Chemistry Unit #4

Biological Catalysts

large proteins, lowers activation energy, unchanged by reactions

Properties of Enzymes

not altered or consumed during reaction, effective in small amounts, accelerates the reaction to equillibrium

Isoform

different forms of the same protein

Isoenzyme

enzyme isoforms that catalyze the same reaction but differ in structure and tissue distribution

Zymogen (proenzyme)

secreted in structurally inactive form to prevent action until needed

Apoenzyme

protein portion and inactive without cofactor

Cofactor

compounds required for enzyme function, bound loose or tight with covalent bond (prosthetic group)

Holoenzyme

apoenzyme and cofactor

Allosteric Sites

where regulatory molecules bind

Active Site

where the substrate binds

Inhibitors

decreases the rate of reaction. May be reversible or permanent

Competitive

similar to substrates and competes for active sites

Noncompetitive

binds to allosteric site and changes structure of enzyme so it can’t bind substrate

Uncompetitive

binds to enzyme-substrate complex and prevents release of product

Increased enzymes

increased synthesis and increased cell injury/destruction causes this

Decreased enzymes

decreased synthesis and inherited deficiency causes this

Temperature affect on enzymes

optimal is 37 C 40-50 denaturation occurs

pH affect on enzymes

extreme pH denaturation occurs, with some exceptions (pepsin)

Substrate Concentration

the less concentration there is the slower the reaction

Enzyme Concentration

enzyme activity is directly proportional to enzyme this

Michaelis-Menten Curve

describes the relationship between substrate concentration and reaction velocity

Michaelis-Menten Curve Formula

V = Vmax(S)/Km + S

Feature of Km

unique to each enzyme and substrate pair and is measured at ½ Vmax

First Order Kinetics

velocity directly proportional to the substrate concentration

Zero Order Kinetics

velocity plateau

Fixed Time/Discontinuous/End Point Assay

reaction stopped at a specific time with addition of a weak acid to measure enzyme

Continuous Monitoring/Kinetic Assay

more common way to measure enzyme, takes multiple points

Activity Unit

amount of enzyme that produces 1 umol of product per minute under standard conditions

Alternative methods for enzyme measurement

electrophoresis and immunoassay are much more specific

Oxidoreductases

catalyze oxidation-reduction reactions between 2 substrates

Transferases

catalyze the transfer of a group other than hydrogen between 2 substrates

Hydrolases

catalyze hydrolytic cleavage of substrate

Example of Oxidoreductases

lactate dehydrogenase

Examples of transferases

creatine kinase

Example of Hydrolases

lipase and amylase

High Specificity

found predominately in one tissue

Moderate specificity

widely distributed in the body

Low specificity

found in most body tissues

Lactate Dehyrogenase Loaction

liver, heart, skeletal muscle, and RBC

Lactate Dehyrogenase Significance

MI, myocarditis, shock, CHF, pernicious anemia, and megalobastic anemia

Lactate Dehyrogenase Measurement

forward reaction measuring pyruvate and NADH at 340nm

Lactate Dehyrogenase Reference Range

100 - 224 U/L

Creatine Kinase Function

transferase involved in ATP regeneration in muscles, allows phosphate to store as energy

Creatine Kinase Location

skeletal muscle, brain, and heart

Creatine Kinase Significane

CK - MM (skeletal and cardiac muscle), CK- BB (brain and CNS), and CK - MB (cardiac muscle)

Creatine Kinase Measurement

reverse reaction that uses ATP, CK, and G-6-PD to measure NADPH at 340nm

Creatine Kinase Reference Range Male

46 - 171 U/L

Creatine Kinase Reference Range Female

34 - 145 U/L

Asparate Aminotransferase (AST) Function

transfer of amino groups between aspartate and keto acids, important in synthesis and degradation of amino acids

Aspartate Aminotransferase (AST) Location

liver, heart, skeletal muscle, and kidney

Asparate Aminotransferase (AST) Measurement

measures absorbance at 340 nm with Malate/NAD and Oxaloacetate and L-Glutamate

Asparate Aminotransferase (AST) Reference Range

5 - 35 U/L

Alanine Aminotransferse (ALT) Function

catalyzes transfer of amino group from alanine to ketoglutarate

Alanine Aminotransferse (ALT) Location

liver

Alanine Aminotransferse (ALT) Measurement

decrease in absorbance at 340nm using L-Lactate and NAD

Alanine Aminotransferse (ALT) Reference Range

7 - 45 U/L

Alkaline Phosphate (ALP) Function

frees inorganic phosphate from organic phosphate

Alkaline Phosphate (ALP) Location

found in almost all tissues

Alkaline Phosphate (ALP) Measurement

increase in absorbance at 405nm (yellow)

Alkaline Phosphate (ALP) Reference Range Male - Female 4-15yrs

54 - 369 U/L

Alkaline Phosphate (ALP) Reference Range Male

53 - 128 U/L

Alkaline Phosphate (ALP) Reference Female

42 - 98 U/L

Gamma Glutamyltransferase (GGT) Function

protein synthesis, regulation of tissue gluthaione, and transport of amino acids across membranes

Alkaline Phosphate (ALP) Location

liver, brain, prostate, pancreas, and kidney

Alkaline Phosphate (ALP) Elevated

alcoholism, hepatobiliary disorders, pancreatitis, and diabetes

Alkaline Phosphate (ALP) Measurement

increased absorbance at 405nm

Alkaline Phosphate (ALP) Reference Range Male

6 -55 U/L

Alkaline Phosphate (ALP) Referenc Range Female

5 - 38 U/L

Amylase Function

hydrolase involved in digestion of starch

Amylase Location

salivary glands and pancreas

Amylase elevation

gastric and duodenal ulcers, renal disease, narcotic use, and mumps

Amylase measurement

product hydrolyzed by amylase and product produces color change measured spectrophotometrically

Amylase Reference Range

30 - 220 U/L

Lipase function

catalyzes hydrolysis of triglycerides

Lipase location

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

Lipase measurement

measures rate of color formation spectrophotometrically

Lipase Reference Range

less than 38 U/L