The reported values for the number of metabolite binding sites vary widely (Table XI). Concensus of opinion would indicate that the mammalian PFK monomer contains three A TP binding sites, one or two F6P binding sites, one adenine binding site, one citrate binding site, and one FBP binding site.
Of the three binding sites for A TP, one is identical to the AMP binding site (Kemp and Krebs, 1 967), one represents the catalytic site (Kemp and Krebs, 1 967), and one is the A TP inhibitory site. The three sites differ from each other with respect to their ligand specificities and biological actions. The catalytic site has a high affrnity for A TP (Kd l �M), while the inhibitory site has a lower affinity (Kd l OO�M)(Wolfman et al. , 1 978; Pettigrew and Frieden, 1979a).
The binding of A TP appears to be biphasic (Roberts and Kellett, 1 9 80). The binding of A TP to PFK has been shown to cause a decrease in the reactivity of the class I thiol (Kemp, 1 9 69a; Mathias and Kemp , 1 972). This suggests that a change in conformation of the enzyme occurs leading to a decrease in the local mobility of the
T A B L E XI
PROPOSED NUMBER OF MET ABO LITE BINDING SITES IN PFK
Binding sites/monomer Ref
ATP 3 (i) 3 (ii) 3 (iii) 3 (iv) 2 (v) 3 (vi) F6P 2 (i) 1 (ii) 1 (vii) 1 .5 (iii) 2 (viii) Adenine 3 (i) 1 (ii) 1 (viii) 1 (ix) Gtrate 1 (i) 1 (x)
S ugar Bisphosphates 1 (xi)
Number of different metabolite binding sites proposed for mammalian PFK by different laboratories.
(i) Garfinkel, 1966 (vii) Hill and Hammes, 1975 (ii) Kemp and Krebs, 1967 (viii)Setlow and Mansour, 1972 (iii) Lorenson and Mansour, 1969 (ix) Pettigrew and Frieden, 1978 (iv) Ogawa and Atkinson, 1985 (x) Colombo et 1975
(v) Wolfman et 1978 (xi) Liou and Anderson, 1978 (vi) Foe et al. , 1 983
class I thiol due to either immobilization or burial of the group (Jones et al., 1 972; 1 973), resulting in a decrease in its reactivity.
Citrate, 3- phosphoglycerate, phosphoenolpyruvate , and creatine phosphate each increase the affinity of PFK for ATP ( Kemp and Krebs, 1 967 ; Colombo et al. , 1 975), while AMP, cAMP and Pi decrease the affinity for A TP (Kemp, 1 969a).
subu";t
Muscle PFK treated with subti lisin resulted in an inactive enzyme of"molecular weight 7 4 000 which still retained its tetrameric structure (Riquelme and Kemp, 1 980). Binding studies revealed that only one adenine binding site remained in contrast to the three present in the native enzyme. Both the A TP and F6P binding sites were lost along with the A TP inhibitory site. cAMP bound to the proteolysed enzyme with the same affinity as the native enzyme. Binding sites for F16BP and A MP were retained (Gottschalk et al. , 1 983). Thesedata suggests a relatively discrete domain essential to the catalytic activity, but structurally distinct from several allosteric regulatory sites. ADP, cAMP and AMP competively bind to the same site with dissociation constants of 0.5, 0.6, and l . 8J..!M respecti vely. The dissociation constant for cAMP was found to decrease in the presence of F6P and F16BP (Kemp and Krebs, 1967). S tructural mapping studies of rabbit muscle PFK have shown that the distance between the cAMP binding site and the most reactive sulfhydryl group of PFK is 28 ± 6A (Craig and Hammes, 1 980).
A tryptic peptide from sheep heart PFK labelled with the affinity label p-fluorosulfonyl [ 14C]- benzoyl- 5'- adenosine, which binds specifically to the allosteric activator site has been i solated and sequenced (Weng et 1980).
� 262 I LASRMGAYA I DLLLAGY 279 � SH RM ULSARLGARAUELLLEGG 673 NFATKMGAK 68 1 * MGAKAMNWMAGK689 *
* Residue labelled with p-fluorosulfonyl [ 14C]-benzoyl-5'-adenosine
It was suggested (Weng et 1980), that the side chain of the labelled lysine (67 7) was i nvolved in the binding of the phosphate moiety of the allosteric activators, AMP,
cAMP and ADP. They also suggested that the C-terminal lysine residue in this sequence, or the lysine or arginine residue preceding this sequence may also be involved in this binding. Evidence in support of this came from the work of Kemp � al. ( 1 987), who isolated a tryptic peptide from rabbit muscle PFK which contained a labelled lysine, corresponding to that at position 68 1 in the sheep heart. Thereby suggesting that both the lysine residues at positions 677 and 68 1 of the rabbit enzyme are involved in a nucleoside phosphate binding site. S ince the adenine moiety in NAD+ has been found to bind to a hydrophobic pocket in several enzymes (Rossman et al., 1 975), the aromatic ring of the phenylalanine at position 674 may play a role in the binding of the adenine moiety if it is properly oriented (Weng et 1 980).
At the time of the publication of the sheep heart PFK adenine nucleotide binding site sequence, the complete amino acid sequence of mammalian PFK was unavailable. Therefore the authors compared their sequence with that of the B ac i l l u s enzyme, and suggested it was homologous to the region 1 7-25 of the B s sequence, which was known to contain two arginine residues which bound to the phosphate groups of allosteric effectors (Evans and Hudson, 1979). Since then, the complete amino acid sequence of rabbit muscle PFK has become available (Lee et al., 1 987), and a comparison of the complete sequence with that of the bacterial enzyme shows that the adenine binding site region is not homologous to the region suggested by Weng et al. ( 1980). The region of the adenine site corresponds to a region of the bacterial enzyme where no contacts to effectors are proposed at all (Hellinga and Evans,
1 985).
Mammalian PFK is thought to have evolved from the bacterial enzyme, of molecular weight 35 000, by gene duplication, fusion, and mutation of duplicated catalytic and regulatory sites to generate additional allosteric sites (Poorman et 1 9 84). The "hinge" region joining the two halves of the molecule is therefore an additional structure w hich must be fitted into the molecule. Since this hinge is located around the region of the adenine binding site, its presence may have necessitated the movement of the site in mammalian PFK to a site distinct from that of the bacterial enzyme. Thereby explaining the lack of complementarity between the binding sites of the mammalian and bacterial
enzymes.
One mole of citrate is bound per monomer of PFK. For tight binding MgA TP is required, in the absence of either Mg2+ or A TP very weak binding is observed. Both phosphoenolpyruvate and 3-phosphoglycerate compete with the binding of citrate, indicating a common binding site on the enzyme for these three inhibitors. Creatine
phosphate does not compete with citrate binding, and an additional site for the binding of creatine phosphate, distinct from the A TP site is indicated (Colombo S1 gl., 1 97 5). The amino acid sequence at the allosteric site for citrate has been determined for rabbit muscle PFK (Kemp et 1 987). Since the citrate binding site is likely to have multiple positive charges, pyridoxal phosphate was used to label the site. The sequence of the peptide isolated from a tryptic digest matches the sequence at positions 555-563 of rabbit muscle PFK (Poorman et al., l984), which is homologous to the Bs sequence
1 53- 1 6 1 .
� 1 53 I RD TATSHE 1 6 1 RM 555 I KQSAAGTK 563
*
* Residue labelled with [3H]-pyridoxal phosphate
The arginine in position 1 54 of the bacterial enzyme has been implicated by crystallography to be important in the allosteric binding site (Hellinga and Evans, 1 985). This residue is homologous to the phosphopyridoxalated lysine, w hich suggests, as predicted by Poorman et al. ( 1 984), that this allosteric site of the bacterial enzyme has evolved into one of the allosteric binding sites of rabbit skeletal muscle PFK.
Sheep heart PFK appears to have two types of binding site for F6P, a low affinity site (Kd 1 1 !-LM) and a high affinity site (Kd 0.2!-LM). The binding of F6P to both these sites is influenced by effectors. Fructose bisphosphates appear to bind to a single site on the enzyme. F26BP binds to the enzyme with greater affinity than F 1 6BP, which in turn binds with greater affinity than G 1 6BP (Foe et 1 983). The authors have suggested that very subtle differences in the conformations of PFKs exist when bound by F26BP, and w hen bound with F 1 6B P, with F26BP inducing a "less inhibitable" subconformation due to a better fit at the sugar bisphosphate binding site. Poorman et al. ( 1 984) have proposed that the sugar bisphosphate binding site has evolved from a mutated F6P binding site.