Enzymes

What types of reactions do enzymes catalyze?

• muscle contraction

• nerve conduction

• respiration

• digestion

• growth

• reproduction

• maintaining body temperature

The presence of enzymes in serum is typically due to:

the normal breakdown of body tissues

• enzymes are biomarkers for certain diseases and conditions (because they can be found in certain tissues)

List 4 important properties of enzymes

• enzymes are never permanently altered or consumed in reactions

• only small amounts of enzymes are needed in reactions (they are reused)

• they accelerate the rate of the reaction, but does not increase the product

• when equilibrium is reached, the same enzyme catalyzes forward and reverse reactions

apoenzyme

the protein component of an enzyme, NOT active without a cofactor

cofactor

a compound required for an enzyme’s function that binds to the apoenzyme

holoenzyme

apoenzyme + cofactor (unit)

active site

where the substrate binds to the enzyme

allosteric site

a specific location on an enzyme where regulatory molecules can bind, influencing the enzyme’s activity

Inhibitors decrease the rate of an enzymatic reaction. What are the 3 types of inhibitors?

• competitive inhibitor

  • very similar to substrates, but competes with the substrate for the active site

  • increased concentrations of substrates can stop these inhibitors

• noncompetitive inhibitor

  • inhibitor binds to an allosteric site, changing the structure of the enzyme so the substrate can’t bind

• uncompetitive inhibitor

  • inhibitor binds to the enzyme-substrate complex and prevents the release of product

How does temperature affect enzyme activity? How is it controlled during lab testing?

• low temperatures (<37) cause slow reactions

• high temperatures (>37) cause denaturation of the enzymes

• how is this controlled in the lab?

  • water baths keep cuvettes with samples at 37 C

How does pH affect enzyme activity? How is it controlled during lab testing?

• pHs below 7 and above 8 lead to denaturation

• how is it controlled?

  • reagents for enzyme tests have buffers to manage pH

How does substrate concentration affect enzyme activity?

• substrate concentration and rate of enzymatic reactions are directly proportional

  • low substrate = slow reaction

  • high substrate = faster reaction

What is the Michaelis-Menton curve?

• a graphical representation of the relationship between substrate concentration and reaction rate for enzymatic reactions

• reaction velocity (Vmax) increases with substrate concentration until it reaches saturation (point when all enzyme binding spots are occupied)

first order kinetics

• velocity of an enzymatic reaction is proportional to substrate concentration

• as the substrate concentration increases, the rate of reaction increases linearly until saturation

zero order kinetics

a plateau that occurs during enzymatic reactions, indicates that enzyme saturation is reached

How are enzymes measured in the lab?

• via absorbance methods

  • product formation (results in color change, which is measured)

  • or substrate depletion

What’s the difference between continuous assays and end-point assays for measuring enzyme activity?

• continuous

  • detector continuously measures the reaction and plots data points

• end-point

  • an initial point and end point are recorded, measurements are made after the reaction is completed

What are the three main classes of enzymes?

• oxidoreductases

  • catalyzes redox reactions between two substrates

• transferases

  • catalyzes the transfer of a group other than hydrogen between two substrates

• hydrolases

  • catalyzes the hydrolytic cleavage of a substrate

What are the three types of enzyme tissue specificity?

• high specificity

  • enzyme is predominantly found in one tissue

• moderate specificity

  • enzyme is widely distributed in the body

• low specificity

  • enzyme is found in most body tissues

isoenzyme

Different forms of an enzyme that catalyze the same reaction but have different properties

What type of enzyme is lactate dehydrogenase (LDH) and where can it be found?

• oxidoreductase involved in anaerobic glycolysis

• mostly found in the liver, heart, RBCs, and skeletal muscle

  • hemolyzed specimens increase LDH

When is LDH increased?

• cardiac isoenzymes increased in:

  • after an MI

  • in myocarditis

  • shock

  • CHF

• levels are also high in patients with megaloblastic anemia and pernicious anemia

How is LDH measured?

• by a forward and reverse reaction

  • forward: lactate to pyruvate and NADH

  • reverse: pyruvate to lactate and NAD+

What type of enzyme is creatine kinase (CK) and where can it be found in the body?

• transferase involved in phosphorylation of ATP

  • addition of phosphate to ATP

• found in skeletal muscle, brain tissue, and heart tissue

List the 3 CK isoenzymes (m=muscle and b=brain)

• CK-MM

  • found in skeletal and cardiac muscle

• CK-BB

  • found in the brain and CNS

• CK-MB

  • mostly found in cardiac tissue

How is CK measured?

assessment of the forward and reverse reaction

What is the purpose of measuring CK-MB?

• to evaluate acute myocardial injury after an MI

• peaks 24 hours after an MI and returns to normal 2-4 days after

In what conditions is CK increased?

• myocardial infarction

• muscular dystrophy

• rhabdomyolysis

What is the function of aspartate aminotransferase (AST) and where in the body can it be found?

• facilitates amino acid metabolism (synthesis and degradation)

• found in the liver (high), heart, skeletal muscles, and kidneys

In what conditions is AST increased?

• hepatocellular disorders

• MI

• muscle injury

• may also be elevated in CHF

Where is alanine aminotransferase (ALT) mostly found?

the liver (small amounts are found in the heart, kidneys, and skeletal muscle)

When is ALT increased?

• viral/toxic hepatitis

• obstructive liver disease

• hepatic cancer

• cirrhosis

Where is alkaline phosphatase (ALP) found and when is it increased?

• found in all tissues but is the highest in liver and bone

• increased in:

  • liver disease

  • bone disease

  • renal disease

  • pregnancy

Where can the enzyme gamma-glutamyltransferase (GGT) be found? When is it increased?

• liver, brain, prostate, pancreas, and kidneys

• increased in:

  • alcoholism

  • hepatobiliary disorders

  • pancreatitis

  • diabetes

  • MI

What is the function of amylase? Where is it formed? What is the purpose of measuring it?

• function: digest starch into smaller carbohydrates

• formed by the salivary glands and pancreas

• purpose: diagnosing pancreatitis

When is amylase increased?

• acute pancreatitis

• gastric and duodenal ulcers

• renal disease

• narcotics use

• mumps

What is lipase and when is it elevated?

• a pancreatic enzyme that hydrolyzes triglycerides into monoglycerol (alcohol and fatty acids)

• elevated in acute pancreatitis, pancreatic carcinoma, kidney disease, duodenal ulcers, and intestinal obstruction

How is lipase measrued?

turbidimetrically and spectrophotometrically

cholenesterase (ChE)

An enzyme that breaks down esters, mainly in the liver

• acetylcholenesterases are found in RBCs

• pseudocholinesterases are found in serum and the liver

Causes of decreased cholinesterase

• pesticide poisoning

• abnormal genetic variants