(14) MEASUREMENT OF CATALYTIC ACTICITY

Question 1

• Dependent on enzyme concentration
• Always performed in zero-order kinetics
• Performed during the linear phase of rxn

Answer 1

MEASUREMENT OF CATALYTIC ACTIVITY

Question 1

↑product conc. = ↓in substrate conc.
↓ coenzyme conc. (NADH) = ↑ altered coenzyme conc.

Answer 1

Ex. ↓ NADH (reduced) = ↑ NAD (oxidized)
↑NADH (reduced) = ↓NAD (oxidized)

Question 2

↑absorbance of coenzyme @340nm

Answer 2

NADH: reduced

Question 3

NAD: oxidized

Answer 3

↓absorbance of coenzyme @340nm

Question 4

NAD: oxidized

Answer 4

↓absorbance of coenzyme @340nm

Question 5

NOTE:

Answer 5

o ↑Enzyme activity to convert substrate to product
o More product is formed = more substrate is converted
o ↑product conc. = ↓substrate conc. (inverse relationship)

Question 6

➢ Reagents are combined, rxn proceeds, rxn is stopped & measurement of rxn is measured
➢ Only 1 measurement.

Answer 6

FIXED-TIME (2-point assay)

Question 7

➢ Multiple enzyme activity is included in procedure
➢ Multiple measurements at specific time intervals or continuous measurement as absorbance changes. (30-60sec interval)
➢ Deviation from zero kinetics can be observed.

Answer 7

CONTINUOUS MONITORING / KINETIC ASSAYS

Question 8

• Enzyme of interest (target) in serum.

Answer 8

Primary enzyme

Question 9

• Its activity relies on primary enzyme activity
• Ex. Karmen method in AST for MI profile

Answer 9

Secondary / Coupling / Indicator enzyme

Question 10

Mathematical representation of relationship between velocity of enzymatic rxn & substrate conc.

Answer 10

MICHAELIS-MENTEN EQUATION

(refer to handout page 5)

Question 11

Amount of enzyme that will catalyze the reaction of 1μmol of substrate per minute (umol/min).

Answer 11

IU (EC)

Question 12

Amount of enzyme that will catalyze the reaction of 1mol of substrate per second (mol/s).

Answer 12

Kat (SI)

Question 13

Quantified by electrophoretic techniques that provides resolution of isoenzymes & isoforms.

Answer 13

Measurement of Enzyme Mass

Question 14

Enzyme-substrate binding would cause enzyme catalytic action to hasten conversion of substrate into a product.

Answer 14

Enzyme theories

Question 15

Shape of key (substrate) must fit into the lock (enzyme).

Answer 15

Emil Fisher’s/ Lock & Key theory

Question 16

Based on substrate binding to the active site of enzyme.

Answer 16

Kochland’s Induced Fit Theory