Sandbox/Judy Voet/PFK: Difference between revisions
No edit summary |
No edit summary |
||
| Line 2: | Line 2: | ||
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, and fructose-2,6-bisphosphate (F2,6P). | Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, PEP and fructose-2,6-bisphosphate (F2,6P). | ||
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /> | <applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /> | ||
PFK from B. stearothermophilus is a tetramer of identical 320-residue subunits. It is an allosteric enzyme that is described using the symmetry model of allosterism whereby there is a concerted transition from its high-activity R state to its low-activity T state. The X-ray structures of both R and T states of the enzyme have been reported.<ref>PMID:2136935</ref> The binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme. | <scene name='Sandbox/Judy_Voet/PFK/4pfk_biol/1'>PFK from B. stearothermophilus</scene> is a tetramer of identical 320-residue subunits. It is an allosteric enzyme that is described using the symmetry model of allosterism whereby there is a concerted transition from its high-activity R state to its low-activity T state. The X-ray structures of both R and T states of the enzyme have been reported.<ref>PMID:2136935</ref> The binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme. | ||
Two of the active sites of the enzyme are located at the interface of subunits A (light Blue) and D (yellow) and two at the interface of subunits B (green) and C (pink). Two of the allosteric sites are located at the interface of subunits A and B and two at the interface of subunits C and D. Here is a closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_closeup/1'>A/D interface active site</scene> of subunit D (Yellow). Note that amino acids from subunit A (light blue) also contribute to the binding of F6P. Also here is a closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_closeup/2'>A/B allosteric site</scene> of subunit B with contributions from both subunits to the binding of ADP. | Two of the active sites of the enzyme are located at the interface of subunits A (light Blue) and D (yellow) and two at the interface of subunits B (green) and C (pink). Two of the allosteric sites are located at the interface of subunits A and B and two at the interface of subunits C and D. Here is a closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_closeup/1'>A/D interface active site</scene> of subunit D (Yellow). Note that amino acids from subunit A (light blue) also contribute to the binding of F6P. Also here is a closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_closeup/2'>A/B allosteric site</scene> of subunit B with contributions from both subunits to the binding of ADP. | ||
| Line 14: | Line 14: | ||
This kinemage shows the two subunits of the tetramer whose interface contains two active sites. | This kinemage shows the two subunits of the tetramer whose interface contains two active sites. | ||
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /> | <kinemage align="right" width="450" height= "450" file="PFK1.kin" /> | ||
The first view, 1. PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are | The first view, 1. PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are part of the F6P binding site in the T and R states, srespectively(see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds. | ||
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme. | Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme. | ||
| Line 30: | Line 30: | ||
KINEMAGE 2 comes up in view 1, The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). | KINEMAGE 2 comes up in view 1, The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). | ||
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the | What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the absence of the positive charge of Arg 162 have on the binding of F6P? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2. Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view. | ||
==Site-Directed Mutagenesis== | ==Site-Directed Mutagenesis== | ||
At one time, the negative charge of Glu 161 was thought to have a negative effect on F6P binding in the T state. This idea has not been supported by site-directed mutagenesis experiments<ref>PMID:10759544</ref>.Several mutant PFKs have been made, including R162A, E161A and R162A/E161A . The R162A mutation caused a 30-fold decrease in F6P binding. The E161A mutation, however, had little effect on the ability of PEP to inhibit F6P binding. | |||
{{clear}} | {{clear}} | ||
== Atomic Coordinates== | == Atomic Coordinates== | ||