Week 7: Enzyme Kinetics

cofactor

1. nonprotein molecule necessary for enzyme activity

2. activator = inorganic (Mg)

3. coenzyme = organic (NAD+)

factors that influence enzymatic reactions

1. substrate concentration

2. enzyme concentration

3. pH

4. temperature

5. cofactors

6. inhibitors

common (concentration) measurements of enzyme activity

1. increased product

2. decreased substrate

3. decreased coenzyme

4. increased concentration of altered coenzyme

enzyme concentrations are always performed in ______-order kinetics

zero

in laboratory reactions, __________ must be lacking and other variables carefully controlled

inhibitors

types of measurement of enzymatic reactions

1. fixed time

2. continuous monitoring (kinetic assay)

creatine kinase CK

1. associated with ATP regeneration in contractile or transport systems

2. storage or high energy creating phosphate

tissue source of creatine kinase

1. skeletal muscle

2. heart muscle

3. brain tissue

creatine kinase CK: assay enzyme activity

catalyzes both forward and reverse reactions involving phosphorylation of creatine or ADP

creatine kinase CK analysis: source of error

1. hemolysis can elevate CK activity

2. CK is inactivated by light → dark storage

reference range of CK

1. male = 15-160 U/L

2. female = 15-130 U/L

enzymatic reaction rate is governed by:

1. enzyme concentration (rate is proportional)

2. substrate concentration

3. temperature

4. pH

michaelis-menten curve axes

1. x = substrate concentration

2. y = reaction velocity

michaelis-menten equation

v = Vmax*[S] / Km + [S]

michaelis constant

[S] at which v = half Vmax

decreased Km = ________ affinity

increased

first order kinetics

rate is proportional to the substrate concentration

zero order kinetics

rate is proportional to enzyme concentration

enzyme reaction phases

1. lag phase

2. linear phase

3. substrate depletion phase

lineweaver-burk plot intercepts

1. y = 1/Vmax

2. x = -1/Km

optimal pH of clinical enzyme activity

7-8

optimal temperature of clinical enzyme activity

37 C or 30 C

competitive inhibition: Km _________, Vmax __________

increases, unchanged

noncompetitive inhibition: Km ____________, Vmax __________

unchanged, decreases

activator ion examples

1. Mg2+

2. Fe2+

3. Ca2+

4. Cl-

activator ions can be ___________ or ___________ bound to enzymes

permanently, transiently

coenzyme examples

1. NAD+

2. NADP+

coenzymes are ___________ bound to enzymes

transiently

prosthetic groups

permanently bound coenzymes

pyridoxal phosphate PP is added to _______ and _______ substrate reagents

AST, ALT

coupled enzyme: primary reaction

1. rate limiting step

2. enzyme whose activity is to be determined

reaction catalyzed by creatine kinase

creatine phosphate + ADP → creatine + ATP (Mg++ cofactor)

coupled enzyme: auxiliary reaction

one or more subsequent reactions

coupled enzyme: indicator reaction

1. final reaction that produces product

2. absorbance measured

3. directly proportional to primary reaction

CK coupled enzymes: primary, auxiliary, and indicator enzymes

1. CK

2. HK

3. GPD

reaction catalyzed by hexokinase

ATP + glucose → 6-glucose-6-phosphate + ADP

reaction catalyzed by GPD

G-6-P + NADP+ → 6-phosphogluconate + NADPH

continuous monitoring can detect loss of __________ over reaction

linearity

steps if result is above linear range

1. reassay

2. less sample volume

3. dilute sample

4. reduce incubation time