and ApL4 in leaves. 87 Sucrose induction of ApL3 and ApL4 transcription in leaves allowed formation of heterotetramers less sensitive to the allosteric effectors, resem-bling the situation in sink tissues, which are regulated by an allosteric mechanism (3PGA/Pi ratio).
2. Relationship Between the Small and Large Subunits:
inactive small subunit in which the catalytic Asp145 was mutated. 63 In this way, the activity of the small (catalytic) subunit was reduced by more than three orders of magnitude ( Table 4.4 ). Co-expression of the large subunit double mutant LargeK44R/
T54K with smallD145N generated an enzyme with considerable activity, 10% and 18% of the wild type enzyme (smallWTLargeWT) in the ADPGlc synthetic and pyrophosphorolytic direction, respectively ( Table 4.4 ). A single mutation (K44R), generated an enzyme (smallD145NLargeK44R) with no signifi cant activity over the control (smallD145NLargeWT). Mutation T54K (smallD145NLargeT54K) provided about 3% of WT activity, but the combination of both mutations in the large subunit (smallD145NLargeK44R/T54K) had the most dramatic effect ( Table 4.4 ). Therefore, it was concluded that Arg44 and Lys54 are needed for restoring catalytic activity to the large subunit. The resurrection of catalytic activity in the large subunit by incor-poration of Arg44 and Lys54 predicted that the homologous residues in the small subunit are critical. Replacement of the homologous two residues with Lys and Thr in the small subunit (by mutations R33K and K43T) decreased the activity by one and two orders of magnitude, respectively, in both directions, confi rming the hypoth-esis ( Table 4.4 ).65 The mutant enzymes were still activated by 3PGA and inhibited by Pi. 65 The wild type enzyme and smallD145NLargeK44R/T54K had very similar kinetic properties, indicating that the substrate site domain has been conserved. The apparent affi nities for the substrates in both directions were in the same range, and the allosteric properties of smallD145NLargeK44R/T54K also resembled those of the wild type ( Table 4.5 ). 65 The presence of 2 mM Pi decreased the apparent affi n-ity of 3PGA for both the wild type and smallD145NLargeK44R/T54K, indicating that this new form has a similar sensitivity to Pi inhibition, and that the activator – inhibitor interaction was the same ( Table 4.5 ).
The fact that only two mutations in the L subunit restored enzyme activity is very strong evidence that the large subunit is derived from a catalytic ancestor. To confi rm
Table 4.3 Sequence comparison of the potato tuber large subunit with ADPGlc PPases shown to be enzymatically active. Residues that are 100% conserved are in bold and the ones conserved in the small subunit but not in the large subunit are underlined a
R. sphaeroides MKAQPPLRLTAQAMAFV L A GG R GSR LKE LT DRRA KP A V Y
R. rubrum MDQITEFQLDINRALKETLALV L A GG R G S R LRD LT NRES KP A V P
A. tumefaciens MSEKRVQPLARDAMAYV L A GG R G S R LKE LT DRRA KP A V Y
E. coli MVSLEKNDHLMLARQLPLKSVALI L A GG R G T R LKD LT NKRA KP A V H
E. coli SG14 MVSLEKNDHLMLARQLPLKSVALI L A GG R G T R LKD LT NKRA KP T V H
G. stearothermophilus MKKKCIAML L A GG Q G S RL RS LT TNIA KP A V P
Anabaena sp. PCC7120 MKKVLAII L G GG A G T RL YP LT KLRA KP A V P
Arabidopsis thaliana small subunit
MAVSDSQNSQTCLDPDASSSVLGII L G GG A G T RL YP LT KLRA KP A V P
H. vulgare endosperm LPSPSKHEQCNVYSHKSSSKHADLNPHAIDSVLGII L G GG A G T RL YP LT KKRA KP A V P 33 43 Small unit MAVSDSQNSQTCLDPDASRSVLGII L G GG A G T RL YP LT KKRA KP A V P Large unit MAYSVITTENDTQTVFVDMPRLERRRANPKDVAAVI L G GG E G T K L FP LT SRA T P A V P
44 54
a The full color version of this table can be found at www.Elsevier.books.com/
that catalysis occurs in the large subunit of smallD145NLargeK44R/T54K, the sub-strate site was disrupted in each subunit and their kinetic properties compared. In previous experiments, replacement of Lys198 in the small subunit of the wild type enzyme decreased the substrate (G1P) affi nity, whereas disruption of the homologous residue (Lys213) in the large subunit did not have the same effect. 63 In smallD145N-LargeK44R/T54K, the mutation K213R on the large subunit severely decreased the apparent affi nity for G1P, whereas mutation of K198R on the small subunit did not ( Table 4.6 ). This indicated that the large subunit double mutant, and not smallD145N, was the catalytic subunit. In the wild type enzyme, Lys213 does not seem to play any important role, but in smallD145NLargeK44R/T54K it recovered its ancestral abil-ity to confer to the enzyme a high apparent affi nabil-ity for G1P. Previous results showed that Asp145 in the small subunit of the wild type is essential for catalysis, whereas
Table 4.4 Activity of small (catalytic) and large (regulatory) subunit ADPGlc PPase mutants a
Subunits Units/mg
Small (catalytic) Large (regulatory) ADPGlc synthesis b ATP synthesis c
WT WT 32 1 49 2
D145N WT 0.017 0.001 0.037 0.002
D145N K44R 0.031 0.001 0.033 0.002
D145N T54K 0.92 0.08 0.56 0.03
D145N K44R/T54K 3.2 0.2 9.0 0.7
R33K WT 3.6 0.2 4.1 0.1
K43T WT 0.32 0.1 0.28 0.01
a The enzyme activities of purifi ed co-expressed small and large subunits were measured for ADPGlc synthetic activity or ADPGlc PPase (ATP synthesis) activity
b For ADPGlc synthesis, 4 mM 3PGA (activator), 2 mM ATP and 0.5 mM Glc-1-P were used
c For pyrophosphorolysis, 4 mM 3PGA, mM ADPGlc and 1.4 mM PPi were used
Table 4.5 Comparison of the kinetic properties of the wild-type ADPGlc PPase from potato tuber and the mutant smallD145NLargeK44R/T54K
ADPGlc synthesis
SmallWT LargeWT Activation SmallD145N/
LargeK44R/T54K
Activation
S0.5 /A0.5 (μ M)
(nH) (-fold) S0.5 ( μ M) (nH) (-fold)
ATP 97 7 1.6 170 13 1.8
G1P 27 2 1.1 11 1 1.2
3PGA 135 11 0.9 60 5 13.6 0.7 1.3 20 2 ADP-glucose pyrophosphorolysis
ADPGlc 200 20 1.4 70 6 1.5
PPi 37 2 1.0 98 10 1.0
3PGA 1.1 0.3 1.1 3.1 0.3 4.0 0.5 0.9 7.6 1.0 3PGA
( 2 mM Pi)
96 19 1.0 101 13 1.1
IV. Properties of the Plant 1,4- α -Glucan-Synthesizing Enzymes 93
the homologous Asp160 in the Large wild type subunit is not. 63 However, mutation to D160N or D160E in the active large subunit, LK44R/T54K, abolished the activity ( Table 4.7 ). This shows the ancestral essential role of this residue and confi rms that the catalysis of smallD145NLarge K44R/T54K does occur in the large subunit.
A comparative model of LK44R/T54K illustrates the predicted role of Arg44 and Lys54 ( Figure 4.1 ). In the model Asp160, which is homologous to the catalytic Asp145 in the small subunit and the catalytic Asp142 in E. coli ADPGlc Ppase, 63,90 interacts with Lys54. This type of interaction (Lys54 with Asp160) has also been observed in crystal structures of enzymes that catalyze similar reactions, e.g. dTDP-glucose pyrophosphorylase (dTDPGlc PPase) and UDP-N-acetyl-glucosamine pyro-phosphorylase (UDPGlcNAc PPase), and is postulated to be important for catalysis by correctly orienting the aspartate residue. 91 – 93 Also, Lys54 interacts with the oxy-gen atom bridging theα - and β -phosphate groups as has been observed in the crystal structure of E. coli dTDPGlc Ppase. 93 That contact may neutralize a negative charge density stabilizing the transition state and making the pyrophosphate a better leaving group. Arg44 interacts in the model with the β - and γ -phosphates of ATP, which cor-respond to the PPi product ( Figure 4.1 ). Likewise, Arg15 in E. coli dTDPGlc PPase was postulated to contribute to the departure of Ppi. 91 The kinetic data agreed with the predicted interaction of PPi with Arg44. A Lys in that position, in both the cata-lytic large subunit mutant and the small subunit, decreased the apparent affi nity for
Table 4.6 Effect of mutations on the glucose 1-P site a
Subunits Glucose 1-P, S0.5 Specifi c activity, U/mg Small Large μ M Increase b
(fold)
ADPGlc synthesis
ADPGlc
pyrophosphorolysis
D145N K44R/T54K/K213R 910 110 82 1.4 4.2
D145N/K198R K44R/T54K 13.2 0.5 1.2 2.2 6.3
a Reaction conditions are as in Table 4.2
b The increase values are the ratio of the values obtained with the mutant enzymes to the wild-type enzyme
Table 4.7 The Effects of mutations of residue Asp160 on large subunit activity a
Subunits Specifi c activity, units/mg Small Large
D145N WT 0.001
D145N K44R/54K 0.35 0.05
D145N K44R/54K/D160N 0.002 D145N K44R/54K/D160E 0.002
a ADP-glucose pyrophosphorylase activity was measured in crude extracts. Saturating concentrations used: for 3PGA, 4 mM, for ADPGlc, 2 mM. The PPi concentration was 1.4 mM
PPi at least 20-fold. 65 In the model of the wild type, noncatalytic large subunit, Lys44 and Thr54 cannot interact as Arg44 and Lys54 ( Figure 4.1 ).
3. Phylogenetic Analysis of the Large and Small Subunits
A phylogenetic tree of the ADPGlc PPases present in photosynthetic eukaryotes also sheds information about the origin of the subunits. 65 This tree showed that plant small and large subunits can be divided into two and four distinct groups, respectively. The two main groups of small subunits are from dicot and monocot plants, whereas large subunit groups correlate better with their documented tissue expression. The fi rst large subunit group is generally expressed in photosynthetic tissues, 65 and comprises large subunits from dicots and monocots. Group II displays a broader expression pat-tern, whereas groups III and IV are expressed in storage organs (roots, stems, tubers and seeds). Subunits from group III are only from dicot plants, whereas group IV are seed-specifi c subunits from monocots. These last two groups stem from the same branch of the phylogenetic tree, and split before monocot and dicot separation. 65,94,95
4. Crystal Structure of Potato Tuber ADPGlc PPase
The crystal structure of the potato tuber homotetrameric small (catalytic) subu-nit ADPGlc PPase has been determined to 2.1 Å resolution. 64 The structures of the
Lys44
Thr54 Asp160
Arg44
Lys54
Asp160 Gly131
Asp166 Asp166
Gly131
Asp31 Asp31
Cys73 Cys73
LWT Noncatalytic
LK44R/T54K Catalytic
Figure 4.1 Involvement of large (regulatory) subunit of mutant K44R/T54K in enzyme catalysis. 90 The WT and double-mutant large subunits were modeled based on the dTDPGlc PPase and UDPGlcNAc PPases as indicated. Portions of residues 31 – 73 and 131 – 136 are shown. The deoxyribose triphosphate portion common to dTTP and ATP is modeled with Mg 2 as a blue sphere. The nitrogen atom of the adeninyl group attached to the ribosyl unit is also in blue. The dotted green lines depict hydrogen bonds. (The full color version of this fi gure can be found at www.Elsevier.books.com/ )
IV. Properties of the Plant 1,4- α -Glucan-Synthesizing Enzymes 95
enzyme in complex with ATP and ADPGlc have been determined to 2.6 Å and 2.2 Å resolution, respectively. Ammonium sulfate was used in the crystallization process and was found to be tightly bound to the crystalline enzyme. It was also found that the small subunit homotetrameric potato tuber ADPGlc PPase was inhibited by inor-ganic sulfate with an I 0.5 value of 2.8 mM in the presence of 6 mM 3PGA. 64 Sulfate is considered as an analog of phosphate, the allosteric inhibitor of plant ADPGlc PPases. Thus, the atomic resolution structure of the ADPGlc PPase probably presents a conformation of the allosteric enzyme in its inhibited state. The crystal structure of potato tuber ADPGlc PPase ( Figure 4.2 ) allows one to determine the location of acti-vator and substrate sites in the three-dimensional structure and their relation to the catalytic residue, Asp145. The structure also provides insights into the mechanism of allosteric regulation.
The overall fold of the potato tuber ADPGlc PPase small subunit catalytic domain is quite similar to that of two other pyrophosphorylases, viz., N -acetylglucosamine 1-phosphate uridylyltransferase (GlmU) from E. coli92,96 and S. pneumoniae97,98 and glucose 1-phosphate thymidylyltransferase (Rffh) from P. aeruginosa91 and E. coli ,93 although their primary sequences have only very low sequence similarities. The cata-lytic domain is composed of a seven-stranded β sheet covered by α helices, a fold reminiscent of the dinucleotide-binding Rossmann fold. 99 At one of its ends, the cen-tralβ -sheet is topped by a two-stranded β -sheet. The catalytic domain makes strong hydrophobic interactions with the C-terminal domain through an α -helix that encom-passes residues 285 – 297 ( Figure 4.2 ). The catalytic domain is connected to the C-ter-minalβ -helix domain by a long loop containing residues 300 – 320. This loop makes numerous interactions with the equivalent region of another monomer.
The C-terminal domain comprises residues 321 – 451 and adopts a left-handed β -helix fold composed of six complete or partial coils with two insertions, one of which encompasses residues 368 – 390. The other encompasses residues 401 – 431.
Figure 4.2 Crystal structure of potato tuber ADP-glucose small (catalytic) subunit monomer. The catalytic domain is in yellow and the beta-helix domain is in pink. ADPGlc is shown in atom type: carbon atoms are green, oxygen atoms are red, nitrogen atoms are blue, phosphorus atoms are magenta, and the sulfate group is orange. (The full color version of this fi gure can be found at www.Elsevier.books.com/ )
This type of left-handed β -helix domain fold has been found in the structures of bacterial acetyltransferases, including E. coli UDP- N -acetylglucosamine 3- O -acyl-transferase,100 Methanosarcina thermophila carbonic anhydrase, 101 Mycobacterium bovis tetrahydrodipicolinate N -succinyltransferase 102 and GlmU, 92 and in other proteins such as T4 bacteriophage gp5. 103 However, the β -helix domain seen in the other structures is an acetyltransferase or succinyltransferase domain. In the present structure of ADPGlc PPase, the β -helix domain is involved in cooperative allosteric regulation with the N-terminal catalytic region and interactions with the N-terminal region within each monomer, and contributes to oligomerization.
Homotetramer Structure
Crystalline potato tuber ADPGlc PPase small subunit is a tetramer with approximate 222 symmetry and approximate dimensions of 80 90 110 Å 3 ( Figure 4.3 ). It can be viewed as a dimer of dimers, labeled A, A , B and B ( Figure 4.3 ). Monomers A and B interact predominantly by end-to-end stacking of their β -helix domains, although there is also a signifi cant interface between the linker loop connecting the two domains ( Figure 4.3 ). This interface buries 2544 Å2 of surface area. The catalytic domains of A and B (and B and A ) also make an extensive interface. Several hydrogen bond and hydrophobic interactions stabilize the interface between A and B , burying a surface area of 1400 Å2 . All residues defi ning dimerization interfaces are identical or similar in the large subunit. Figure 4.3 delineates all oligomerization interactions seen within
T295O S321N S321O M323N M323O M323O L324O A326N A326O
K322NZ I333O I333N S331O S331N D330N D330N V328O V328N Interactions between monomer A and B
Y305 Y312 T313 P320
P319 Y317F*
L318 Y372 I333
Interactions between monomer A and Bⴕ A79N
N82ND2 R87NE A107O Q109O K*Q126NE2
E133OE2 E103OE1 E99OE1D*
Q109NE2 W129NE1 Q109OE1
N77 W129
Interaction between monomer A and Aⴕ
C12SG C12SG
Burying 2588Å2surface area
Burying1404Å2surface area
*Residues that are not identical but similar in potato tuber ADP-glucose pyrophosphorylase large subunit.
A
A B
B
Figure 4.3 ADPGlc PPase tetramer and interactions between the monomers in the tetramer. The fi gure shows amino acid interactions between the monomers and the ADPGlc PPase catalytic (small) subunit tetramer. (The full color version of this fi gure can be found at www.Elsevier.books.com/ )
IV. Properties of the Plant 1,4- α -Glucan-Synthesizing Enzymes 97
the tetramer in the asymmetric unit. Cys12 of monomer A and the equivalent cysteine residue of monomer A make a disulfi de bond, as do equivalent cysteine residues of monomers B and B . The inter-subunit disulfi de bond between the small (catalytic) subunit is preserved in the heterotetramer. However, there is no disulfi de bond between the large (regulatory) subunits, as Cys12 is not conserved. This disulfi de bond estab-lishes the relative orientation of the small subunits in the heterotetramer to be like A and A in the α4 -homotetramer structure. The disulfi de bond is the only interaction made between A and A (or B and B subunits). Potato tuber ADPGlc PPase is redox-regulated by reduction and oxidation of the intermolecular disulfi de bond between the two small subunits. 60,62 This covalent regulatory modifi cation is discussed later.
Sulfate Binding Mimics Phosphate Inhibition
Sulfate is an inhibitor of potato tuber ADPGlc PPase small subunit homotetramer with I 0.52.8 mM in the presence of 6 mM 3-PGA. 64 The electron density map for potato tuber ADPGlc PPase small subunit suggests that there are three sulfate ions tightly bound to the enzyme. Most probably, this is due to the high sulfate con-centration (150 mM) in the crystallization solution. Two sulfate ions bind within 7.5 Å of each other in a crevice located between the N- and C-terminal domains of the enzyme. 64 A third sulfate ion binds between the two subunits of the enzyme.
The sulfate ions make numerous interactions with residues shown to be involved in the allosteric activator binding site, as demonstrated by chemical modifi cation 104,105 and site-directed mutagenesis studies. 59 The structures contain 12 sulfate ions within a tetramer in the asymmetric unit (three per monomer) and are all, therefore, repre-sentative of the inhibited conformation of the enzyme.
Sulfate 1 makes hydrogen bond interactions with R41, R53, K404 and K441 ( Figure 4.4 ). 64 The side-chain nitrogen atom of R41 makes hydrogen bond tions with one of the sulfate ion oxygen atoms, and D403 makes a salt bridge interac-tion with R41 to facilitate the binding. D413 in the potato tuber enzyme large subunit (D403 in the small subunit) was identifi ed as important for activation by 3PGA. 106 All these residues are conserved in virtually all plant ADPGlc PPases, and four of the fi ve (all but K441) are strongly conserved in bacterial ADPGlc PPases ( Figure 4.5 ). Site-directed mutagenesis studies have identifi ed residues K441 and K404 in the small subunit of potato tuber as important for 3PGA activation. 59 The enzyme’s affi nity for 3PGA was lowered and the inhibition by Pi diminished when mutations at these residues were Ala (neutral) or Glu (negative). The kinetic parameters for the substrates, ADPGlc, PPi and the cofactor Mg 2 were not affected. Mutations on the homologous residues in the large subunit showed lesser or no effects on regulation of the enzyme. 63 Therefore, it was concluded that K404 and K441 in potato tuber ADPGlc PPase small subunit are important for the binding of 3PGA and Pi, and the main role of the large subunit is to interact with the small subunit and modulate its activation mechanism. 59 These studies indicate that the activator 3PGA binds at or near the inhibitor binding site defi ned in the structure by sulfate 1.
Sulfate 2 makes similar interactions with surrounding positively-charged residues, R53, R83, H84, Q314 and R316 ( Figure 4.4 ). Site-directed mutagenesis studies have
shown that H83 of the E. coli enzyme (H84 in the potato tuber enzyme small sub-unit) is involved in activator binding. 107 Chemical modifi cation with phenylglyoxyl has identifi ed R294 in the Anabaena sp. enzyme (R316 in the potato tuber enzyme small subunit) as an important residue for inhibition by Pi, as mutations of this resi-due lowered the apparent affi nity for Pi more than 100-fold. 108 Mutations of this Arg residue to Ala, Gln or Lys caused a change in inhibitor selectivity such that these mutants were inhibited by NADPH or FBP. 109 Taken together, these studies con-fi rm the importance of the sulfate ion-binding site in the allosteric regulation of the enzyme, and indicate that 3PGA may also bind near the sulfate 2 binding site.
Sulfate 3 is located between two subunits, viz., A and B . This sulfate ion interacts with R83 of one monomer and K69, H134 and T135 of the other subunit. K69 and R83 are conserved in both the small and large subunits of all plant ADPGlc PPases. H134 is conserved in all small subunits, and T135 is conservatively replaced by Asn in other plant small subunits. The precise role of this location is not yet clear. Sulfate binding may be non-specifi c or it may interfere with the dimerization of the subunits, thus caus-ing the R → T equilibrium to be more toward the T (inhibited) state. Current structural results strongly support previous data on the allosteric regulation of this enzyme, and provide some insights on how the binding of allosteric effectors could affect catalysis.
B
b helix domain A
Catalytic domain
R316 Q314
H84
R83
D370
K441 K404 R53
H134 R41 T135
K69 B
D403 A
Figure 4.4 ADPGlc PPase monomer showing: (a) the sulfate binding region between the catalytic and beta-helix domains; and (b) the amino acid residues interacting with sulfate. The sulfate residues are yellow and the interacting residues are green in one subunit. The neighboring subunit and its residues are purple.
(The full color version of this fi gure can be found at www.Elsevier.books.com/ )
IV. Properties of the Plant 1,4- α -Glucan-Synthesizing Enzymes 99
H1 S1
H9 H10
H1 H2 H3 H4
S10 S11
H5 H5 H6
S2 S3 S4 S5
H2 H3 H4 H5
S6 S7 H6 S8 H7 H8 S9
ADPGlc interaction ATP interaction
M
Figure 4.5 Sequence alignment of ADPGlc PPase from different species. Secondary stucture of the potato tuber enzyme is shown above the sequence. Cylinders, helices; straight block arrows, beta strands; curved block arrows, turns in the beta-helix domain. Green stars are residues interacting with ADPGlc and red stars are residues interacting with ATP. Residues that are identical are shaded in purple. Abbreviations are: Stu_s, potato tuber Small subunit; Ath_S_APS1, Arabidopsis thaliana Small subunit; Zma_S_endosp, maize Small subunit; Hvu_S_endosp, Hydra vulgaris Small subunit; Stu_L, potato tuber Large subunit; Eco_glgC, E. coli ADPGlc PPase;
Rsp_glgC, R. spheroides ADP-Glc PPase; Bst_glgC, Bacillus stearothermophilus ADPGlc PPase; Cre_S, Chlamydomonas reinhardtii Small subunit; Ana_glgC, Anabaena ADP-Glc PPase; Atu_glgC, Agrobacterium tumefaciens ADP-Glc PPase. (The full color version of this fi gure can be found at www.Elsevier.books.com/ )
ATP Binding
When ATP binds to the enzyme, both A and A monomers undergo almost identi-cal conformational changes. Several regions move signifi cantly, viz., a loop region from residue 27 to 34, another loop region from residue 106 to 119, and residues K40, R41, Q75 and F76. 64 Both loop regions make direct interactions with the ade-nine portion of the nucleotide. Specifi cally, the main chain nitrogen atom of G28 makes a hydrogen bond with N3 of the adenine ring; several hydrophobic interac-tions are established between the adenine ring and L26 and G29; the side chain of Q118 makes a hydrogen bond with N6 of the adenine ring; and the main-chain oxy-gen atom of Q118 makes a hydrooxy-gen bond with N6. These residues all undergo cor-related conformational change upon ATP binding. Interactions of Q75 with both G30 and W116, and interactions of the K40 side chain with P111 couple the motions of the Q75, G30 and 106 – 119 regions. 64
Furthermore, ATP binding in the A and A subunits drives conformational change in the B and B subunits, as P111 of A and A is packed snugly against W129 in B and B, respectively. 64 Motion of P111 accompanying ATP binding leads directly to motion of W129 and, in fact, the entire region from 165 to 231 in the B/B subunits.
ADP-Glucose Binding
Three of the four subunits (A, A and B), bind ADPGlc in the ADPGlc PPase/ADP-Glc complex. The B subunit binds neither ATP nor ADPGlc, and is conformationally more rigid than the other three subunits. A and A bind ADPGlc identically, and ADPGlc binding produces conformational changes in A and A identical to that which occurs when ATP binds (described above). The adenyl and ribosyl units of ADPGlc in A and A are positioned identically to the adenyl unit of ATP, and the interactions between the enzyme and ADPGlc are also identical to those seen in the ATP complex. No elec-tron density is seen for the glucosyl moiety of ADPGlc in the A and A active-sites, indicating it to be disordered. This indicates that the conformational changes seen in A and A on ATP or ADPGlc binding are due almost exclusively to the adenyl and ribo-syl moieties. Both phosphate groups are also ordered. In contrast, the entire ADPGlc molecule is well-ordered in the B subunit active-site. The adenyl and ribosyl positions are very similar to those seen in the A and A subunits through the region 112 – 117, which undergoes conformational change upon ATP or ADPGlc binding in A and A , and is disordered in B and B both with and without ADPGlc in the active-site. The two phosphate groups and the glucosyl units are very well-ordered in the B active-site and adopt positions and conformations similar to that seen in other sugar nucleotide pyrophosphorylase complex structures ( Figure 4.6 ). There are several direct interac-tions between the enzyme and the glucosyl unit of ADPGlc. These include hydrogen bonds between E197, S229, D280 and the glucosyl unit ( Figure 4.6 ). In addition K198 makes a salt bridge with the phosphate group attached to the glucosyl unit.
The B subunit undergoes a very large subdomain movement in response to ADPGlc binding. Two residues, E197 and K198, are critical for binding the glucosyl and phos-phate moieties of ADPGlc; K198 has been characterized as a Glc1P binding residue
IV. Properties of the Plant 1,4- α -Glucan-Synthesizing Enzymes 101
by site-directed mutagenesis 58 and both are part of a motif present in many sugar – nucleotide PPases. These two residues are shifted out of the binding pocket in the B subunit of the ATP-bound structure, while they are pulled more inward in the unbound B subunit and are pulled in signifi cantly in the ADPGlc-bound molecule ( Figure 4.6 ).
Implication for Catalysis
Detailed kinetic studies on ADPGlc PPase have shown that a sequential bi bi mech-anism fi ts the data with ATP binding fi rst. 75,89,110 Structural data from two related enzymes, GlmU and Rffh, indicate the presence of a metal ion in the active-site. 93,96 A Co 2 ion (in GlmU) and an Mg 2 ion (in GlmU and Rffh) are located in almost identical locations in the two distinct enzymes when the active-sites are aligned.
In GlmU, both the Mg 2 and the Co 2 are chelated to two conserved residues (Asp105 and Asn227), and to the two phosphate groups of the product acetylglucosamine). In Rffh, the Mg 2 is also bound to two conserved carboxylate residues (Asp223 and Asp108) and to the α -phosphate group of TTP. When the ADPGlc PPase active-site is aligned with these active-sites, two acidic residues, Asp145 and Asp280, are spatially close to the metal chelating residues in Rffh and GlmU. Mutation of Asp145 residue to Asn in the potato tuber enzyme and the equiv-alent Asp 142 in the E. coli enzyme results in a reduction in catalytic activity by four orders of magnitude. 63,90 Taken together, it is concluded that the metal-mediated catalytic mechanism proposed for RffH and GlmU is also used by ADPGlc PPase.
Also concluded is that the metal ion is chelated by the residues equivalent to D145 and D280 in all ADPGlc PPases, and that the mutational sensitivity of D145 is due to the requirement for metal ion in the reaction. The absolute requirement for a metal ion has been biochemically demonstrated for ADPGlc PPase from several organ-isms.13,14,111 Structural data from GlmU, RmlA and RffH have shed considerable light on the mechanism of sugar nucleotide pyrophosphorylases, and the similarity
D145
K198 D280
Q148 G28
E197 F181 K43
Figure 4.6 Hydrogen bond interactions between ADP-glucose and the ADPGlc PPase catalytic subunit.
Protein carbon bonds are green; ADPGlc carbon bonds are yellow; oxygen atoms are red; nitrogen atoms are blue; and phosphate atoms are purple. (The full color version of this fi gure can be found at www.
Elsevier.books.com/ )
between the active-site of α4 -ADP-Glc PPase and these enzymes indicates a similar mechanism for all of these enzymes. The structure of the RffH/dTTP complex is par-ticularly informative, because it identifi es the GXGXRL loop, which is strongly con-served in all of these enzymes, to be the site of the triphosphate moiety of ATP and shows that the residue equivalent to R33 in the α subunit of ADPGlc PPase is critical for triphosphate binding. Conformational change of this loop will therefore have pro-found effects on the activity of the enzyme. In addition to R33, D145, D280, K43, E197 and K198 (potato tuber α4 numbering, Figure 4.6 ) are also conserved in the other sugar nucleotide pyrophosphorylases of known structures, are in similar loca-tions in the active-sites, and make similar interacloca-tions with the sugar nucleotide.
Based on results with other sugar nucleotide pyrophosphorylase structures, 91,93 the following is postulated for catalysis. The β - and γ -phosphate groups of ATP bind to the conserved loop around R33, folding back over the nucleotide and leaving the space opposite the pyrophosphate entity free for G1P binding. The location of the phosphate group in G1P and that of the α -phosphate group of ATP are close to the same posi-tions seen for the phosphate groups in the B subunit ADPGlc complex. K198 is an absolutely conserved amino acid in ADPGlc PPases and in other sugar nucleotidyl-transferases. K198 stabilizes the negative charge on the phosphate group of G1P in this model, increasing the nucleophilicity of the O1P atom. Electrostatic repulsions between the negative charges on the phosphate group of G1P and the phosphate groups of ATP are compensated for by chelation between the phosphate groups of ATP, G1P and Mg 2 . A number of conserved basic side chains also surround the phosphate groups at the active-site (R33, K43, K198). Additional counterbalancing charges come from the N-terminal dipole of the helical turn at R33 and the main chain amide nitro-gen pocket formed by residues G27 to T32. In fact, R33 makes a close hydronitro-gen bond with a phosphate oxygen atom of ADPGlc in the ADPGlc-bound structure. In muta-genesis studies of GlmU, 92 mutation of R15 (equivalent to R33 of ADPGlc PPase) reduced kcat almost 6000-fold, while Km was doubled, confi rming that this residue has an important role in orienting and charge compensation of the pyrophosphate group.
P36 within this fl exible loop adopts a cis-peptide bond. The loop is located at the interface between the N-terminal catalytic domain and the C-terminal β -helix domain within the immediate vicinity of the regulator-binding site, suggesting a possible route for crosstalk between the active-site and the allosteric regulation site. While most of the residues in the active-site are conserved in the catalytically inactive large subunit, there are two changes: R33 is a lysine residue; and K43 is a threonine residue. Since K43 interacts with ADPGlc and R33 is critical for proper ATP triphosphate binding, both are likely to be important in catalytically deactivating the large subunit.
Allosteric Regulation
Crystal structure analysis identifi ed three allosteric inhibitor-binding sites, sulfate 1, sulfate 2 and sulfate 3, two of which (sulfate 1 and sulfate 2) have been shown by numerous biochemical experiments to be involved in allosteric regulation of the enzyme. The high sequence homology of the residues in the sulfate 3 binding site suggests that it too may represent an important allosteric binding site. The major question is how these allosteric binding sites communicate with the active-site to
IV. Properties of the Plant 1,4- α -Glucan-Synthesizing Enzymes 103