Sandbox/Judy Voet/PFK: Difference between revisions

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Judy Voet

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 	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).
-	The symmetry model of allosterism requires that an oligomeric (multisubunit) protein maintain its molecular symmetry in undergoing a transition from its high-activity R state to its low-activity T state. Hence, this transition must be concerted, with no intermediate states. The X-ray <ref>PMID:2136935</ref>and enzymological evidence indicates that PFK, a tetramer of identical subunits, is an allosteric enzyme that follows the symmetry model. Hence, 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.
+	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.
+The Active sites of the enzyme are located at the interface of subunits A (yellow) and D (light blue) and at the interface of subunits B and C. The allosteric sites are located at the interface of subunits A and B and at the interface of subunits C and D. Here is a <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_closeup/1'>Closeup</scene> of the active site on subunit A (yellow). Note that amino acids from subunit D (light blue) also contribute to the binding of F6P.
 	This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.
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-	PFK from B. stearothermophilus is a tetramer of identical 320-residue subunits. 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" />
 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 important participants in PFK's allosterically facilitated conformational change (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.