From the
Glucose-6-phosphatase (G6Pase) is the enzyme deficient in
glycogen storage disease type 1a, an autosomal recessive disorder. We
have previously identified six mutations in the G6Pase gene of glycogen
storage disease type 1a patients and demonstrated that these mutations
abolished or greatly reduced enzymatic activity of G6Pase, a
hydrophobic protein of 357 amino acids. Of these, four mutations (R83C,
R295C, G222R, and Q347X) are missense and one (Q347X)
generates a truncated G6Pase of 346 residues. To further understand the
roles and structural requirements of amino acids 83, 222, 295, and
those at the carboxyl terminus in G6Pase catalysis, we characterized
mutant G6Pases generated by near-saturation mutagenesis of the
aforementioned amino acids. Substitution of Arg-83 with amino acids of
diverse structures including Lys, a conservative change, yielded mutant
G6Pase with no enzymatic activity. On the other hand, substitution of
Arg-295 with Lys (R295K) retained high activity, and R295N, R295S, and
R295Q exhibited moderate activity. All other substitutions of Arg-295
had no G6Pase activity, suggesting that the role of Arg-295 is to
stabilize the protein either by salt bridge or hydrogen-bond formation.
Substitution of Gly-222, however, remained functional unless a basic
(Arg or Lys), acidic (Asp), or large polar (Gln) residue was
introduced, consistent with the hydrophobic requirement of codon 222,
which is predicted to be in the fourth membrane-spanning domain. It is
possible that Arg-83 is involved in stabilizing the enzyme
(His)-phosphate intermediate formed during G6Pase catalysis. There
exist 9 conserved His residues in human G6Pase. His-9, His-119,
His-252, and His-353 reside on the same side of the endoplasmic
reticulum membrane as Arg-83. Whereas H119A mutant G6Pase had no
enzymatic activity, H9A, H252A, and H353A mutant G6Pases retained
significant activity. Substitution of His-119 with amino acids of
diverse structures also yielded mutant G6Pase with no activity,
suggesting that His-119 is the phosphate acceptor in G6Pase catalysis.
We also present data demonstrating that the carboxyl-terminal 8
residues in human G6Pase are not essential for G6Pase catalysis.
Glucose-6-phosphatase (G6Pase,
In order to gain insight into the mechanism of G6Pase
catalysis and the pathogenesis of the type 1a disorder, we analyzed the
G6Pase gene of 12 unrelated GSD type 1a patients and identified six
mutations, including one insertion (459insTA), one codon deletion
(
Studies have shown that an enzyme-phosphate
intermediate is formed during G6Pase catalysis and the phosphate
acceptor in G6Pase is a His residue
(9, 10) . Sequence
analysis and alignment reveal the presence of 9 conserved His residues
in human
(5) , mouse
(6) , and rat
(11) G6Pase.
His-9, His-119, His 252, and His-353 are predicted to be on the same
side of the ER membrane as Arg-83, Arg-295, and
Gln-347
(5, 6, 8) . In this study, we examined
the role of these 4 His residues in G6Pase catalysis by altering each
of the His residues by site-directed mutagenesis and analyzing G6Pase
activity after transient expression of WT and mutant G6Pase cDNA in
COS-1 cells.
The
phG6Pase-DraIII construct, which retains the primary amino
acid sequence of WT G6Pase but contains an additional DraIII
site at nucleotides 614-622, was constructed by site-directed
mutagenesis using the G6Pase-WT as a template. The outside primers are
O1 and O2, and the two inside mutant primers are I1
(5`-CCTCACCAAGTGGTTGCTGGAGTC-3`, nucleotides 611-634, sense) and
I2 (5`-AACCACTTGGTGAGGAAAATGAGC-3`, nucleotides 625-602,
antisense). The amplified fragments were digested with XhoI
and XbaI and ligated into a pSVL vector. The
pSVLhG6Pase-DraIII construct exhibits WT G6Pase enzymatic
activity as demonstrated by transient expression assays in COS-1 cells
(see ). The pSVLhG6Pase-DraIII-5` fragment
(containing nucleotides 77-619 of hG6Pase) or the
pSVLhG6Pase-DraIII-3` fragment (containing nucleotides
621-1156 of hG6Pase) was obtained by digestion of
pSVLhG6Pase-DraIII with XbaI/DraIII or
XhoI/DraIII, respectively, and purified on a low
melting agarose gel.
We also employed a modification of cassette
mutagenesis
(12) to generate multiple mutations of codons 83,
222, and 295. The two outside polymerase chain reaction primers for
codon 83 (nucleotides 326-328) mutants (R83M, R83N, R83T) are O1
and I2. The sense strand of the degenerate inside primers (nucleotides
316-336) is 5`-CTTTGGACAG(A/C/G)(A/C/G/T)(G/T)CCATACTG-3`. The
amplified fragments were digested with DraIII and
XhoI and ligated into the pSVLhG6Pase-DraIII-3`
fragment. Mutants were identified by DNA sequencing.
The two outside
polymerase chain reaction primers for codon 222 (nucleotides
743-745) and codon 295 (nucleotides 962-964) mutants are I1
and O2. The sense strand of the degenerate inside primers (nucleotides
731-754) for mutants G222K, G222L, G222M, G222N, G222Q, G222R,
G222T, and G222V is 5`-AGCTTCGCCATC(A/C/G)(A/C/G/T)(C/G)TTTTATCTG-3`
and for mutants G222D, G222S, and G222Y is
5`-AGCTTCGCCATC(C/G/T)(A/C)TTTTTATCTG-3`. The sense strand of the
degenerate inside primers (nucleotides 953-976) for mutants
R295H, R295I, R295K, R295M, R295N, R295P, R295Q, and R295S is
5`-CTCCCATTC(A/C/G)(A/C/G/T)(C/G)CTCAGCTCTATT-3` and for mutants R295E
and R295Y is 5`-CTCCCATTC(G/T)(A/G)(A/T)CTCAGCTCTATT-3`. The amplified
fragments were digested with DraIII and XbaI and
ligated into the pSVLhG6Pase-DraIII-5` fragment.
The
5`-primer for H9A mutant is
5`-ATGGAGGAAGGAATGAATGTTCTCGCTGACTTTGGGATC-3` (nucleotides
80-118, sense) and the 3`-primer is O2. The 5`-primer for H353A
mutant is O1, and the 3`-primer is 5`-TTACAACGACTTCTTGGCCG-3`
(nucleotides 1153-1134, antisense). The outside primers used in
codons 119 and 252 are O1 and O2. The sense strand of the inside
primers for mutant H119A is 5`-CCTCTGGCGCTGCCATGGGCACAG-3` (nucleotides
426-449), and mutant H252A is 5`-GAATGGGTCGCCATTGACACC-3`
(nucleotides 824-844). The amplified fragments were digested with
XhoI and XbaI and ligated into a pSVL vector.
The
two outside polymerase chain reaction primers for codon 119
(nucleotides 434-436) mutants (H119I, H119K, H119M, H119N, H119R,
and H119T) are O1 and I2. The sense strand of the degenerate inside
primers is 5`-CCTCTGGC(A/C)(A/G/T/C)(G/T)GCCATGGGCACAG-3` (nucleotides
426-449). The amplified fragments were digested with
DraIII and XhoI and ligated into the
pSVLhG6Pase-DraIII-3` fragment.
The 5`-primer for
carboxyl-terminal G6Pase mutants is O1, and the 3`-antisense primers
are H353X (5`-TTACGGCTGGCCCAGGACCTG-3`, nucleotides
1118-1138), Q351X (5`-TTAGCCCAG GACCTGGGCGAG-3`,
nucleotides 1112-1132), G350X (5`-TTACAGGACCTGGGCGAGGCA-3`, nucleotides 1109-1129), and
L349X (5`-TTAGACCTGGGCGAGGCAGTA-3`, nucleotides
1106-1129). All constructs were confirmed by DNA sequencing.
RNA was
isolated by the guanidinium thiocyanate/CsCl method
(14) ,
separated by electrophoresis in 1.2% agarose gels containing 2.2
M formaldehyde, and transferred to Nytran membranes. The
filters were hybridized at 42 °C in the presence of the phG6Pase-1
probe as described previously
(5) .
Phosphohydrolase
activity was determined essentially as described by Burchell et
al.(15) . Reaction mixtures (100 µl) contained 50
mM cacodylate buffer, pH 6.5, 10 mM glucose
6-phosphate, 2 mM EDTA, and appropriate amounts of cell
homogenates and were incubated at 30 °C for 10 min. Sample
absorbance was determined at 820 nm and is related to the amount of
phosphate released using a standard curve constructed by a stock of
inorganic phosphate solution. Essentially the same enzymatic activity
was obtained in control or deoxycholate (0.2%)-treated homogenates.
In earlier
studies
(5, 7, 8) , we sequenced each G6Pase
mutant construct generated by site-directed mutagenesis. It is
time-consuming and impractical to analyze the large number of mutants
generated by near-saturation mutagenesis. In the present study, we
created a DraIII site at nucleotides 614-622 of the
human G6Pase cDNA. The G6Pase-DraIII construct retains the
primary amino acid sequence of WT G6Pase and exhibits WT G6Pase
enzymatic activity (). Therefore, mutagenesis and sequence
analysis of the resulting mutant constructs can be performed using the
smaller G6Pase-DraIII-5` or G6Pase-DraIII-3`
fragment.
To study the role of Arg-83 in G6Pase catalysis, this
amino acid was substituted with amino acids of diverse structures
including Glu (R83E), Lys (R83K), Leu (R83L), Met (R83M), Asn (R83N),
Gln (R83Q), Ser (R83S), and Thr (R83T) (). Like the
enzymatically inactive R83C mutant, phosphohydrolase activity was not
detectable in COS-1 cells transfected with any of the G6Pase codon 83
mutants, including a conservative substitution of Lys for Arg.
G6Pase is an ER protein containing the ER
transmembrane protein retention signal, KK, at residues 354 and
355
(5, 6) . In earlier studies, we showed that the
K355X mutant lacking the KK motif remained in the microsomal
fractions of the transfected cells. Therefore, we examined
phosphohydrolase activities in microsomal and soluble fractions of
COS-1 cells transfected with either WT, H353X, Q351X,
G350X, or L349X mutant (). G6Pase
activities were associated primarily with microsomal preparations as
low or undetectable levels of enzyme activity were found in the soluble
fractions of WT or mutant G6Pase transfected cells. Moreover,
microsomal G6Pase in WT as well as mutant transfected cells exhibited
similar latencies and heat sensitivities ().
WT G6Pase also contains an Arg at codon 295
residing at the same side of the ER membrane as His-119 (Fig. 1).
As opposed to Arg-83, replacement of Arg-295 with a Lys retained 67% of
wild-type activity, suggesting that codon 295 needs to be basic for
optimal G6Pase catalysis. However, the low phosphohydrolase activity of
the R295H mutant (3.9% of wild-type activity) implies that a weakly
basic residue at codon 295 is not sufficient to confer significant
enzymatic activity. This is supported by the moderate but significant
(11.1 and 14% of WT activity) G6Pase activity exhibited by the R295N
and R295Q mutants, respectively. At the present time, we cannot rule
out the possibility that Arg-295 may stabilize the negatively charged
G6Pase-phosphate intermediate formed during G6Pase
catalysis
(9, 10) . However, the fact that substituting
Arg-295 with an Asn or a Gln yielded mutant G6Pases with moderate
enzymatic activity suggests that Arg-295 may play a stabilizing role
either by salt bridge formation or hydrogen-bonding
(22, 23) with another amino acid in human G6Pase.
Using
near-saturation mutagenesis, we established the structure-function
requirements of codon 222 in G6Pase catalysis. Codon 222 is located in
the fourth putative membrane-spanning domain in human G6Pase
(Fig. 1). When the native Gly is replaced with an Arg (the
mutation identified in GSD type 1a), a Lys, a Gln, or an Asp, the
resulting G6Pase mutants exhibited 4, 19.5, 26.3, and 27.1% of WT
enzyme activity, respectively. On the other hand, when the Gly is
replaced with a Val, a Leu, an Asn, a Met, a Tyr, a Thr, or a Ser, the
mutants retain 53-83% of WT G6Pase activity. Our results suggest
that codon 222 cannot tolerate basic (Arg or Lys), acidic (Asp), or
large polar (Gln) amino acids, which is consistent with the hydrophobic
requirement of a membrane-spanning segment. Recently, Shiang et
al.(24) showed that a Gly to Arg mutation in the
transmembrane domain of the fibroblast growth factor receptor 3 causes
the most common form of dwarfism. Although the molecular mechanisms
underlying the dominant dwarfism phenotype is currently unknown, it is
clear that the Gly to Arg mutation has a profound effect on the
function of the receptor. It is possible that the G222R mutation in the
membrane-spanning domain of G6Pase inhibiting enzymatic activity is
mediated through a similar mechanism.
In earlier studies, we showed
that the Q347X mutant of 346 residues is devoid of G6Pase
activity, whereas the K355X mutant of 354 residues is
enzymatically active
(7) . Moreover, the K355X mutant
lacking the ER protein retention signal (KK at residues 354 and 355)
remained in the microsomal fraction of the transfected cells. This
suggests that the KK motif in human G6Pase is neither essential for
enzymatic activity nor for microsomal retention. In the present report,
we sequentially deleted the carboxyl-terminal residues in human G6Pase
and analyzed the phosphohydrolase activity and microsomal association
of each mutant. Our data demonstrated that G6Pase mutants lacking
3-8 carboxyl-terminal residues retained over 40% catalytic
activity, whereas deletion of 9 terminal amino acids (L349X)
reduced activity to 5% of WT G6Pase. WT and the carboxyl-terminal
truncation mutants remained associated with the microsomes of
transfected cells. Therefore, the carboxyl-terminal 8 amino acids in
human G6Pase are not essential for G6Pase catalytic activity or
membrane retention.
Phosphohydrolase activity in whole homogenates was assayed in
reactions containing 10 mM glucose 6-phosphate using two
independent isolates of each construct in two separate transfections.
The activity is expressed as nmol/min/mg of protein, and data are
presented as the mean ± S.D.
We thank Drs. Margaret Chamberlin and Ida Owens for
critical reading of the manuscript.
(
)
E.C.
3.1.3.9) catalyzes the terminal step in gluconeogenesis and
glycogenolysis and is the key enzyme in glucose
homeostasis
(1, 2) . Deficiency of G6Pase causes glycogen
storage disease (GSD) type 1a, an autosomal recessive disorder with
clinical manifestations of severe hypoglycemia, hepatomegaly, lactic
acidemia, hyperlipidemia, hyperuricemia, and growth
retardation
(3, 4) . To understand the molecular basis of
GSD type 1a, we characterized cDNAs and genes encoding human and murine
G6Pase
(5, 6) . Sequence analysis of the deduced G6Pase
indicates that both enzymes are hydrophobic proteins containing six
putative membrane-spanning segments
(5, 6) . This is
consistent with the observations that mammalian G6Pase is tightly
associated with the endoplasmic reticulum (ER) and nuclear membranes
(1, 2).
F327), and four missense
(5, 7, 8) .
Site-directed mutagenesis followed by transient expression assays
demonstrated that each mutation abolished or greatly reduced G6Pase
activity
(5, 7, 8) . The four missense mutations
consist of a C to T transition at nucleotide 326 (Arg to Cys at codon
83, R83C) (5), a G to C transversion at nucleotide 743 (Gly to Arg at
codon 222, G222R)
(8) , a C to T transition at nucleotide 962
(Arg to Cys at codon 295, R295C)
(6) , and a C to T transition at
nucleotide 1118 (Gln to stop at codon 347, Q347X)
(7) .
Of the 24 G6Pase alleles characterized, 9 alleles (37.5%) contain the
R83C mutation, indicating that this mutation is probably the most
prevalent mutation in GSD type 1a
(5, 7, 8) . In
the present study, we examined the structural requirements of codons
83, 222, and 295 in G6Pase catalysis by constructing mutant G6Pases
with nearly all possible substitutions and examined phosphohydrolase
activity after transient expression of wild-type (WT) or mutant
constructs in COS-1 cells. We also constructed a series of truncated
G6Pase mutants to determine which of the 11 terminal amino acids was
essential for activity.
Construction of G6Pase Mutants
The phG6Pase-1
cDNA (G6Pase-WT) containing nucleotides 77-1156 of the entire
coding region of the human G6Pase cDNA
(5) was used as a
template for mutant construction by polymerase chain reaction. The
outside primers used in codon 83 are O1 (5`-AGGATGGAGGAAGGAATGAA-3`,
nucleotides 77-96, sense) and O2 (5`-TTACAACGACTTCTTGTGCGGCTG-3`,
nucleotides 1153-1130, antisense), which contain an additional
XhoI or XbaI linker, respectively. Nucleotides
326-328 of codon 83 mutant primers (nucleotides 319-337)
are R83E, GAA; R83K, AAG; R83L, CTA; R83Q, CAG; and R83S, TCG. The
amplified fragments were digested with XhoI and XbaI
and ligated into a pSVL vector (Pharmacia Biotech Inc.).
Expression in COS-1 Cells and Northern Blot Hybridization
Analysis
COS-1 cells were grown at 37 °C in HEPES-buffered
Dulbecco's modified minimal essential medium supplemented with
streptomycin, penicillin, and 4% fetal bovine serum. The WT or mutant
G6Pase cDNA in a pSVL vector was transfected into COS-1 cells by the
DEAE-dextran/chloroquine method
(13) . Mock transfections of
COS-1 cells with the pSVL vector alone were used as controls. After
incubation at 37 °C for 3 days, the transfected cultures were
harvested for G6Pase assays or lysed for RNA isolation.
Phosphohydrolase Assay
Cells were disrupted by
sonication or three consecutive freeze-thaw cycles. Microsomal
membranes were isolated by the method of Burchell et al. (15)
from freshly prepared homogenates of WT or mutant G6Pase-transfected
COS-1 cells. Disrupted microsomal membranes were prepared by incubating
intact membranes in 0.2% deoxycholate for 20 min at 0 °C. The
latency or intactness of microsomal preparations was assessed by
assaying mannose 6-phosphosphate hydrolysis in intact versus detergent-disrupted microsomes
(16) .
RESULTS
The Role of Arg-83 in G6Pase Catalysis
Analysis
of the G6Pase genes of 12 GSD type 1a patients uncovered six mutations
including four missense, R83C, G222R, R295C, and
Q347X(5, 7, 8) . R83C, R295C, and
Q347X mutant G6Pases are enzymatically inactive (5, 7),
whereas the G222R mutant has a very low phosphohydrolase
activity
(8) . To examine the structural requirements for these
amino acids as well as the length of carboxyl terminus essential for
G6Pase catalysis, we constructed a series of codon 83, 222, 295, and
carboxyl-terminal deletion mutants. Phosphohydrolase activity was
examined in whole homogenates after transient transfection of WT or
mutant G6Pase into COS-1 cells.
Structural Requirements of Codon 295 in G6Pase
Catalysis
Mutation of another arginine, Arg-295, in the G6Pase
gene also causes the GSD type 1a disorder
(5) . To characterize
the structure-function relationship of this amino acid, we altered
Arg-295 to either Glu (R295E), His (R295H), Ile (R295I), Lys (R295K),
Met (R295M), Asn (R295N), Pro (R295P), Gln (R295Q), Ser (R295S), or Tyr
(R295Y) and analyzed phosphohydrolase activity of the mutant G6Pases
(). In addition to the inactive naturally occurring R295C
mutant, substitution of Arg-295 with either Ile, Pro, or Tyr abolished
G6Pase catalytic activity. However, as opposed to mutations of Arg-83,
R295M, R295E, or R295H mutant G6Pase exhibited low phosphohydrolase
activity; R295N, R295S, or R295Q mutants exhibited moderate activity,
and the R295K mutant retained high G6Pase activity ().
Structural Requirements of Codon 222 in G6Pase
Catalysis
Secondary structural analysis of human G6Pase predicts
that codon 222 would be located in the fourth membrane-spanning domain
(Fig. 1). To determine the structural requirement of codon 222 in
G6Pase catalysis, we constructed codon 222 mutants by semi-saturation
mutagenesis. In addition to the natural G222R mutant, the Gly-222 was
also substituted with Asp (G222D), Lys (G222K), Leu (G222L), Met
(G222M), Asn (G222N), Gln (G222Q), Ser (G222S), Thr (G222T), Val
(G222V), or Tyr (G222Y) (I). G222M, G222Y, R222T, or G222S
retained over 70% of WT G6Pase catalytic activity, and G222V, G222L, or
G222N retained at least 50% of WT G6Pase activity. However,
substitution of Gly-222 with Gln (large polar), Asp (acidic), or
Arg/Lys (basic) residues greatly inhibited G6Pase phosphohydrolytic
activity (I).
Figure 1:
The predicted secondary structure of
human G6Pase and the locations of the four missense mutations
identified in GSD type 1a patients. Transmembrane spanning domains were
identified by the method of Klein et al. (25) using the
PC/Gene Program. The four mutations are highlighted and
denoted by arrows, and the His residues are highlighted and numbered.
His-119 May Be the Phosphate Acceptor in G6Pase
It
has been shown that the phosphate acceptor in G6Pase is a His
residue
(9, 10) . The stringent structural requirement of
codon 83 suggests that Arg-83 in G6Pase may be involved in positioning
the phosphate. 4 conserved His residues at codons 9, 119, 252, and 353
are predicted to reside on the same side of the ER membrane as Arg-83
(Fig. 1). Therefore, we altered each of the 4 His residues
individually to Ala, an amino acid to which a phosphate group cannot be
transferred. Phosphohydrolase activity was analyzed after transient
expression of WT or mutant G6Pase cDNAs in COS-1 cells ().
H252A and H353A mutant G6Pases retained over 60% WT activity, and H9A
mutant retained 13% activity. On the other hand, the H119A mutant
G6Pase had no enzymatic activity. To establish the vital role of
His-119 in G6Pase catalysis, we substituted this amino acid with either
Ile (H119I), Lys (H119K), Met (H119M), Asn (H119N), Arg (H119R), or Thr
(H119T) (). Like the enzymatically inactive H119A mutant,
none of the other codon 119 mutants had detectable phosphohydrolase
activity.
The Role of Carboxyl-terminal Residues in G6Pase
Catalysis
The Q347X mutant, lacking the 11
carboxyl-terminal amino acids, is enzymatically inactive, in contrast
to the K355X mutant, which retains significant G6Pase
activity
(7) . Therefore, residues 355-357 in human G6Pase
are nonessential amino acids, and residues 348-354 may play
important role in G6Pase catalysis. To further examine the length of
the carboxyl terminus required for G6Pase activity, we examined
phosphohydrolase activity of mutant G6Pases containing sequential
deletion of carboxyl-terminal residues (). Deletion of
residues 355-350 yielded mutant G6Pases retaining at least 40% of
WT enzyme activity. However, a G6Pase of 348 residues (L349X)
exhibited only 5% of WT G6Pase activity, demonstrating that the 8
carboxyl-terminal residues beyond this codon that contain an ER protein
retention signal
(5, 17) are not necessary for its
catalytic activity.
Northern Blot Hybridization Analysis
Northern blot
hybridization analysis of G6Pase transcripts from transfected cells
showed that WT as well as the various mutant G6Pase mRNAs were
expressed at similar levels (data not shown). This indicates that the
reduction in enzymatic activity was due to the defect in the G6Pase
protein and not due to a decrease in transfection efficiency.
DISCUSSION
The characterization of mutations in the G6Pase gene of GSD
type 1a patients that abolish or greatly reduce G6Pase activity has
pinpointed a number of amino acid residues important in G6Pase
catalysis
(5, 7, 8) . In the present report, we
examine the structure-function relationship of amino acids 83, 222, and
295 and the role of carboxyl-terminal residues 348-354 in the
catalytic activity of G6Pase. Replacement of Arg-83 with amino acids of
diverse structures including a Lys (a conservative change) yielded a
G6Pase devoid of enzymatic activity, demonstrating that the Arg at
codon 83 is absolutely required for G6Pase catalysis. This is
reminiscent of the case in human placental alkaline phosphatase where
it has been shown that substitution of the Arg at position 166 with a
Lys markedly inhibits phosphatase activity
(18) . Based on x-ray
crystallography studies of E. coli alkaline
phosphatase
(19) , it was proposed that Arg-166 is involved in
positioning the phosphate, which binds to a Ser residue at the active
center of placental alkaline phosphatase
(18) . The stringent
structural requirement of codon 83 suggests that Arg-83 in G6Pase may
be also involved in positioning the phosphate, which binds a His at the
active center of G6Pase
(9, 10) . 4 His residues, His-9,
His-119, His-252, and His-353 are predicted to be on the same side of
the ER membrane as Arg-83 (Fig. 1). In the present study, we show
that His-119, like Arg-83, is absolutely required for G6Pase activity,
suggesting that His-119 is the phosphate acceptor during G6Pase
catalysis. Hepatic 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase
(20) and acid phosphatases
(21) also form
enzyme-phosphate intermediates during catalysis. An Arg residue has
been shown to reside adjacent to the His residue that accepts the
phosphate in both families of enzymes. It is possible that these
adjacent Arg residues also play a role in positioning the phosphate
during catalysis.
Table:
Analysis of phosphohydrolase activity of G6Pase
WT, DraIII, and codon 83 mutant constructs in COS-1 cells
Table:
Analysis of
phosphohydrolase activity of G6Pase WT and codon 295 mutant constructs
in COS-1 cells
Table:
Analysis of phosphohydrolase activity
of G6Pase WT and codon 222 mutant constructs in COS-1 cells
Table:
Analysis of phosphohydrolase activity of
G6Pase WT and His mutant constructs in COS-1 cells
Table:
The role of carboxyl-terminal amino acids
in G6Pase catalysis
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.