal activity at 20% ethanol. At 17% ethanol the ATPase activities of all
three enzymes were similar (Fig. 6A), but at
25% ethanol the ATPase activities of the cw15 and
E83D enzymes were
increased significantly whereas that of the
E83K CF1
remained the same (Fig. 6B). At both ethanol concentrations
the rates of all three CF1s were linear for several minutes
showing that the
E83K CF1 is in a less active
conformation at 25% ethanol and is not subject to rapid
denaturation.
The sensitivity of certain CF0CF1 ATP
synthases to tentoxin has been attributed to the presence of aspartic
acid in position 83 of the subunit. This was demonstrated by Avni
et al. (7) by analysis of resistant and sensitive tobacco
strains and subsequent mutagenesis studies with the C. reinhardtii atpB gene. The C. reinhardtii atpB sequence
from codons 74-91 (VRAVSMNPTEGLMRGMEV) was replaced by the
corresponding tobacco sequences of the resistant (VRAV
M
TEGL
RGMEV) or sensitive
lines
(VRAV
M
T
GL
RGMEV) by changing four or five amino acids, respectively (underlined). The
resulting mutant enzyme containing Glu in position 83 was resistant to
tentoxin whereas that of the mutant with Asp in position 83 was
inhibited by tentoxin.
In this work we have exchanged a single codon of the C. reinhardtii atpB gene, Glu83 by Asp, to determine
whether in fact this change alone determines tentoxin sensitivity. The
Mg-ATPase activity of the isolated CF1 containing the Asp
residue in position 83 (E83D) was indeed inhibited by tentoxin at
concentrations similar to those required to inhibit enzymes from
sensitive plants and from the sensitive cyanobacterium Anacystis
nidulans (Fig. 4B) (7, 8, 25). Effective inhibition by
tentoxin required preincubation of the enzyme with tentoxin as reported
previously (Fig. 4C) (8, 25). Photophosphorylation of
isolated thylakoids of the
E83D mutant was also inhibited by
tentoxin with 50% inhibition obtained at 4 µM (Fig.
4A). The difference of 1 order of magnitude between the
sensitivity to tentoxin of photophosphorylation and
CF1-ATPase activity (Fig. 4) might be explained by
different accessibility of tentoxin to its binding site. It is known
that the conformation of isolated CF1 differs from the
conformation of the CF1 sector in the membrane-bound CF0CF1 complex (26, 27). On the other hand,
photophosphorylation of the mutant enzyme produced by Avni et
al. (7) containing a tobacco/C. reinhardtii hybrid
subunit was rather sensitive to tentoxin even after a relatively short
preincubation time. The apparent higher affinity of this mutant enzyme
to tentoxin could be the result of the additional changes made in the
sequence of the tobacco/C. reinhardtii hybrid gene (7).
The fact that the enzymes with the bulkier amino acid side chains (Glu
and Lys) in position 83, irrespective of their charge, are resistant to
tentoxin whereas the enzyme with Asp in position 83 is sensitive
indicates that in the resistant species the access of tentoxin to its
binding site is sterically hindered. According to the three-dimensional
structure of F1 (28), the amino acid corresponding to
position 83 of the subunit of C. reinhardtii is located
in the lower region of the crown structure at the interface where the
alternating
and
subunits are in close contact. Tentoxin contains several hydrophobic residues that might be important for the
interaction with a hydrophobic pocket partly screened by amino acid
83. The spatial structure of tentoxin is wedge shaped and could fit
into the
interface domain. The more extended Glu and Lys
residues in position
83 may sterically hinder the access of tentoxin
to this binding domain inducing resistance to tentoxin. Tentoxin was
shown to inhibit the AMP-PNP-induced release of tightly bound ADP from
CF1 (10), suggesting that tentoxin may bind at the
interface between an
and
subunit and thereby preclude
conformational changes required for catalytic turnover. Tentoxin acting
as a wedge can prevent the transfer of information between different
nucleotide binding sites on the ATP synthase and inhibit the enzymatic
activity.
The transformants where the highly conserved tetrapeptide
T82-E83-G84-L85 was deleted could not grow photoautotrophically probably because the CF0CF1 complex was not
assembled. As revealed by the crystal structure of mitochondrial
F1 (28), the four amino acids deleted in the
subunit
form a loop connecting two
sheets of the
subunit in the
stabilizing crown structure, and their deletion would prevent proper
folding of the mutant
subunit. Changing only one residue, a Glu to
a Lys, in the
E83K mutant yielded an active ATP synthase which was
not sensitive to tentoxin and exhibited similar rates of
photophosphorylation to those of the wild type and the
E83D
mutant.
The Mg-ATPase activities of the cw15, E83D, and
E83K
CF1's were quite similar at elevated assay temperature
(Table II) or in the presence of octylglucoside (Fig. 5A),
but significant differences were found with the alcohol-enhanced
Mg-ATPase activity (Fig. 5, B and C). The higher
methanol versus ethanol concentrations required to attain
maximal rates of ATP hydrolysis indicate that the dielectric properties
of the assay medium plays an important role in the activation process
by alcohols. Denaturation by alcohols was ruled out (Fig. 6,
A and B) as a possible reason for the lower activity of
the
E83K enzyme as compared with the cw15 and
E83D enzymes. We
propose that the presence of a Lys residue in position 83, which
according to the three-dimensional structure of F1 is in
close vicinity to residue Arg
52, alters the active
conformation of the CF1-ATPase induced with alcohols. On
the other hand, the octylglucoside-induced activation of the
CF1-ATPase does not seem to be affected in the same manner by the introduction of Lys in position 83.
It is very interesting that both the sensitivity of CF1 to
tentoxin and the ATPase activity enhanced by alcohols are affected by
mutations at codon 83. It is likely that the primary effect of organic
solvents is to alter the conformation of the enzyme by lowering the
dielectric constant of the medium (29, 30). The release of tightly
bound, inhibitory ADP, which is a rate-limiting step in catalysis, is
stimulated by alcohols (31) and by octylglucoside (32). In the presence
of ethanol the Mg-ATPase activity of CF1 is no longer
inhibited by ADP (29). Ethanol also removes the subunit from the
CF1 complex and stimulates the ATPase in this way (33). It
will be interesting to determine which of these effects is affected by
the mutations in codon 83. Our results demonstrate that the stimulatory
effects of octylglucoside and ethanol are based on different
mechanisms. The ATPase activity of the
E83K enzyme is stimulated to
the same extent as that of the cw15 and
E83D enzymes by
octylglucoside but affected differently by methanol and ethanol.
Tentoxin inhibits multisite catalysis by binding at one of three
/
interfaces of the enzyme (probably near amino acid
83),
interferes with the interactions between catalytic sites, and inhibits
the release of ADP from the catalytic site (10). The results presented
here demonstrate that the domain around amino acid
83 in the crown
domain participates in activation and inhibition of the catalytic
activity of CF1, probably via long range conformational
effects that affect the affinity of the tight sites for ADP. Other
mutants in the crown domain (
E38K (34) and
C63W (35)) were shown
to affect the coupling efficiency of ATP synthase, probably by
interfering with conformational signaling. These results imply that the
crown domain is involved in determining the conformational states of
the catalytic sites by transmitting conformational signals essential
for catalysis.