What are isozymes (isoenzymes)?

Isozymes, also called isoenzymes, are different forms of an enzyme that catalyze the same reaction but differ in amino acid sequence and in properties such as Km, regulation, heat stability and tissue distribution. Classic examples are hexokinase and glucokinase, the creatine kinase isoenzymes CK-MM, CK-MB and CK-BB, the five LDH isoenzymes, alkaline phosphatase, aldolase A and B, and COX-1 and COX-2. Because tissues carry their own isozymes, the pattern released after injury can point to the damaged organ.

What are some examples of isoenzymes?

Hexokinase in most tissues and glucokinase in the liver and pancreatic beta cells both phosphorylate glucose. Creatine kinase has CK-MM in skeletal muscle, CK-MB in heart and CK-BB in brain. Lactate dehydrogenase runs from LDH-1 in heart and red cells to LDH-5 in liver and skeletal muscle. Others include tissue-nonspecific (liver and bone), placental and intestinal alkaline phosphatase, aldolase A, B and C, COX-1 and COX-2, and the muscle and liver forms of glycogen phosphorylase and pyruvate kinase.

What are the types of isoenzymes?

Isoenzymes arise in several ways. Separate genes make hexokinase and glucokinase, aldolase A, B and C, and COX-1 and COX-2. Hybrid isozymes form when subunits from two genes combine, as in the CK dimers and LDH tetramers. One gene can give two forms by alternative splicing, as with pyruvate kinase M1 and M2, and different alleles give allozymes such as G6PD variants. Forms made by modifying one sequence, such as phosphorylase a and b, are not true isozymes.

What is the difference between hexokinase and glucokinase?

Both phosphorylate glucose to glucose-6-phosphate. Hexokinase, found in most tissues, has a high affinity (low Km) for glucose and is inhibited by glucose-6-phosphate, so it keeps supplying tissues when glucose is low. Glucokinase, found in the liver and pancreatic beta cells, has a weak affinity (UniProt lists a Km of about 6 mM), is not inhibited by glucose-6-phosphate and is controlled by a regulatory protein. It acts as the beta cell's glucose sensor, and losing one copy causes MODY2.

Which isoenzymes rise after a heart attack?

Classically, CK-MB, the heart-rich dimer of one M and one B subunit, and LDH-1, which rises above LDH-2 in the flipped pattern. CK-MB returns to normal within about 2 to 3 days, so a new rise once signaled reinfarction, while the LDH flip appears later and lasts longer. Today the Fifth Universal Definition of Myocardial Infarction (2026) uses cardiac troponin I or T above the sex-specific 99th percentile, keeping a CK-MB mass assay only where troponin is unavailable.

How do you tell if a high alkaline phosphatase comes from the liver or bone?

Check gamma-glutamyl transferase (GGT) or 5'-nucleotidase, which rise with liver and bile duct disease but not with bone disease. A raised alkaline phosphatase with a raised GGT points to a hepatobiliary cause such as cholestasis; with a normal GGT it points away from the liver, most often to bone, as in Paget disease. Bone alkaline phosphatase is also the least heat-stable fraction. Growth in children, late pregnancy and a fatty meal can raise alkaline phosphatase normally.

Isozymes (Isoenzymes): Definition, Examples, CK-MB & LDH

Isozymes (isoenzymes) are different forms of one enzyme that catalyze the same reaction but differ in structure, kinetics (Km), regulation and tissue distribution. Classic examples are hexokinase vs glucokinase, creatine kinase (CK-MM, CK-MB, CK-BB), lactate dehydrogenase (LDH-1 to LDH-5), alkaline phosphatase, aldolase A vs B and COX-1 vs COX-2.

Step 1 uses them two ways. Physiology: each tissue gets a version tuned to its job, so liver glucokinase stores glucose after meals while hexokinase keeps brain and muscle supplied when glucose is low. Diagnosis: injured tissue leaks its own isozymes, so CK-MM points to skeletal muscle, and a raised alkaline phosphatase (ALP) with a normal GGT points away from the liver, usually to bone.

One update matters: myocardial infarction (MI) is now diagnosed with cardiac troponin I or T, not CK-MB or the LDH "flip" (Fifth Universal Definition of MI, 2026). Learn those two as classic teaching.

What is an isozyme? Definition and meaning

The National Library of Medicine defines isoenzymes as structurally related forms of an enzyme with the same mechanism and classification that differ in chemical, physical or immunological characteristics. "Isozyme" and "isoenzyme" mean the same thing.

The formal nomenclature (IUPAC-IUB, 1976) is stricter:

"Multiple forms of an enzyme" is the umbrella term for all proteins in one species that catalyze the same reaction.
"Isoenzyme" or "isozyme" is reserved for forms with genetically determined differences in primary structure (amino acid sequence), not forms made by modifying one sequence.
Allozymes (alloenzymes) are isozymes made by different alleles of one gene (MeSH); the nomenclature's example is the many variants of glucose-6-phosphate dehydrogenase (see G6PD deficiency).

Isozymes can differ in Km and catalytic rate, inhibitors and activators, location inside the cell, electrophoretic mobility, heat stability and antibody binding. They cannot differ in the reaction: if an answer choice says isozymes catalyze different reactions, it is wrong.

"Isoform" is looser: StatPearls calls the CK-MM forms that blood carboxypeptidases make by clipping a lysine "isoforms", and troponin assays target the "cardiac isoforms" of troponin I and T, which are not enzymes at all.

Examples of isoenzymes

Enzyme	Isozymes	How they differ	Exam hook
Hexokinase (glucose to glucose-6-phosphate)	Hexokinase vs glucokinase (hexokinase IV, GCK)	Most tissues vs liver and pancreatic β cells; glucokinase has weak affinity and no product inhibition	GCK loss of function = MODY2; red-cell hexokinase deficiency = hemolytic anemia
Creatine kinase	CK-MM, CK-MB, CK-BB (plus mitochondrial CK)	M and B subunit dimers: skeletal muscle, heart, brain	Rhabdomyolysis (MM); classic MI marker (MB)
Lactate dehydrogenase	LDH-1 (H4) to LDH-5 (M4)	H and M subunit tetramers: heart and red cells to liver and skeletal muscle	LDH-1 > LDH-2 "flip" after MI or hemolysis
Alkaline phosphatase	Tissue-nonspecific (liver, bone, kidney), intestinal, placental, germ cell	Four genes; liver and bone forms share one gene	GGT or 5′-nucleotidase sorts liver from bone; persistently low ALP suggests hypophosphatasia
Aldolase	A, B, C	A: embryo and adult muscle; B: adult liver, kidney, intestine; C: brain (with A)	Aldolase B splits fructose-1-phosphate; its loss = hereditary fructose intolerance
Cyclooxygenase	COX-1, COX-2	Constitutive (stomach lining, kidney, platelets) vs induced by inflammation	Aspirin inactivates COX irreversibly; celecoxib is COX-2 selective
Pyruvate kinase	L and R (one gene, PKLR); M1 and M2 (one gene, PKM)	Liver and red cells vs adult muscle and brain; M2 in the embryo and in cancer cells	PK deficiency, the most common glycolytic cause of hemolysis; the L form, unlike M, is switched off by phosphorylation
Phosphofructokinase-1	Tetramers of M, L and P subunits	Muscle M4, liver L4, red cells a random M and L mix	Muscle PFK deficiency (Tarui, GSD VII): exercise myopathy plus compensated hemolysis
Glycogen phosphorylase	Muscle (myophosphorylase, PYGM) and liver (PYGL)	Separate genes, same glycogen-to-glucose-1-phosphate reaction	McArdle disease (GSD V) vs Hers disease (GSD VI)
Malate dehydrogenase	Cytosolic and mitochondrial	Separate genes, separate compartments	The nomenclature's model of genetically independent isozymes

Types of isoenzymes: how they arise and how labs separate them

Type	How it happens	Examples
Separate genes	Related genes make proteins for the same reaction	Hexokinase vs glucokinase; aldolase A, B, C; COX-1 vs COX-2; cytosolic vs mitochondrial malate dehydrogenase
Hybrid (subunit) isozymes	Subunits from two genes assemble in different mixes	CK dimers (M gene on 19q13, B gene on 14q32); LDH tetramers from LDHA (M, 11p15.4) and LDHB (H, 12p12.1); PFK-1 tetramers
One gene, two transcripts	Alternative splicing or alternative promoters	PKM gives PKM1 and PKM2; PKLR gives the R and L forms; alternative GCK promoters appear to let insulin regulate liver glucokinase and glucose the β-cell form
Allelic variants	Different alleles of one gene (allozymes)	G6PD variants
Not isozymes (strict definition)	Same sequence, modified or reshaped	Phosphorylase a vs b (phosphorylation); chymotrypsins cut from chymotrypsinogen; allosteric conformations

How labs tell them apart:

Electrophoresis. The 1976 rules number isozymes by mobility toward the anode, form 1 fastest: LDH-1 is the fastest LDH band, and CK-BB, the most anodal CK, is CPK-1 (MB is CPK-2, MM is CPK-3).
Heat. ALP fractions differ in heat stability: placental most stable, then intestinal, liver and bone (least).
Antibodies. CK-MB mass assays use an antibody that recognizes only the MB dimer, and troponin assays target sequences found only in the cardiac isoforms.

Hexokinase vs glucokinase

	Hexokinase	Glucokinase (hexokinase IV)
Where	Most tissues, including brain and muscle	Liver and pancreatic β cells
Affinity for glucose	High (low Km)	Weak (high Km; UniProt lists about 6 mM); effective only when glucose is abundant
Inhibited by glucose-6-phosphate	Yes	No
Other control	Follows PFK-1: when PFK-1 slows, fructose-6-phosphate and then glucose-6-phosphate build up and inhibit hexokinase	Glucokinase regulatory protein holds it inactive in the hepatocyte nucleus when glucose is low and fructose-6-phosphate is high; fructose-1-phosphate releases it
Job	Keeps glucose flowing into tissues even when blood glucose is low	β-cell glucose sensor for insulin release; supplies glucose-6-phosphate for liver glycogen after meals
Linked disease	Hexokinase deficiency: autosomal recessive nonspherocytic hemolytic anemia (red cells use an erythrocyte-specific hexokinase 1 isoform)	Heterozygous loss: GCK-MODY (MODY2); loss of both copies: permanent neonatal diabetes; activating variants: familial hyperinsulinemic hypoglycemia

Why a weak enzyme helps: glucokinase's low affinity gives the brain and muscles first call on glucose when supply is limited (Berg).
GCK-MODY in numbers (StatPearls): GCK variants cause 30-60% of MODY. The glucose threshold for insulin release is reset higher, so fasting glucose sits around 99-144 mg/dL, HbA1c is typically 5.6-7.3% before age 40, complications are rare and drug treatment is usually unnecessary outside pregnancy.

How these enzymes interact with PFK-1 and pyruvate kinase is in glycolysis regulation; reading Km off a graph is Lineweaver-Burk territory.

Creatine kinase isoenzymes: CK-MM, CK-MB, CK-BB

Isozyme	Subunits	Main tissue	Typical causes of a rise
CK-MM (CPK-3)	M + M	Skeletal muscle (about 98% of its CK) and most heart CK (70-85%)	Rhabdomyolysis, crush injury, myositis, muscular dystrophy, strenuous exercise, intramuscular injections, seizures; hypothyroidism
CK-MB (CPK-2)	M + B	Heart (15-30% of cardiac CK, by source); 1-3% of skeletal muscle CK	MI (classic marker), myocarditis, defibrillation, heart surgery, electrical injury
CK-BB (CPK-1)	B + B	Brain (MedlinePlus adds lung)	Brain injury, stroke, seizures, pulmonary infarction; brain-damaged or low-birth-weight newborns

Timing after MI: CK-MB rises about 3-6 hours after the infarct, peaks at about 12-24 hours and clears within 2-3 days (48-72 hours; MedlinePlus, StatPearls), so a new CK-MB rise was the classic sign of reinfarction.
CK-MB is not purely cardiac. Sustained training raises the CK-MB content of skeletal muscle (StatPearls calls it fetal reversion), and chronic renal failure can too. Labs therefore use a relative index, CK-MB mass divided by total CK: 3% or less is physiological, 3-5% equivocal, over 5% consistent with myocardial necrosis.
Lab trap: macro-CK (often CK-BB bound to IgG) can falsely raise CK-MB measured by immunoinhibition; mass assays using antibodies to the MB dimer have largely replaced that method.

Total CK as a muscle-injury marker is drawn out in rhabdomyolysis-induced AKI; troponin and CK-MB timelines side by side are in the cardiac biomarkers guide.

LDH isoenzymes and the "flipped" LDH-1:LDH-2 ratio

LDH interconverts pyruvate and lactate. Its five isoenzymes are tetramers of H (heart, LDHB) and M (muscle, LDHA) subunits:

Isoenzyme	Subunits	Highest in
LDH-1	H4	Heart muscle and red cells
LDH-2	H3M	Reticuloendothelial system and red cells (StatPearls); white cells (MedlinePlus)
LDH-3	H2M2	Lung
LDH-4	HM3	Kidney (MedlinePlus adds placenta and pancreas)
LDH-5	M4	Liver and skeletal muscle

Why two subunits: the M subunit has higher affinity for pyruvate and favors pyruvate to lactate; the H subunit has higher affinity for lactate and favors lactate to pyruvate, which suits the heart.
The flip: normally LDH-2 exceeds LDH-1. After MI, LDH-1 exceeds LDH-2 (ratio above 1). Total LDH rises 6-12 hours after MI, peaks at 24-72 hours and normalizes within 8-14 days (StatPearls), so the flip outlasts CK-MB.
Not only MI: hemolytic anemia can flip the ratio too, mainly when many lysed cells are reticulocytes (Kazmierczak 1990), as can megaloblastic anemia (Jaswal 2000). A hemolyzed sample falsely raises LDH because red cells are full of it.
Where total LDH still earns its keep: hemolysis, staging nonseminomatous testicular tumors and sorting exudates from transudates (Light's criteria). Routine isozyme testing is usually unavailable, so labs use CK for muscle, ALT for liver and troponin for the heart (StatPearls).

Troponin vs CK-MB and LDH: what current practice uses

The Fifth Universal Definition of Myocardial Infarction (ESC, ACC, AHA and World Heart Federation, August 2026) sets the current rules:

Acute myocardial injury is a rise and/or fall of cardiac troponin I or T with at least one value above the sex-specific 99th percentile upper reference limit. MI is that injury caused by ischemia: ischemic symptoms, new ischemic ECG changes or new Q waves make it likely, and imaging or angiography confirms it.
Biomarker hierarchy: high-sensitivity troponin is the most sensitive and specific test. If it is not available, a conventional troponin assay is preferred over CK-MB, which has lower sensitivity and specificity; a CK-MB mass assay is a fallback only when no troponin assay exists.
Kinetics: troponin is detectable within 2-3 hours of symptom onset, peaks at 12-24 hours and stays elevated for days or weeks.
Reinfarction: because troponin can stay high for weeks, suspected recurrence is judged on serial troponin (a new rise suggests it), often with angiography; the document says CK-MB "adds cost without providing diagnostic clarity."
US guidance agrees: the 2021 AHA/ACC chest pain guideline calls high-sensitivity troponin the preferred standard and found adding CK-MB or myoglobin to troponin not beneficial.

The isozyme idea survives inside the modern test: troponin assays work because they target amino acid sequences specific to the cardiac isoforms of troponin I and T. Complications after MI are covered in acute MI complications.

Alkaline phosphatase isoenzymes: liver, bone, placenta, intestine

ALP is a family of isoenzymes from four genes: tissue-nonspecific ALP (ALPL: liver, bone, kidney), intestinal, placental and germ cell ALP. More than 80% of serum ALP comes from liver and bone.

Liver and bone ALP come from one gene. They share one amino acid sequence and differ in sugar and lipid side chains added after translation, so under the strict 1976 definition they are forms of one isozyme (tissue-nonspecific ALP), not separate isoenzymes; GeneReviews calls them isoforms, StatPearls and labs isoenzymes.
Rises without liver disease: growing children and adolescents (bone), late pregnancy (placental ALP), a fatty meal in people with blood group O or B (intestinal ALP, for up to 12 hours, so test fasting) and chronic renal failure.
Liver or bone? Check GGT or 5′-nucleotidase, which rise with hepatobiliary ALP but not with bone disease. GGT also stays normal in childhood and pregnancy (5′-nucleotidase can rise in pregnancy), but alcohol and enzyme-inducing drugs raise it; a high GGT with a normal ALP can be seen in alcohol-associated hepatitis (Merck).
Isolated high ALP in an older adult usually comes from bone, for example Paget disease of bone. Other bone causes: metastases, osteosarcoma, healing fractures, hyperparathyroidism, hyperthyroidism and osteomalacia.
Heat test: after 56 °C for 10 minutes, residual activity under 20% points to bone ALP and over 20% to liver ALP. Memory aid: bone burns first. The tumor-made Regan isoenzyme is the most heat-stable of all.
Low ALP is a clue too: hypophosphatasia (loss of ALPL function; high pyridoxal 5′-phosphate, rickets or osteomalacia, baby teeth lost with the root attached; treated with asfotase alfa), Wilson disease with hemolysis, zinc deficiency, hypothyroidism and pernicious anemia.
Half-life about 7 days, so ALP can stay high for about a week after a biliary obstruction clears.

USMLE traps

Trap	What is actually true
"Isozymes catalyze different reactions"	Same reaction, different sequence, kinetics, regulation or tissue
"Phosphorylase a and b are isozymes"	They are one sequence, interconverted by phosphorylation; the strict definition excludes them
"Glucokinase has the lower Km"	Glucokinase has the higher Km (weak affinity) and no glucose-6-phosphate inhibition
"CK-MB is heart-specific"	About 1-3% of skeletal muscle CK is MB, more in trained athletes and renal failure; troponin is preferred
"Use CK-MB to spot reinfarction"	Classic teaching; the 2026 Universal Definition uses serial troponin, with CK-MB only when no troponin test exists
"An LDH flip means MI"	Also seen in hemolytic anemia (especially with reticulocytosis) and megaloblastic anemia; hemolyzed samples raise LDH
"High ALP means liver disease"	Check GGT: bone, growth, pregnancy, a fatty meal and renal failure also raise ALP
"Celecoxib can replace low-dose aspirin"	It lacks platelet effects, is not a substitute for cardiovascular prophylaxis and carries the NSAID boxed warning
"McArdle disease is liver phosphorylase deficiency"	McArdle is muscle (PYGM); liver phosphorylase deficiency is Hers disease (GSD VI)
"Hereditary fructose intolerance is fructokinase deficiency"	It is aldolase B deficiency; fructokinase deficiency causes benign essential fructosuria (fructose and galactose mnemonic)

Practice vignettes

Vignette 1: the family with mild diabetes. A 16-year-old girl has fasting plasma glucose levels of 118 and 124 mg/dL on two occasions; her HbA1c is 6.3%. She has a normal BMI and no islet autoantibodies. Her mother and maternal grandfather have "mild diabetes" that has never needed treatment. Genetic testing finds a heterozygous loss-of-function variant in the enzyme that phosphorylates glucose in pancreatic β cells and hepatocytes. Compared with the isozyme that does the same reaction in skeletal muscle, the affected enzyme normally has which property?

A. A lower Km for glucose. B. Inhibition by glucose-6-phosphate. C. A higher Km for glucose and no inhibition by glucose-6-phosphate. D. Inactivation by phosphorylation after glucagon. E. Conversion of glucose to glucose-1-phosphate.

Answer: C. This is GCK-MODY (MODY2): one faulty GCK copy resets the β-cell glucose threshold, giving mild, stable fasting hyperglycemia. A and B describe hexokinase. D describes the liver (L) form of pyruvate kinase, not glucokinase, whose brake is its regulatory protein. E: isozymes catalyze the same reaction, and glucose-1-phosphate comes from glycogen phosphorylase.

Vignette 2: chest pain on day 4. A 64-year-old man had an anterior ST-elevation MI treated with angioplasty. His high-sensitivity troponin I peaked on day 1 and is falling but still above the 99th percentile on day 4, when crushing chest pain returns. Which laboratory approach best supports reinfarction?

A. A single troponin I value above the 99th percentile. B. Serial troponin measurements showing a new rise. C. CK-MB activity. D. The LDH-1:LDH-2 ratio. E. Total CK.

Answer: B. Troponin can stay high for weeks, so a new rise on serial testing is what suggests recurrence (Fifth Universal Definition, 2026). A cannot separate the old infarct from a new one. C is the classic answer because CK-MB normalizes within 2-3 days, but current guidance says it adds cost without clarity. D: the flip from the first MI can persist for up to about 2 weeks, and hemolysis confounds it. E: total CK is mostly skeletal muscle CK-MM.

Vignette 3: the isolated alkaline phosphatase. A 71-year-old man has an ALP of 390 U/L on routine testing. AST, ALT, bilirubin and GGT are normal. After heating the serum to 56 °C for 10 minutes, 12% of the ALP activity remains. Which is the most likely source?

A. Bone. B. Liver and bile ducts. C. Placenta. D. Small intestine. E. A tumor-derived Regan isoenzyme.

Answer: A. A normal GGT argues against the liver, and residual activity under 20% after heating points to bone ALP, the least heat-stable fraction; in an older adult, think of Paget disease. B: hepatobiliary ALP rises with GGT and keeps over 20% of its activity. C: placental ALP is the most heat-stable normal fraction and rises in late pregnancy. D: intestinal ALP rises after a fatty meal and is more heat-stable than bone ALP. E: the Regan isoenzyme is the most heat-stable of all.

Vignette 4: cramps in the gym. A 19-year-old man gets painful cramps within minutes of sprinting or lifting weights and once passed dark urine after a workout. If he slows down for a few minutes, he can keep jogging. Resting CK is 2,100 U/L. On a forearm exercise test, lactate barely rises while ammonia rises normally. Hemoglobin, reticulocytes, bilirubin, uric acid, fasting glucose and liver size are normal. Which enzyme is deficient?

A. Muscle glycogen phosphorylase. B. Liver glycogen phosphorylase. C. Muscle phosphofructokinase. D. Aldolase B. E. Glucokinase.

Answer: A. McArdle disease (GSD V): myophosphorylase is the muscle isoenzyme of glycogen phosphorylase, and the "second wind", high resting CK, myoglobinuria and low lactate-to-ammonia ratio fit (more in the McArdle disease breakdown). B (Hers disease) causes hepatomegaly and ketotic hypoglycemia. C (Tarui disease) looks similar in muscle but adds compensated hemolysis and hyperuricemia, because red-cell PFK contains M subunits. D causes vomiting and hypoglycemia after fructose. E is a liver and β-cell enzyme.

Key takeaways

Isozymes = same reaction, different protein: separate genes, subunit mixes, splice variants or alleles; post-translational variants are not isozymes in the strict sense.
Glucokinase (liver, β cells): high Km, no glucose-6-phosphate inhibition, held by its regulatory protein; heterozygous loss = MODY2. Hexokinase: low Km, product-inhibited.
CK is an M/B dimer (MM muscle, MB heart, BB brain); LDH is an H/M tetramer (LDH-1 heart and red cells, LDH-5 liver and muscle).
MI today = troponin: a rise and/or fall with a value above the sex-specific 99th percentile, plus evidence of ischemia (2026 Universal Definition); CK-MB re-rise and the LDH flip are classic teaching.
ALP: GGT or 5′-nucleotidase decides liver vs bone; bone ALP is heat-labile; low ALP suggests hypophosphatasia.
Pairs to know: aldolase B, COX-1 vs COX-2, PK and PFK, muscle vs liver phosphorylase. More on the Biochemistry hub.