F2,6BP (fructose-2,6-bisphosphate) is a signal molecule that controls glycolysis; it is not a glycolytic intermediate. PFK-2 makes it by phosphorylating fructose-6-phosphate, and FBPase-2 breaks it back down, two activities of one bifunctional enzyme. F2,6BP activates PFK-1, the rate-limiting enzyme of glycolysis, and inhibits fructose-1,6-bisphosphatase, the matching gluconeogenic enzyme. In the liver its level is high in the fed state and low in starvation, so it sets the direction of glucose metabolism.

Does F2,6BP activate PFK-1, and how?

Yes. Fructose-2,6-bisphosphate is a potent allosteric activator of phosphofructokinase-1. It increases PFK-1's affinity for fructose-6-phosphate, turning the enzyme's sigmoidal velocity curve into a hyperbolic one, and it diminishes the inhibitory effect of ATP, which otherwise binds a regulatory site and lowers substrate affinity. F2,6BP also inhibits fructose-1,6-bisphosphatase, so glycolysis speeds up while gluconeogenesis slows. In the liver, insulin raises F2,6BP and glucagon lowers it.

How is F2,6BP regulated?

F2,6BP is regulated mainly by phosphorylation of the bifunctional PFK-2/FBPase-2 enzyme that makes and breaks it. In the liver, glucagon raises cAMP, protein kinase A phosphorylates a single serine, PFK-2 is inhibited and FBPase-2 activated, so F2,6BP falls in fasting. Insulin activates a protein phosphatase, PFK-2 activity returns and F2,6BP rises after meals. Abundant fructose-6-phosphate also speeds its synthesis, and over time insulin increases, and glucagon decreases, expression of the bifunctional enzyme.

What are the three regulated steps of glycolysis?

Glycolysis is regulated at its three irreversible steps. Hexokinase, or glucokinase in the liver and pancreatic β cells, phosphorylates glucose; hexokinase is inhibited by glucose-6-phosphate, while glucokinase is held by its regulatory protein. PFK-1, the rate-limiting enzyme, is activated by AMP and fructose-2,6-bisphosphate and inhibited by ATP, citrate and low pH. Pyruvate kinase is activated by fructose-1,6-bisphosphate and inhibited by ATP and alanine, and its liver form is switched off by glucagon-triggered phosphorylation.

What is the difference between hexokinase and glucokinase?

Both phosphorylate glucose to glucose-6-phosphate. Hexokinase, present in all cells, has high affinity for glucose and is inhibited by glucose-6-phosphate. Glucokinase, found in the liver and pancreatic β cells, has about 50-fold lower affinity, is not inhibited by glucose-6-phosphate and works mainly when glucose is abundant, which gives brain and muscle first call when supply is limited. It is the β-cell glucose sensor, and a variant in one GCK copy causes GCK-MODY, with mild fasting hyperglycemia.

Glycolysis Regulation: PFK-1, F2,6BP & USMLE Question

Q: Does PFK-2 stimulate glycolysis?

Yes, indirectly. PFK-2 makes fructose-2,6-bisphosphate, which activates PFK-1 and inhibits fructose-1,6-bisphosphatase, so more PFK-2 activity means faster glycolysis. Whether PFK-2 is active depends on the tissue isoform. In the liver, glucagon acts through cAMP and protein kinase A to phosphorylate the bifunctional enzyme, switching PFK-2 off and FBPase-2 on; insulin reverses this. In the heart, phosphorylation does the opposite and activates PFK-2, for example by AMP-activated protein kinase when oxygen supply is restricted.

Glycolysis is regulated at its three irreversible steps (hexokinase, or glucokinase in the liver and pancreatic β cells; phosphofructokinase-1, PFK-1; and pyruvate kinase), and PFK-1, the rate-limiting enzyme, is activated by AMP and fructose-2,6-bisphosphate (F2,6BP) and inhibited by ATP, citrate and a falling pH. F2,6BP is the liver's hormonal lever: one bifunctional enzyme makes it (its PFK-2 activity) and destroys it (its FBPase-2 activity). Insulin leaves that enzyme dephosphorylated, so F2,6BP rises, PFK-1 speeds up and gluconeogenesis slows; glucagon, through cAMP and protein kinase A (PKA), phosphorylates it, so F2,6BP falls and the liver switches to making glucose. Pyruvate kinase is activated feed-forward by fructose-1,6-bisphosphate and slowed by ATP, alanine and, in the liver form only, glucagon-triggered phosphorylation.

The vignette

A 19-year-old woman with type 1 diabetes is admitted with diabetic ketoacidosis after missing several insulin doses: glucose 540 mg/dL, pH 7.12, bicarbonate 10 mEq/L, positive serum ketones. She is treated with IV fluids, potassium and a continuous insulin infusion. Several hours later her glucose has fallen, dextrose is added to her fluids and the insulin infusion continues. Her hepatocytes are now shifting from gluconeogenesis toward glycolysis.

Which change most directly increases glycolytic flux in her hepatocytes?

A. Phosphorylation of the PFK-2/FBPase-2 bifunctional enzyme by protein kinase A
B. Increased ATP binding to the allosteric site of PFK-1
C. Increased fructose-2,6-bisphosphate concentration
D. Increased citrate concentration
E. Increased fructose-1,6-bisphosphatase activity
F. Increased acetyl-CoA activation of pyruvate carboxylase
G. Increased glucose-6-phosphate inhibition of glucokinase

Answer: C, increased fructose-2,6-bisphosphate. Insulin activates a protein phosphatase that dephosphorylates the hepatic bifunctional enzyme, so its kinase (PFK-2) activity wins and F2,6BP rises. F2,6BP is a potent activator of PFK-1: it raises the enzyme's affinity for fructose-6-phosphate and blunts ATP inhibition. It also inhibits fructose-1,6-bisphosphatase (FBPase-1), so glycolysis speeds up while gluconeogenesis slows. More slowly, through gene expression, insulin raises glucokinase and pyruvate kinase and lowers the gluconeogenic enzymes PEPCK, FBPase-1 and glucose-6-phosphatase. The clinical side of the crisis is in the DKA vs HHS question.

Sneaky variant: move the question to sprinting skeletal muscle and ask what speeds PFK-1. The answer becomes rising AMP: as contraction lowers the ATP/AMP ratio, AMP reverses ATP's inhibition of PFK-1.

Why every other choice is wrong

A. PKA phosphorylation of PFK-2/FBPase-2: this is the glucagon (fasting) signal. In the liver isoform, phosphorylation of a single N-terminal serine inhibits the kinase and activates the bisphosphatase, so F2,6BP falls, PFK-1 loses its activator and FBPase-1 is released. It describes the fasting liver, not the liver after insulin and dextrose.
B. ATP on PFK-1's allosteric site: ATP is a substrate of PFK-1, but at high levels it also binds a regulatory site distinct from the catalytic site and lowers the enzyme's affinity for fructose-6-phosphate, slowing glycolysis. "Substrate and inhibitor" is the classic trap.
D. Citrate: an early citric acid cycle intermediate whose buildup signals that biosynthetic precursors are plentiful. It inhibits PFK-1 by enhancing ATP's inhibition (see the TCA cycle question).
E. FBPase-1 activity: FBPase-1 is the rate-limiting enzyme of gluconeogenesis, the enzyme that reverses the PFK-1 step. More activity opposes glycolysis; F2,6BP and AMP inhibit it, and insulin lowers its expression.
F. Acetyl-CoA activating pyruvate carboxylase: pyruvate carboxylase turns pyruvate into oxaloacetate in mitochondria, the first step of gluconeogenesis from pyruvate. Acetyl-CoA activates it allosterically when acetyl-CoA backs up during fasting, so it signals gluconeogenesis, not glycolysis.
G. G6P inhibiting glucokinase: a false premise. Hexokinase is inhibited by its product, glucose-6-phosphate; glucokinase is not. Glucokinase's brake is the glucokinase regulatory protein (GKRP), which binds it and moves it into the nucleus when glucose is low and fructose-6-phosphate is high. After dextrose and insulin, glucokinase is working harder, not less.

The three irreversible steps at a glance

Step	Enzyme	Activated by	Inhibited by	Clinical tie-in
Glucose → glucose-6-phosphate	Hexokinase (all cells)	No key allosteric activator; high affinity keeps it working when glucose is scarce	Glucose-6-phosphate, its product	Hexokinase deficiency: hemolytic anemia
Glucose → glucose-6-phosphate	Glucokinase (liver, β cells)	High glucose (released from GKRP); insulin raises its expression	GKRP when glucose is low; not inhibited by G6P	GCK variants: GCK-MODY, neonatal diabetes, hyperinsulinemic hypoglycemia
Fructose-6-phosphate → fructose-1,6-bisphosphate (rate-limiting)	PFK-1	AMP, F2,6BP	ATP, citrate, low pH	Muscle PFK deficiency (Tarui disease)
Phosphoenolpyruvate → pyruvate	Pyruvate kinase	Fructose-1,6-bisphosphate (feed-forward); insulin raises its expression	ATP, alanine; glucagon-triggered phosphorylation of the liver (L) form	Pyruvate kinase deficiency: hemolytic anemia

Hexokinase follows PFK-1. When PFK-1 is inhibited, fructose-6-phosphate and glucose-6-phosphate build up, and G6P inhibits hexokinase.
Gluconeogenesis has its own enzymes for all three steps: pyruvate carboxylase and PEPCK (pyruvate → oxaloacetate → phosphoenolpyruvate), FBPase-1 and glucose-6-phosphatase. Drill them in the gluconeogenesis key enzymes question, and see every pathway's control step in the rate-limiting enzymes table.

Does F2,6BP activate PFK-1? How it works

Yes: fructose-2,6-bisphosphate is a potent allosteric activator of PFK-1. It does two things:

It raises PFK-1's affinity for fructose-6-phosphate. Without F2,6BP, PFK-1's velocity rises along a sigmoidal curve as fructose-6-phosphate increases; in Berg's classic figure, 1 μM F2,6BP makes that curve hyperbolic.
It diminishes ATP inhibition, the brake that high ATP applies through PFK-1's regulatory site.

F2,6BP also inhibits FBPase-1, which runs the same step backward in gluconeogenesis, so one molecule pushes the step forward and blocks its reversal. Its level, high in the fed state and low in starvation, therefore sets the liver's direction.

F2,6BP is a signal, not a pathway intermediate. Glycolysis runs through fructose-1,6-bisphosphate (F1,6BP). F2,6BP is made on a side branch when PFK-2 phosphorylates fructose-6-phosphate at carbon 2, and FBPase-2 hydrolyzes it back to fructose-6-phosphate. Abundant fructose-6-phosphate, as in a liver facing high blood glucose, speeds its synthesis, a process Berg calls feedforward stimulation.

	Fructose-1,6-bisphosphate (F1,6BP)	Fructose-2,6-bisphosphate (F2,6BP)
Made by	PFK-1, the committed step of glycolysis	PFK-2, the kinase half of the bifunctional enzyme
Role	Pathway intermediate	Signal molecule, off the pathway
Main target	Activates pyruvate kinase (feed-forward)	Activates PFK-1; inhibits FBPase-1
Removed by	Aldolase, the next glycolytic step; FBPase-1 in gluconeogenesis	FBPase-2, the phosphatase half of the same enzyme
Hormonal control	Indirect	Insulin raises it; glucagon lowers it (liver)

The general principles are in the allosteric regulation guide.

PFK-1: the rate-limiting enzyme and its regulators

PFK-1 catalyzes the committed step of glycolysis, the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate, which makes it the pathway's most important control element. Each regulator reports something different:

Regulator	Effect on PFK-1	What it signals	Where it matters most
AMP	Activates; reverses ATP inhibition	Low energy charge	Contracting skeletal muscle
F2,6BP	Activates; raises affinity for fructose-6-phosphate and diminishes ATP inhibition	Fed state, high glucose, insulin	Liver
ATP	Inhibits by binding a regulatory site separate from the catalytic site, which turns the hyperbolic substrate curve sigmoidal	High energy charge	Contracting muscle; Berg notes it matters less in liver
Citrate	Inhibits by enhancing ATP inhibition	Biosynthetic precursors are plentiful	Liver
H⁺ (falling pH)	Inhibits by augmenting ATP inhibition; prevents excessive lactic acid formation	Lactic acid buildup	Fast-twitch muscle working anaerobically

Why AMP rather than ADP: adenylate kinase salvages some ATP from ADP, and the AMP this produces becomes the low-energy signal. Muscle PFK-1 listens mainly to energy charge; liver PFK-1 also listens to hormones through F2,6BP.

Does PFK-2 stimulate glycolysis? Liver vs muscle vs heart

Yes, indirectly: PFK-2 makes F2,6BP, and F2,6BP activates PFK-1, so when PFK-2 activity outweighs FBPase-2 activity, glycolysis speeds up. The catch is that both activities sit in one polypeptide chain, after an N-terminal regulatory domain, and whether phosphorylation turns the kinase on or off depends on the isoform. Mammals have four PFK-2/FBPase-2 isoenzymes, each coded by a different gene that can produce several isoforms. Three tissues show the contrast:

Tissue	Enzyme form	What phosphorylation does	Main trigger	Glycolysis
Liver	L isoform	PKA phosphorylates an N-terminal serine: kinase off, phosphatase on, so F2,6BP falls	Glucagon (cAMP) in fasting; insulin's phosphatase reverses it after meals	Down in fasting, up when fed
Skeletal muscle	M isoform, spliced from the same gene as L	Its N-terminus differs and lacks the serine PKA phosphorylates in the liver form	Energy charge: AMP rising during contraction activates PFK-1 directly	Up during exercise
Heart	Separate heart isoenzyme	Several protein kinases phosphorylate its C-terminus: kinase on, so F2,6BP rises	AMP-activated protein kinase (AMPK) when oxygen supply is restricted, among other signals	Up

One-line rule for the liver: phosphorylation means glucagon, and glucagon means less glycolysis. Check the tissue first: AMPK phosphorylates and activates heart PFK-2, and in perfused rat hearts oxygen lack activated both and raised F2,6BP, a mechanism thought to help stimulate heart glycolysis during ischemia.

Insulin vs glucagon: the fed and fasting switch

Glucagon binds a G protein-coupled receptor that activates adenylyl cyclase, and the cAMP cascade leads to phosphorylation of the bifunctional enzyme (by PKA) and of liver pyruvate kinase. Insulin activates a protein phosphatase that dephosphorylates the bifunctional enzyme. Both hormones also change enzyme amounts, more slowly.

	Fed state: insulin	Fasting state: glucagon
Signal in the hepatocyte	Protein phosphatase activated	cAMP → PKA
Bifunctional enzyme	Dephosphorylated: PFK-2 active	Phosphorylated: FBPase-2 active
F2,6BP	High	Low
PFK-1 (glycolysis)	Activated	Loses its activator
FBPase-1 (gluconeogenesis)	Inhibited	Released
Liver pyruvate kinase	Dephosphorylated, active	Phosphorylated, less active
Gene expression	More glucokinase, PFK-1, pyruvate kinase and bifunctional enzyme; less PEPCK, FBPase-1 and glucose-6-phosphatase	Less of the regulated glycolytic enzymes; more PEPCK and FBPase-1
Net effect	Glycolysis, plus glucose-6-phosphate for glycogen and fatty acid synthesis	Gluconeogenesis and glucose output

Local signals are reciprocal too: AMP stimulates PFK-1 but inhibits FBPase-1, and citrate inhibits PFK-1 but activates FBPase-1.

Hexokinase vs glucokinase

Both enzymes phosphorylate glucose to glucose-6-phosphate, but they are tuned for opposite jobs.

Feature	Hexokinase	Glucokinase (hexokinase IV)
Where	Present in all cells	Liver and pancreatic β cells
Affinity for glucose	High (low Km): works even when glucose is scarce	About 50-fold lower; UniProt lists a Km near 6 mM for the human enzyme (measured at 30 °C)
Inhibited by glucose-6-phosphate	Yes	No
Other control	Follows PFK-1 through G6P buildup	GKRP binds it and moves it into the nucleus when glucose is low and fructose-6-phosphate is high; insulin increases its expression
Job	Captures glucose even when supply is limited	β cells: glucose sensor for insulin release; liver: glucose-6-phosphate for glycogen and fatty acid synthesis after meals
Disease	Hexokinase deficiency: hemolytic anemia	GCK-MODY (one variant copy), permanent neonatal diabetes (recessive), familial hyperinsulinemic hypoglycemia (dominant)

The low affinity is the point: glucokinase activity tracks blood glucose across its physiological range, so β cells match insulin release to glucose and the liver phosphorylates glucose mainly when it is abundant, leaving brain and muscle first call when supply is limited.

Pyruvate kinase: feed-forward activation and the liver isoform

Pyruvate kinase converts phosphoenolpyruvate (PEP) to pyruvate, making ATP, in the last irreversible step of glycolysis. It comes in an L form in the liver, an M form in muscle and brain, and an R form in red cells, the form mutated in pyruvate kinase deficiency.

Control	Effect	Form affected
Fructose-1,6-bisphosphate	Activates, so pyruvate kinase keeps pace with the flux coming from PFK-1 (feed-forward)	L, R and M2; not the adult M1 form
ATP	Inhibits when energy charge is high	L and M
Alanine	Inhibits; made from pyruvate in one step, it signals plentiful building blocks	Liver form
PKA phosphorylation (glucagon, cAMP)	Inactivates; dephosphorylation when blood glucose is adequate reactivates it	L only; the M form is not controlled by phosphorylation
Insulin	Increases the amount of enzyme	Liver

Isoform caveat: Berg says F1,6BP activates the muscle (M) form too, but UniProt splits M into M1, found in adult tissues and highly active without F1,6BP, and M2, the embryonic and proliferating-cell form, which needs it.

Switching off liver pyruvate kinase matters in fasting: gluconeogenesis spends energy turning pyruvate into PEP through pyruvate carboxylase and PEPCK, and an active pyruvate kinase would turn that PEP straight back into pyruvate. Pyruvate's next step is covered in the pyruvate dehydrogenase complex tips.

Pyruvate kinase deficiency and other clinical correlations

Pyruvate kinase deficiency is the most common glycolytic enzyme defect causing nonspherocytic hemolytic anemia. It is autosomal recessive and caused by variants in PKLR (chromosome 1q22). Red cells lack mitochondria, so lactic acid fermentation is their sole source of ATP, and the pyruvate kinase step yields 50% of red cell ATP. Without it, red cells lose deformability and are trapped in splenic and hepatic capillaries; young red cells are most affected. Raised 2,3-BPG (2,3-DPG) shifts the oxygen dissociation curve to the right, which helps compensate for the anemia.

Presentation: from the neonatal period, when hyperbilirubinemia may be the first sign, into adult life; jaundice, hepatosplenomegaly, pigment gallstones (at least 30% of patients) and iron overload.
Labs: nonspecific smear (echinocytes, polychromasia) without spherocytes; Coombs test and osmotic fragility are negative.
Diagnosis: red cell PK activity, which recent transfusion, reticulocytosis or white cell interference can confound; PKLR genetic testing.
Treatment: folic acid, transfusions and iron chelation as needed; splenectomy for severe or transfusion-dependent disease, after which hemolysis persists but may improve. Mitapivat, an oral pyruvate kinase activator that binds the PK tetramer allosterically, is approved for hemolytic anemia in adults with PK deficiency; abrupt interruption can trigger acute hemolysis.
How common: an estimated 3.2 to 8.5 cases per million in Western populations, although a prevalence of 1 in 20,000 has been reported.

Other red cell glycolytic defects: hexokinase, glucose phosphate isomerase and phosphofructokinase deficiencies also cause hemolytic anemia. All are autosomal recessive, so anemia appears only when both gene copies are mutated (homozygous or compound heterozygous).

Glucokinase variants: a variant in one GCK copy causes GCK-MODY (MODY2), 30% to 50% of MODY cases: slightly raised blood glucose, especially fasting, often no symptoms and extremely rare complications. Recessive GCK variants cause permanent neonatal diabetes, and other dominant variants cause familial hyperinsulinemic hypoglycemia.

Tarui disease (GSD VII): PFKM variants disable the muscle subunit of PFK-1. Muscle PFK is a tetramer of four PFKM subunits, while red cells mix PFKM and PFKL subunits. Patients have exercise intolerance with cramps, nausea and vomiting, myoglobinuria after vigorous exercise and compensated hemolysis. Compare the McArdle disease question.

How USMLE tests glycolysis regulation: traps and look-alikes

Trap	What's actually true
Pyruvate kinase or hexokinase is the rate-limiting enzyme of glycolysis	PFK-1 is; it catalyzes the committed step
F1,6BP and F2,6BP do the same job	F1,6BP is the intermediate and activates pyruvate kinase; F2,6BP is an off-pathway signal that activates PFK-1
Phosphorylation always switches PFK-2 off	In liver, PKA phosphorylation turns the kinase off; in heart, phosphorylation turns it on
Glucose-6-phosphate inhibits glucokinase	Only hexokinase is product-inhibited; glucokinase is held by GKRP
Glucagon activates liver pyruvate kinase	PKA phosphorylation inactivates the L form; the M form is not regulated this way
Acetyl-CoA activating pyruvate carboxylase points to glycolysis	It points to gluconeogenesis in the fasting state

PK deficiency's main look-alike is a membrane defect:

Feature	PK deficiency	Hereditary spherocytosis
Defect	Glycolytic enzyme	Red cell membrane
Inheritance	Autosomal recessive	Autosomal dominant in about 75% of cases; positive family history usually found
Smear	No spherocytes; echinocytes may be seen	Spherocytes easily seen
Osmotic fragility	Negative	Osmotic lysis present

G6PD deficiency, by contrast, is X-linked recessive, with hemolysis triggered by infections, medications and oxidant stress, and Coombs positivity points to antibody-mediated hemolysis. See hereditary spherocytosis and G6PD deficiency; more pathway guides are on the Biochemistry hub.

Worked vignettes

Vignette 1. A 61-year-old man is brought in with crushing chest pain, and angiography shows an occluded left anterior descending artery. In the ischemic myocardium, glycolytic flux rises as oxygen delivery falls. Which change in his cardiomyocytes most directly raises fructose-2,6-bisphosphate?

Answer: Phosphorylation of the heart PFK-2/FBPase-2 isoenzyme by AMP-activated protein kinase (AMPK), which activates its kinase.

Reasoning: AMPK, an energy-state sensor, is activated when oxygen supply is restricted. It phosphorylates the heart isoenzyme and activates PFK-2, so F2,6BP rises and PFK-1 speeds up. "Phosphorylation lowers F2,6BP" is the liver isoform's rule, not the heart's.

Vignette 2. A 6-year-old boy has had jaundice since the newborn period and has needed several transfusions. His spleen is palpable 4 cm below the costal margin, and ultrasound shows gallstones. Hemoglobin is low and reticulocytes are high. The smear shows echinocytes but no spherocytes, the Coombs test is negative, osmotic fragility is normal and G6PD activity is normal. His parents are healthy first cousins. Which enzyme is most likely deficient, and what can confound the enzyme assay?

Answer: Red cell pyruvate kinase (autosomal recessive PKLR variants); recent transfusion, reticulocytosis or white cell interference can confound the assay.

Reasoning: Chronic hemolysis since birth with no spherocytes, normal osmotic fragility and a negative Coombs test fits a glycolytic enzyme defect, and consanguinity fits recessive inheritance. Without pyruvate kinase, ATP-starved red cells lose deformability and are trapped in the spleen. PKLR testing confirms the diagnosis when the assay is unreliable.

Vignette 3. A 24-year-old man has a fasting plasma glucose of 118 mg/dL at a pre-employment checkup and a similar value on repeat. He has no symptoms, his BMI is 22 and islet autoantibodies are negative. His mother and maternal grandfather have had mildly raised fasting glucose for decades without complications. Which enzyme is most likely affected?

Answer: Glucokinase, from a variant in one copy of GCK (GCK-MODY, also called MODY2).

Reasoning: Glucokinase is the β-cell glucose sensor. A GCK variant limits its ability to sense a rise in blood glucose, so glucose stays mildly raised, especially fasting. MODY is autosomal dominant, GCK-MODY accounts for 30% to 50% of cases, and complications are extremely rare. Compare the common forms in diabetes type 1 vs type 2.

Key takeaways

PFK-1, the rate-limiting enzyme, is activated by AMP and F2,6BP and inhibited by ATP, citrate and low pH.
F2,6BP is a signal, not an intermediate: it raises PFK-1's affinity for fructose-6-phosphate, relieves ATP inhibition and inhibits FBPase-1.
PFK-2 stimulates glycolysis by making F2,6BP. Liver: glucagon (cAMP, PKA) phosphorylation switches PFK-2 off, insulin switches it back on. Heart: phosphorylation (for example by AMPK) switches PFK-2 on.
Pyruvate kinase: feed-forward activation by F1,6BP; inhibited by ATP and alanine; only the liver (L) form is inactivated by phosphorylation.
Glucokinase (liver, β cells): low affinity, not inhibited by G6P, held by GKRP. Hexokinase: high affinity, product-inhibited.
Clinical: PK deficiency (Coombs-negative nonspherocytic hemolysis, mitapivat), GCK-MODY (mild fasting hyperglycemia), Tarui disease (muscle PFK).